RU2787595C2 - Highly concentrated compositions of antibodies to c5 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to stable aqueous solutions containing an antibody to C5 in a high concentration and methods for the production of these solutions. The stable aqueous solution contains an antibody to C5 at a concentration of 100 mg/ml ± 5%, 50 mM ± 5% of a phosphate buffer, 5% ± 5% of sucrose. In another option, the solution also contains 0.05% ± 5% of polysorbate 80. The antibody to C5 contains a heavy chain with SEQ ID NO: 14 and a light chain with SEQ ID NO: 11. Methods for the treatment or prevention of disorders associated with C5 complement system, such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic-uremic syndrome (aHUS), using these solutions, are also proposed. Therapeutic kits containing the specified solution and a means for administration of the solution to a patient who needs treatment are also presented.

EFFECT: invention provides a stable highly concentrated aqueous solution of an antibody to C5, which remains for at least 97% monomeric and preserves at least 90% of its C5-binding activity during storage at 2-8°C for at least six months.

27 cl, 53 dwg, 23 tbl, 3 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under U.S. Provisional Application Serial No. 62/537,741, filed July 27, 2017. The entire contents of the aforementioned Provisional Patent Application are incorporated herein by reference.

SEQUENCE LIST

This application contains a sequence listing that has been filed electronically in ASCII format and is incorporated herein by reference in its entirety. The specified ASCII copy created on July 26, 2018 is named AXJ-226PC_SL.txt and is 32987 bytes in size.

BACKGROUND OF THE INVENTION

The complement system works in conjunction with other immune systems in the body to protect against invading cellular and viral pathogens. At least 25 complement proteins are known and have been found as a complex array of plasma proteins and membrane cofactors. Plasma proteins make up approximately 10% of the globulins in the blood serum of vertebrates. The components of the complement system perform their immune defense function through interactions in a series of complex but precise enzymatic cleavage and membrane binding events. The resulting cascade of the complement system leads to the formation of products with opsonic, immunoregulatory and lytic function. A brief description of the forms of biological activity associated with the activation of the complement system is presented, for example, in The Merck Manual, 16th ^Edition .

Although a properly functioning complement system provides strong protection against microbial challenge, inappropriate regulation or activation of complement system pathways has been implicated in the pathogenesis of a number of disorders, including paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) (see, e.g., Socié G, et al. , French Society of Haematology Haematet Lancet 1996;348(9027):573-577; Brodsky, R., Blood 2014;124(18):2804-2811); Hillmen, P., et al. , Am. J. Hematol. 2010;85(8):553-559; Caprioli et al. (2006) Blood 108 :1267-1279; and Kavanagh et al. (2006) British Medical Bulletin 77 and 78:5-22).

Patients with disorders associated with the complement system, such as PNH or aHUS, were at risk of significant morbidity and mortality. Accordingly, it is an object of the present invention to provide improved compositions and methods for the treatment of patients with disorders associated with the complement system.

BRIEF DESCRIPTION OF THE INVENTION

Provided herein are stable, highly concentrated aqueous solutions of anti-C5 antibodies, as well as methods for making and using the formulations. The present invention contemplates, among other aspects, formulation conditions suitable for maintaining the physical and functional stability of an anti-C5 antibody (e.g., ravulizumab, also known as "antibody BNJ441" and "ALXN1210") for a significant time in highly concentrated solutions. For example, the present invention provides formulation conditions capable of maintaining an anti-C5 antibody in predominantly monomeric form for up to 2 years at 2°C-8°C, even when the antibody is present in solutions at concentrations of about 100 mg/mL or higher. In addition, as described herein and illustrated in the illustrative examples, such formulations also minimize aggregation, fragmentation, or degradation of an anti-C5 antibody (e.g., ravulizumab) in highly concentrated solutions. For example, the present invention contemplates formulation conditions capable of maintaining an anti-C5 antibody in a highly concentrated form for two years without detectable antibody fragmentation or degradation (as determined by a size exclusion chromatography (SEC-HPLC) technique such as gel-penetrating HPLC) and with no more than 2% aggregates. Also provided herein are conditions suitable for formulating solutions of an anti-C5 antibody, such as ravulizumab, at concentrations greater than 200 mg/mL.

The advantages of stable highly concentrated aqueous solutions of anti-C5 antibody are numerous. First, for therapeutic applications requiring administration of an antibody to a patient in a small volume, therapeutic efficacy often depends on the amount of antibody that can be administered in that small volume. In the absence of the ability to formulate antibodies to C5 at high concentrations, the use of, for example, subcutaneous, intravitreal and/or intra-articular delivery routes will often be ruled out. Similarly, highly concentrated antibody formulations provide the patient with greater choice of routes of administration. In therapeutic routes that require frequent, continuous administration and/or self-delivery, administration is made possible by the high concentration of the formulations and may be more attractive to patients than intravenous infusion. For example, highly concentrated anti-C5 antibody formulations may allow the patient to self-administer the antibody, for example, by subcutaneous or intravenous injection. Thus, the ability to formulate antibodies at high concentrations may improve administration compliance by providing a simple home administration alternative for patients with complement disorders.

In addition, the methods for preparing aqueous solutions described herein do not require a freeze-drying step, just as the highly concentrated aqueous solutions presented do not require reconstitution from lyophilized material. The highly concentrated antibody solutions of the present invention provide several advantages over reconstituted lyophilized antibody formulations. First, healthcare professionals must reconstitute lyophilized antibody solutions on site under sterile conditions, which increases the possibility of microbial contamination of the solution prior to administration. In addition, the recovery requires considerable care to ensure that all solids contained in the recovery vessel are properly dissolved in the solution. The highly concentrated aqueous solutions provided herein thus provide the healthcare professional, caregiver, and/or patient with a quick, simple, safe, and effective means to deliver a therapeutic antibody to a patient in need thereof.

Other benefits of highly concentrated formulations include, for example, reducing the cost of production by reducing the space for bulk storage and / or the amount of product waste. In addition, the ability to produce a product with a longer shelf life ultimately requires fewer production runs, which ultimately reduces costs for the manufacturer and consumer of the highly concentrated therapeutic antibody.

An exemplary anti-C5 antibody is ravulizumab (also known as BNJ441 and ALXN1210) containing heavy and light chains having the sequences shown under SEQ ID NOs: 14 and 11, respectively, or antigen-binding fragments and variants thereof. In other embodiments, the antibody comprises heavy and light chain complementarity determining regions (CDRs) or variable regions (VR) of ravulizumab. Accordingly, in one embodiment, the antibody comprises the ravulizumab heavy chain variable region (VH) CDR1, CDR2 and CDR3 domains having the sequence shown under SEQ ID NO: 12 and the ravulizumab light chain variable region (VL) CDR1, CDR2 and CDR3 domains, having the sequence shown under SEQ ID NO: 8. In another embodiment, the antibody comprises the heavy chain CDR1, CDR2, and CDR3 sequences shown under SEQ ID NO: 19, 18, and 3, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown under SEQ ID NO: 4, 5 and 6, respectively.

In another embodiment, the antibody contains VH and VL regions having the amino acid sequences shown under SEQ ID NO: 12 and SEQ ID NO: 8, respectively.

In another embodiment, the antibody comprises the heavy chain constant region listed under SEQ ID NO: 13.

In another embodiment, the antibody comprises a human Fc constant region variant that binds to the neonatal human Fc receptor (FcRn), wherein the CH3 human Fc constant region variant contains Met-429-Leu and Asn-435-Ser substitutions at residues corresponding to methionine 428 and asparagine 434 of the native human IgG Fc constant region, each of which is listed according to EU numbering.

In another embodiment, the antibody comprises the heavy chain sequences CDR1, CDR2, and CDR3 listed under SEQ ID NOs: 19, 18, and 3, respectively, and the light chain sequences CDR1, CDR2, and CDR3, listed under SEQ ID NOs: 4, 5, and 6, respectively. , and a human Fc constant region variant that binds to the human neonatal Fc receptor (FcRn), wherein the human CH3 Fc constant region variant contains Met-429-Leu and Asn-435-Ser substitutions at residues corresponding to methionine 428 and asparagine 434 of native human IgG Fc constant region, each of which is identified according to the EU numbering.

In another embodiment, the antibody competes for binding and/or binds to the same epitope on C5 as the aforementioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with SEQ ID NO: 12 and SEQ ID NO: 8).

In another embodiment, the antibody binds to human C5 at pH 7.4 and 25°C with an affinity dissociation constant (K _D ) that is in the range 0.1 nm ≤ K _D ≤ 1 nm. In another embodiment, the antibody binds to human C5 at pH 6.0 and 25° C. with a K _D ≥ 10 nm. In yet another embodiment, [(K _D of the antibody or antigen-binding fragment thereof relative to human C5 at pH 6.0 and at 25°C)/(K _D of the antibody or antigen-negative fragment thereof relative to human C5 at pH 7.4 and at 25 °C)] antibody is more than 25.

In one aspect, a stable aqueous solution is provided (for example, sterile solution), where the solution contains an antibody to C5 at a concentration of approximately 100 mg/ml, where the antibody to C5 contains a heavy chain CDR1 containing the amino acid sequence listed under SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence listed under SEQ ID NO: 18, Heavy chain CDR3 containing the amino acid sequence listed under SEQ ID NO: 3, Light chain CDR1 containing the amino acid sequence listed under SEQ ID NO: 4, Light chain CDR2 containing the amino acid sequence listed under SEQ ID NO: : 5, and a light chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 6. In another embodiment, the solution contains an anti-C5 antibody (e.g., ravulizumab) at a concentration that is or is approximately 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205 .

In another embodiment, the stable aqueous solution contains one or more additional agents (for example, stabilizing agents, buffering agents, surfactants and/or preservatives). For example, in one embodiment, the stable aqueous solution contains a stabilizer. Illustrative stabilizers include, without limitation, polyols, sugars (e.g., sucrose or trehalose), amino acids (for example, arginine), amines and salting out salts. In one embodiment, the solution contains at least one stabilizing agent at a concentration of 2-10% inclusive. In one embodiment, the solution contains 5% sucrose. In another embodiment, the solution contains at least one or more stabilizing agents at a concentration of 10 mM to 50 mM, inclusive. In another embodiment, the stabilizing agent is present in solution at a concentration of at least or equal to 20 mM. In another embodiment, the stabilizing agent is present in solution at a concentration of at least or equal to 25 mM. In another embodiment, the stabilizing agent is present in solution at a concentration of at least or equal to 50 mM. In another embodiment, the solution contains 25 mM arginine.

In another embodiment, the solution contains at least one or more buffer agents. Non-limiting examples of typical buffers that may be contained in the wash(s) solution(s) include Tris (tris(hydroxymethyl)methylamine), bis-Tris, bis-Tris-propane, histidine, triethanolamine, diethanolamine, formate, acetate, MES ( 2-(N-morpholino)ethanesulfonic acid), phosphate, HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), citrate, MOPS (3-(N-morpholino)propanesulfonic acid), TAPS (3{[tris(hydroxymethyl )methyl]amino}propanesulfonic acid), bicine (N,N-bis(2-hydroxyethyl)glycine), tricine (N-tris(hydroxymethyl)methylglycine), TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), PIPES (piperazine-N, N'-bis(2-ethanesulfonic acid), cacodylate (dimethylarsic acid), SSC (sodium citrate saline), and sodium phosphate. In another embodiment, the buffering agent is an amino acid. The amino acid may be, for example, an amino acid selected from the group consisting of histidine (for example, L-histidine), serine (for example, L-serine) and glycine (for example, L-glycine). In another embodiment, the solution contains two or more buffer agents. In a particular embodiment, the buffering agent is sodium phosphate.

In another embodiment, the solution contains at least one or more buffering agents at a concentration of 10 mM to 300 mM, inclusive. In another embodiment, the solution contains at least one buffering agent at a concentration of 10 mM to 200 mM, inclusive. In another embodiment, the solution contains at least one buffering agent at a concentration of 10 mM to 100 mM, inclusive. In another embodiment, the solution contains at least one buffering agent at a concentration of 10 mM to 50 mM, inclusive. In another embodiment, the solution contains at least one buffering agent at a concentration of 20 mM to 50 mM, inclusive. In another embodiment, the buffering agent is present in solution at a concentration of at least or equal to 20 mM. In another embodiment, the buffering agent is present in solution at a concentration of at least or equal to 25 mM. In another embodiment, the buffering agent is present in solution at a concentration of at least or equal to 50 mM.

In another embodiment, the solution contains a carbohydrate excipient at a concentration of 0.1 to 5%. In one embodiment, the carbohydrate excipient is present in the solution at a concentration of at least or equal to 1.5%. In another embodiment, the carbohydrate excipient is present in solution at a concentration of at least or equal to 3%. The carbohydrate filler can be, for example, selected from the group consisting of sorbitol and mannitol. In another embodiment, the solution contains two or more carbohydrate excipients.

In another embodiment, the solution contains a surfactant. Surfactants suitable for use in the compositions of the present invention include, without limitation, fatty acid esters (for example, sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate), sorbitan trioleate, esters of glycerol and fatty acids (for example, glycerol monocaprylate, glycerol monomyristate, glycerol monostearate), polyglycerol fatty acid esters (e.g., decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate), polyoxyethylene esters of sorbitan fatty acids (for example, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate), polyoxyethylene sorbitan fatty acid esters (for example, polyoxyethylene sorbitan tetrastearate, polyoxyethylene sorbitan tetraoleate), polyoxyethylene esters of glycerol and fatty acids (for example, polyoxyethylene glyceryl monostearate), polyethylene glycol fatty acid esters (e.g. polyethylene glycol distearate), polyoxyethylene alkyl ethers (for example, polyoxyethylene lauryl ether), polyoxyethylene-polyoxypropylene alkyl ethers (for example, polyoxyethylene-polyoxypropylene glycol, polyoxyethylene-polyoxypropylene propyl ether, polyoxyethylene-polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ethers (for example, polyoxyethylene nonylphenyl ether), polyoxyethylene hydrogenated castor oils (for example, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylene wax derivatives (for example, polyoxyethylene sorbitol wax), polyoxyethylene derivatives of lanolin (for example, polyoxyethylenelanolin) and polyoxyethylene fatty acid amides (for example, polyoxyethylene stearic acid amide); C12-C18-alkyl sulfates (for example, sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate), polyoxyethylene-C10-C18-alkyl ether sulfate with the addition of an average of 2 to 4 moles of ethylene oxide units (for example, sodium polyoxyethylene lauryl sulfate) and salts of C10-C18 alkyl sulfosuccinate esters (e.g. sodium salt of lauryl sulfosuccinate ester); as well as natural surfactants such as lecithin, glycerophospholipid, sphingophospholipids (for example, sphingomyelin) and esters of sucrose and C12-C18 fatty acids.

In one embodiment, the surfactant in the formulation is a nonionic surfactant. In certain embodiments, the surfactant in the formulation is a polyoxyethylene sorbitan fatty acid ester, such as polysorbate 20, 40, 60, 80, or a combination of one or more of these. In one embodiment, the surfactant in the formulation is polysorbate 80 (Tween 80). In another embodiment, the surfactant in the formulation is Polysorbate 60. In another embodiment, the surfactant in the formulation is Polysorbate 40. In another embodiment, the surfactant in the formulation is Polysorbate 20 (Tween 20). The concentration of the surfactant in the solution may be, for example, from 0.001% to 0.02% inclusive. For example, the surfactant may be present in the composition in an amount of from about 0.001% to about 1%, or from about 0.001% to about 0.5%, or from about 0.01% to about 0.2%. In one embodiment, the aqueous solutions contain a surfactant concentration of at least or about 0.001 (e.g., at least or about 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0 .08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2 , 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0 .33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45 , 0.46, 0.47, 0.48, 0.49, 0.5 or more)%. In another embodiment, the aqueous solution contains no more than 0.2 (for example, not more than 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, 0.002 or 0.001) % pharmaceutically acceptable surface -active substance. In a specific embodiment, the surfactant is 0.05% polysorbate 80.

In another embodiment, the solution contains a preservative. Illustrative preservatives include, without limitation, benzyl alcohol, m-cresol, and phenol.

In one embodiment, the stable aqueous solution contains no more than five agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than four agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than three agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than two agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than one agent in addition to the anti-C5 antibody.

In another embodiment, the stable aqueous solution comprises an anti-C5 antibody comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, a heavy chain CDR3 comprising the amino acid sequence shown under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence shown under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence shown under SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence given under SEQ ID NO: 6 at a concentration of 100 ± 20 (for example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120) mg/ml; 50 ± 15 (for example, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 mM phosphate buffer; 5 ± 3 (for example, 2, 3, 4, 5, 6, 7 or 8)% sucrose and 25 ± 10 (e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34 or 35) mM arginine; where the solution has a pH of 7.4 ± 0.5 (for example, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9).

In another embodiment, the stable aqueous solution consists of an anti-C5 antibody that contains a heavy chain CDR1 containing the amino acid sequence listed under SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence listed under SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence listed under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence listed under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence listed under SEQ ID NO: 5, and a light chain CDR3, containing the amino acid sequence listed under SEQ ID NO: 6, at a concentration of 100 ± 20 (for example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120) mg/ml; 50 ± 15 (for example, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 mM phosphate buffer; 5 ± 3 (for example, 2, 3, 4, 5, 6, 7 or 8)% sucrose and 25 ± 10 (e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34 or 35) mM arginine; where the solution has a pH of 7.4 ± 0.5 (for example, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9).

In another embodiment, the stable aqueous solution comprises an anti-C5 antibody comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, a heavy chain CDR3 comprising the amino acid sequence shown under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence shown under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence shown under SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence given under SEQ ID NO: 6 at a concentration of 100 ± 20 (for example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120) mg/ml; 50 ± 15 (for example, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 mM phosphate buffer; 5 ± 3 (for example, 2, 3, 4, 5, 6, 7 or 8)% sucrose; 25 ± 10 (e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) mM arginine ; as well as 0.05 ± 0.03 (for example, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 and 0.08)% polysorbate 80, where the solution has a pH of 7.4 ± 0.5 (for example, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9).

In another embodiment, the stable aqueous solution consists of an anti-C5 antibody that contains a heavy chain CDR1 containing the amino acid sequence listed under SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence listed under SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence listed under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence listed under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence listed under SEQ ID NO: 5, and a light chain CDR3, containing the amino acid sequence listed under SEQ ID NO: 6, at a concentration of 100 ± 20 (for example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120) mg/ml; 50 ± 15 (for example, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 mM phosphate buffer; 5 ± 3 (for example, 2, 3, 4, 5, 6, 7 or 8)% sucrose; 25 ± 10 (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) mM arginine; as well as 0.05 ± 0.03 (for example, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 and 0.08)% polysorbate 80, where the solution has a pH of 7, 4 ± 0.5 (for example, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9).

In another embodiment, a stable aqueous solution is provided (for example, sterile solution), where the solution contains (a) an anti-C5 antibody (for example, ravulizumab), (b) about 50 mM phosphate buffer; (c) about 5% sucrose; and (d) about 25 mM arginine. In another embodiment, a stable aqueous solution is provided (for example, sterile solution), where the solution contains (a) an anti-C5 antibody (for example, ravulizumab) at a concentration of approximately 100 mg/ml, (b) approximately 50 mm phosphate buffer; (c) about 5% sucrose; and (d) about 25 mM arginine. In another embodiment, the stable aqueous solution contains a) an anti-C5 antibody (e.g., ravulizumab), (b) 50 mM phosphate buffer; (c) 5% sucrose; and (d) 25 mM arginine. In another embodiment, the stable aqueous solution contains (a) an anti-C5 antibody (e.g., ravulizumab) at a concentration of 100 mg/ml, (b) 50 mm phosphate buffer; (c) 5% sucrose; and (d) 25 mM arginine.

In another embodiment, the stable aqueous solution contains (a) anti-C5 antibody, (b) about 50 mM phosphate buffer, (c) about 5% sucrose, (d) about 0.05% polysorbate 80, and (e) about 25 mM arginine . In another embodiment, the stable aqueous solution contains (a) about 100 mg/ml anti-C5 antibody, (b) about 50 mM phosphate buffer, (c) about 5% sucrose, (d) about 0.05% polysorbate 80, and (e) approximately 25 mM arginine. In another embodiment, the stable aqueous solution contains (a) anti-C5 antibody, (b) 50 mM phosphate buffer, (c) 5% sucrose, (d) 0.05% polysorbate 80, and (e) 25 mM arginine.

In another embodiment, the stable aqueous solution contains (a) anti-C5 antibody, (b) 50 mM phosphate buffer, (c) 5% sucrose, (d) 0.05% polysorbate 80, and (e) 25 mM arginine. In another embodiment, the stable aqueous solution contains (a) 100 mg/ml anti-C5 antibody, (b) 50 mM phosphate buffer, (c) 5% sucrose, (d) 0.05% polysorbate 80, and (e) 25 mM arginine. In another embodiment, the stable aqueous solution contains (a) 100 mg/ml anti-C5 antibody, (b) 50 mM phosphate buffer, (c) 5% sucrose, (d) 0.05% polysorbate 80, and (e) 25 mM arginine.

In another embodiment, the stable aqueous solution contains no more than four agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than three agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than two agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than one agent in addition to the anti-C5 antibody.

In another embodiment, the stable aqueous solution consists of (a) about 100 mg/ml anti-C5 antibody, (b) about 50 mM phosphate buffer, (c) about 5% sucrose, and (d) about 25 mM arginine. In another embodiment, the stable aqueous solution consists of (a) 100 mg/ml anti-C5 antibody, (b) 50 mM phosphate buffer; (c) 5% sucrose; and (d) 25 mM arginine.

In another embodiment, the stable aqueous solution consists of (a) about 100 mg/ml anti-C5 antibody, (b) about 50 mM phosphate buffer, (c) about 5% sucrose; (d) about 0.05% polysorbate 80; and (e) about 25 mM arginine. In another embodiment, the stable aqueous solution consists of (a) 100 mg/ml anti-C5 antibody, (b) 50 mM phosphate buffer, (c) 5% sucrose, (d) 0.05% polysorbate 80, and (e) 25 mM arginine.

In one embodiment, the stable aqueous solution contains (a) an anti-C5 antibody at a concentration of approximately 100 mg/mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 comprising the amino acid sequence shown under SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence , shown under SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence shown under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence shown under SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 6, (b) approximately 50 mm phosphate buffer, (c) approximately 5% sucrose and (d) approximately 25 mm arginine.

In another embodiment, the stable aqueous solution contains (a) an anti-C5 antibody at a concentration of 100 mg/mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 comprising the amino acid sequence shown under SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence, listed under SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence listed under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence listed under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence listed under SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 6, (b) 50 mM phosphate buffer, (c) 5% sucrose, and (d) 25 mM arginine.

In another embodiment, the stable aqueous solution comprises (a) an anti-C5 antibody at a concentration of approximately 100 mg/mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 comprising the amino acid sequence shown under SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence , shown under SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence shown under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence shown under SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 6, (b) approximately 50 mm phosphate buffer, (c) approximately 5% sucrose, (d) approximately 0.05% polysorbate 80 and (e) approximately 25 mM arginine.

In another embodiment, the stable aqueous solution contains (a) an anti-C5 antibody at a concentration of 100 mg/mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 comprising the amino acid sequence shown under SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence, listed under SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence listed under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence listed under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence listed under SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 6, (b) 50 mM phosphate buffer, (c) 5% sucrose, (d) 0.05% polysorbate 80, and (e ) about 25 mM arginine.

In another embodiment, the stable aqueous solution consists of (a) an anti-C5 antibody at a concentration of approximately 100 mg/mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 comprising the amino acid sequence shown under SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid the sequence shown under SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence shown under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence, listed under SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence listed under SEQ ID NO: 6, (b) approximately 50 mm phosphate buffer, (c) approximately 5% sucrose, (d) approximately 0.05% polysorbate 80 and (e) about 25 mM arginine.

In another embodiment, the stable aqueous solution consists of (a) an anti-C5 antibody at a concentration of 100 mg/mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 containing the amino acid sequence shown under SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence , shown under SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence shown under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence shown under SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 6, (b) 50 mm phosphate buffer, (c) 5% sucrose, (d) 0.05% polysorbate 80 and ( e) 25 mM arginine.

In one embodiment, the pH is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9. In another embodiment, the pH of the solution is from 7.0 to 7.4. In another embodiment, the pH of the solution is from 7.2 to 7.8. In another embodiment, the pH of the solution is from 7.2 to 7.6. In a specific embodiment, the pH of the solution is 7.4.

The solutions described herein can be formulated for any suitable route of administration. In one embodiment, the solution is formulated for parenteral administration (e.g., by intravenous, subcutaneous, intraperitoneal or intramuscular injection). In a particular embodiment, the solution is formulated for subcutaneous administration. For example, in one embodiment, the stable aqueous solution contains an anti-C5 antibody at a concentration of 100 mg/mL and is formulated for subcutaneous administration. In another specific embodiment, the solution is formulated for intravenous administration. For example, in one embodiment, the stable aqueous solution contains an anti-C5 antibody at a concentration of 100 mg/ml and is formulated for intravenous administration.

In one embodiment of any of the solutions described herein, an anti-C5 antibody (e.g., ravulizumab) remains at least 95% (e.g., at at least 96, 97, 98, or 99%) monomeric when stored at 2°C-8°C for at least six months as determined by SEC-HPLC (e.g., gel permeation HPLC). In another embodiment, the anti-C5 antibody remains at least 95% (e.g., at least 96, 97, 98 or 99%) monomeric during storage at 2°C-8°C for at least nine months as determined by SEC-HPLC. In another embodiment, the anti-C5 antibody remains at least 95% (e.g., at least 96, 97, 98 or 99%) monomeric during storage at 2°C-8°C for at least one year as determined by SEC-HPLC. In another embodiment, the anti-C5 antibody remains at least 95% (e.g., at least 96%, 97%, 98%, or 99%) monomeric during storage at 2°C-8°C for at least 18 months at determination by SEC-HPLC. In another embodiment, the anti-C5 antibody remains at least 95% (e.g., at least 96%, 97%, 98%, or 99%) monomeric during storage at 2°C-8°C for at least two years at determination by SEC-HPLC.

In another embodiment of any of the solutions described herein, less than 5% of the anti-C5 antibodies (eg ravulizumab ) in the solution are aggregated when determined by SEC-HPLC (eg gel permeation HPLC). In another embodiment, less than 4% of the anti-C5 antibodies in solution are aggregated as determined by SEC-HPLC. In another embodiment, less than 3% of the anti-C5 antibodies in solution are aggregated as determined by SEC-HPLC. In another embodiment, less than 2% of the anti-C5 antibodies in solution are aggregated as determined by SEC-HPLC. In another embodiment, less than 1% of the anti-C5 antibodies in solution are aggregated as determined by SEC-HPLC.

In another embodiment of any of the solutions described herein, an anti-C5 antibody (e.g. , ravulizumab) retains at least 80% (e.g. , at least 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of its C5 binding activity during storage at 2°C-8°C for at least six months compared to a reference antibody to C5, corresponding to the antibody to C5 before storage. In another embodiment, an anti-C5 antibody (e.g. ravulizumab ) retains at least 80% (e.g. at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of its C5 binding activity during storage at 2°C-8°C for at least nine months compared to a C5 reference antibody corresponding to an anti-C5 antibody prior to storage . In another embodiment, an anti-C5 antibody (e.g. ravulizumab ) retains at least 80% (e.g. at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of its C5 binding activity during storage at 2°C-8°C for at least one year compared to a reference anti-C5 antibody corresponding to an anti-C5 antibody prior to storage . In another embodiment, an anti-C5 antibody (e.g. ravulizumab ) retains at least 80% (e.g. at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of its C5 binding activity during storage at 2°C-8°C for at least eighteen months compared to a C5 reference antibody corresponding to an anti-C5 antibody prior to storage . In another embodiment, an anti-C5 antibody (e.g. ravulizumab ) retains at least 80% (e.g. at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of its C5 binding activity during storage at 2°C-8°C for at least two years compared to a C5 reference antibody corresponding to an anti-C5 antibody prior to storage . In another embodiment, an anti-C5 antibody (e.g. ravulizumab ) retains at least 80% (e.g. at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of its C5 binding activity during storage at 2°C-8°C for at least three years compared to a C5 reference antibody corresponding to an anti-C5 antibody prior to storage .

In another embodiment of any of the solutions described herein, an anti-C5 antibody (e.g. , ravulizumab) retains at least 80% (e.g. , at least 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of its ability to inhibit hemolysis during storage at 2°C-8°C for at least nine months compared to a reference antibody to C5 corresponding to the anti-C5 antibody before storage. In another embodiment of any of the solutions described herein, an anti-C5 antibody (e.g. , ravulizumab) retains at least 80% (e.g. , at least 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of its ability to inhibit hemolysis during storage at 2°C-8°C for at least six months compared to a reference antibody to C5 corresponding to the anti-C5 antibody before storage. In another embodiment of any of the solutions described herein, an anti-C5 antibody (e.g. , ravulizumab) retains at least 80% (e.g. , at least 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of its ability to inhibit hemolysis during storage at 2°C-8°C for at least one year compared to a reference antibody to C5 corresponding to the anti-C5 antibody before storage. In another embodiment of any of the solutions described herein, an anti-C5 antibody (e.g. , ravulizumab) retains at least 80% (e.g. , at least 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of its ability to inhibit hemolysis during storage at 2°C-8°C for at least 18 months compared to a reference antibody to C5 corresponding to the anti-C5 antibody before storage. In another embodiment of any of the solutions described herein, an anti-C5 antibody (e.g. , ravulizumab) retains at least 80% (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of its ability to inhibit hemolysis during storage at 2°C-8°C for at least two years compared to a reference antibody to C5 corresponding to the anti-C5 antibody before storage. In another embodiment of any of the solutions described herein, an anti-C5 antibody (e.g. , ravulizumab) retains at least 80% (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of its ability to inhibit hemolysis during storage at 2°C-8°C for at least three years compared to a reference antibody to C5 corresponding to the anti-C5 antibody before storage.

In another aspect, methods are provided for preparing a stable concentrated antibody solution containing an anti-C5 antibody at a concentration of 100 mg/ml, 50 mM phosphate buffer, 5% sucrose and 25 mM arginine, the method comprising:

i) obtaining a first aqueous solution containing an antibody to C5, while the first aqueous solution has a first composition and contains no more than 10 mg/ml of antibody to C5;

ii) diafiltering the first aqueous solution to obtain a composition containing 50 mM phosphate buffer, 5% sucrose and 25 mM arginine at pH 7.4, thereby obtaining a second aqueous solution, while the second aqueous solution has a second composition as a result of diafiltration ; and

iii) concentration of the second aqueous solution to obtain a stable concentrated antibody solution containing 100 mg/ml anti-C5 antibody, 50 mM phosphate buffer, 5% sucrose and 25 mM arginine.

In another embodiment, a method is provided for preparing a stable concentrated antibody solution containing an antibody to C5 at a concentration of 100 mg/ml, 50 mm phosphate buffer, 5% sucrose; 25 mM arginine and 0.05% polysorbate 80, the method comprising:

i) obtaining a first aqueous solution containing an antibody to C5, while the first aqueous solution has a first composition and contains no more than 10 mg/ml of antibody to C5;

ii) diafiltration of the first aqueous solution to obtain a composition containing 50 mM phosphate buffer, 5% sucrose, 25 mM arginine and 0.05% polysorbate 80 at pH 7.4, thereby providing a second aqueous solution, while the second aqueous the solution has a second composition as a result of diafiltration; and

iii) concentration of the second aqueous solution to obtain a stable concentrated antibody solution containing 100 mg/ml anti-C5 antibody, 50 mM phosphate buffer, 5% sucrose, 25 mM arginine and 0.05% polysorbate 80.

Also provided are methods of treating a human patient with a condition associated with the complement system, comprising administering to the patient a stable aqueous solution (e.g. , subcutaneously or intravenously) described herein in an amount effective to treat the condition associated with the complement system. Illustrative conditions associated with the complement system include, but are not limited to, rheumatoid arthritis, antiphospholipid antibody syndrome, lupus nephritis, ischemic/reperfusion injury, atypical hemolytic uremic syndrome (aHUS), typical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria (PNH), dense disease sediment, neuromyelitis optica, multifocal motor neuropathy, multiple sclerosis, macular degeneration, HELLP syndrome, spontaneous abortion, thrombotic thrombocytopenic purpura, immune-deficient vasculitis, epidermolysis bullosa, recurrent miscarriage, traumatic brain injury, myocarditis, cerebrovascular disorder, peripheral vascular disorder, renovascular disorder , mesenteric/enteric vascular disorder, vasculitis, Henoch-Schonlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis, immune complex vasculitis, Takayasu's disease, dila cardiomyopathy, diabetic angiopathy, Kawasaki disease, venous gas embolism, restenosis after stenting, rotational atherectomy, percutaneous transluminal coronary angioplasty, myasthenia gravis, cold agglutinin disease, dermatomyositis, paroxysmal cold hemoglobinuria, antiphospholipid syndrome, Graves' disease, atherosclerosis, septicemia systemic inflammatory response, septic shock, spinal cord injury, glomerulonephritis, transplant rejection, Hashimoto's thyroiditis, type I diabetes mellitus, psoriasis, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpasture's syndrome, Degos' disease, and catastrophic antiphospholipid syndrome. In a specific embodiment, the condition associated with the complement system is atypical hemolytic uremic syndrome (aHUS). In another embodiment, the condition associated with the complement system is paroxysmal nocturnal hemoglobinuria (PNH).

Additionally, kits are provided that contain the stable aqueous solution described herein in a therapeutically effective amount adapted for use in the methods described herein. In one embodiment, the kit contains: (i) any of the solutions described herein; and (ii) a means for delivering the solution to a patient in need thereof (eg , a syringe). In one embodiment, the agent is suitable for subcutaneous delivery of a solution to a patient. In one embodiment, the agent is suitable for intravenous delivery of a solution to a patient. In another embodiment, the kits further comprise at least one additional active agent for use in the treatment of a complement disorder in a subject.

BRIEF DESCRIPTION OF GRAPHICS

Figure 1 shows the results of dynamic light scattering for the titration of ravulizumab (ALXN1210) at 50 mg/ml salt with histidine buffer exchange.

Figure 2 shows the results of dynamic light scattering for the titration of ravulizumab (ALXN1210) at 50 mg/ml L-arginine with buffer exchange.

Figure 3 shows dynamic light scattering results for a titration of ravulizumab (ALXN1210) at 50 mg/mL salt with phosphate buffer exchange.

Figure 4 shows differential scanning fluorimetry results of ravulizumab (ALXN1210) at 50 mg/mL with buffer exchange.

Figure 5 shows dynamic light scattering results for ravulizumab (ALXN1210) at 10 mg/mL and 114 mg/mL without L-arginine and at 114 mg/mL with L-arginine added.

Figure 6 shows stability data for ravulizumab (ALXN1210) (T=0 to T=2 weeks inclusive at 2-8°C).

Figure 7 shows stability data for ravulizumab (ALXN1210) (from T=3 weeks to T=2 months inclusive at 2-8°C).

Figure 8 shows stability data for ravulizumab (ALXN1210) (T=0 to T=3 weeks inclusive at 23-27°C).

Figure 9 shows stability data for ravulizumab (ALXN1210) (from T=1 month to T=2 months inclusive at 23-27°C).

Figure 10 shows stability data for ravulizumab (ALXN1210) (T=1 week to T=3 weeks inclusive at 37°C).

Figure 11 shows stability data for ravulizumab (ALXN1210) (from T=1 month to T=2 months inclusive at 37°C).

Figure 12 shows stability data obtained by size exclusion chromatography (SEC) as % monomers for ravulizumab (ALXN1210) (T=0 to T=2 months inclusive at 2-8°C).

13 shows stability data obtained by size exclusion chromatography (SEC) as % monomers for ravulizumab (ALXN1210) (T=0 to T=2 months inclusive at 23-27°C).

Figure 14 shows stability data obtained by size exclusion chromatography (SEC) as % monomers for ravulizumab (ALXN1210) (T=0 to T=2 months inclusive at 37°C).

Figure 15 shows dynamic light scattering stability data for ravulizumab (ALXN1210) in samples with histidine (T=0).

Figure 16 shows dynamic light scattering stability data for samples of ravulizumab AS in histidine buffer (ALXN1210) (T=0).

Figure 17 shows dynamic light scattering stability data for ravulizumab samples in phosphate buffer (ALXN1210) (T=0).

Figure 18 shows dynamic light scattering stability data for samples of ravulizumab (ALXN1210) in phosphate buffer (T=2 months, 2-8°C).

Figure 19 shows freeze-thaw (ALXN1210) ravulizumab stability data (from T=0 to cycle 2 inclusive at T=1 month, -20°C).

The figure 20 shows the stability data of ravulizumab (ALXN1210) obtained during cycles 3-5 freeze-thaw inclusive at T=1 month, -20°C.

The figure 21 shows the stability data obtained using size exclusion chromatography (SEC), as % monomers for ravulizumab (ALXN1210) (freeze-thaw at T=1 month, -20°C).

The figure 22 shows the stability data of the prototypes for ravulizumab (ALXN1210) (from T=0 to T=2 months inclusive at 2-8°C).

The figure 23 shows the stability data of the prototypes for ravulizumab (ALXN1210) (c T=3 months to T=6 months inclusive at 2-8°C).

The figure 24 shows the stability data of the prototypes for ravulizumab (ALXN1210) (from T=9 months to T=12 months inclusive at 2-8°C).

The figure 25 shows the stability data of the prototypes for ravulizumab (ALXN1210) (from T=1 month to T=2 months inclusive at 23-27°C).

The figure 26 shows the stability data of the prototypes for ravulizumab (ALXN1210) (from T=3 months to T=6 months inclusive at 23-27°C).

The figure 27 shows the stability data of the prototypes for ravulizumab (ALXN1210) (from T=9 months to T=12 months inclusive at 23-27°C).

Figure 28 shows the stability data of the prototypes for ravulizumab (ALXN1210) (from T=2 weeks to T=2 months inclusive at 37°C).

Figure 29 shows the stability data of the prototypes for ravulizumab (ALXN1210) (from T=1 month to T=3 months inclusive at -20°C).

Figure 30 shows the stability data of the prototypes for ravulizumab (ALXN1210) (from T=6 months to T=12 months inclusive at -20°C).

Figure 31 shows the results of a stability study of prototypes for ravulizumab (ALXN1210) (from T=3 months to T=6 months inclusive at -80°C).

Figure 32 shows the results of a stability study of prototypes for ravulizumab (ALXN1210) (T=12 months at -80°C).

Figure 33 shows the stability data of the prototypes obtained using size exclusion chromatography (SEC), as % monomers for ravulizumab (ALXN1210) (T=0 to T=12 months inclusive at 2-8°C).

Figure 34 shows SEC stability data for prototypes as % monomers for ravulizumab (ALXN1210) (T=0 to T=12 months inclusive at 23-27°C).

Figure 35 shows SEC stability data for prototypes as % monomers for ravulizumab (ALXN1210) (T=0 to T=12 months inclusive at 37°C).

Figure 36 shows the stability data of the prototypes obtained using size exclusion chromatography (SEC), as % monomers for ravulizumab (ALXN1210) (from T=0 to T=12 months inclusive at -20°C).

Figure 37 shows the stability data of the prototypes obtained using size exclusion chromatography (SEC), as % monomers for ravulizumab (ALXN1210) (from T=0 to T=12 months inclusive at -80°C).

Figure 38 shows dynamic light scattering test sample stability data for samples of ravulizumab ALXN1210 at 75 mg/mL in phosphate buffer (T=0).

Figure 39 shows dynamic light scattering test sample stability data for samples of ravulizumab ALXN1210 at 75mg/ml in phosphate buffer (T=1 month at 2-8°C).

Figure 40 shows dynamic light scattering test sample stability data for samples of ravulizumab ALXN1210 at 100 mg/ml in phosphate buffer (T=1 month at 2-8°C).

Figure 41 shows SEC stability data for prototypes as % monomers for ravulizumab (ALXN1210) in freeze-thaw cycles 1-3 at T=1 month, -20°C.

Figure 42 shows SEC stability data for prototypes as % monomers for ravulizumab (ALXN1210) in cycles 4-5 freeze-thaw at T=1 month, -20°C.

Figure 43 shows SEC stability data for prototypes as % monomers for ravulizumab (ALXN1210) in freeze-thaw cycles 1-3 at T=3 months, -80°C.

Figure 44 shows SEC stability data for prototypes as % monomers for ravulizumab (ALXN1210) in cycles 4-5 freeze-thaw at T=3 months, -80°C.

Figure 45 shows SEC stability data for prototypes as % monomers for ravulizumab (ALXN1210) in freeze-thaw cycles 1-5 at T=1 month, -20°C.

Figure 46 shows SEC stability data for prototypes as % monomers for ravulizumab (ALXN1210) in freeze-thaw cycles 1-5 at T=3 months, -80°C.

Figure 47 is an outline of a phase 1 study designed to evaluate the safety, tolerability, PK, PD, and immunogenicity of a single dose of 400 mg of ravulizumab (ALXN1210) given subcutaneously versus a single dose of 400 mg of ravulizumab (ALXN1210) given intravenously, or placebo administered subcutaneously in 42 healthy subjects.

Figure 48 provides a general description of the distribution of all subjects.

Figure 49 is a graph showing individual serum concentrations of ALXN1210 versus nominal time using a linear scale.

Figure 50 is a graph showing individual serum concentrations of ALXN1210 versus nominal time using a linear-log scale.

Figure 51 is a graph showing the percentage change in mean (±SD) serum free C5 concentration from baseline over time for subjects treated with placebo SC, ALXN1210 SC and ALXN1210 IV.

Figure 52 is a graph showing the percentage change in mean (± SD) serum free C5 concentrations from baseline over time for subjects treated with placebo SC, ALXN1210 SC and ALXN1210 IV.

Figure 53 is a graph showing the percent change in mean (± SD) degree of hemolysis of chick erythrocytes (cRBC) from baseline over time for subjects treated with placebo SC, ALXN1210 SC and ALXN1210 IV.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides stable aqueous solutions containing a high concentration of an anti-C5 antibody (eg ravulizumab ). The solutions can be used in various therapeutic applications, such as methods for the treatment or prevention of disorders associated with the complement system. Illustrative solutions, formulations, therapeutic kits, and methods for preparing and using any of the above are disclosed below and illustrated in the illustrative examples, and are not intended to be limiting in any way.

I. Definitions

Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as is commonly understood by a person skilled in the art. While any methods and compositions similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and compositions are described herein.

The singular forms include references to the plural unless the context clearly requires otherwise.

The term "approximately", in particular in relation to a given amount or number, is meant to cover variations within plus or minus ten percent (± 10%) , (eg ± 5%).

The term "pharmaceutical formulation" refers to preparations that are in such a form as to provide a uniquely effective biological activity of the active ingredients, and which do not contain additional components that have significant toxicity to the subjects to whom the formulation will be administered.

In the context of this document, an "aqueous" pharmaceutical composition is a composition suitable for pharmaceutical use, where the aqueous carrier is water. A composition suitable for pharmaceutical use may be sterile, uniform and/or isotonic. Aqueous pharmaceutical compositions may be prepared directly in aqueous form and/or may be reconstituted from a lyophilisate.

An "isotonic" formulation is one that has substantially the same osmotic pressure as human blood. Isotonic formulations will typically have an osmotic pressure of about 275 to 350 mOsm/kg. The term "hypotonic" describes a formulation with an osmotic pressure lower than that of human blood. Accordingly, the term "hypertonic" is used to describe a formulation with an osmotic pressure higher than that of human blood. Isotonicity can be measured, for example, with a vapor pressure osmometer or with a freezing point osmometer. A "tonicity agent" is a compound that renders the formulation isotonic.

In the context of this document, the "osmolality" of a solution is the number of osmoles of a solute per kilogram of solvent. Osmolality is a measure of the number of particles present in a solution and is independent of particle size or weight. It can only be measured using the property of the solution, which depends only on the concentration of the particles. These properties are vapor pressure reduction, freezing point depression, boiling point increase, and osmotic pressure, and are collectively referred to as colligative properties.

A "sterile" formulation is aseptic or free or substantially free of any living microorganisms and their spores.

A "stable" formulation, as used herein, is one in which an antibody substantially retains its physical stability and/or chemical stability and/or biological activity upon storage. Various analytical procedures for measuring protein stability are available in the art and are discussed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). The stability of anti-C5 antibody formulations can be measured at selected temperatures after selected time periods. For example, an increase in the formation of aggregates after storage is an indicator of the instability of the aqueous composition of antibodies to C5. In addition to aggregation, retention of original clarity, color, and odor throughout shelf life are indicators used to monitor the stability of aqueous solutions of the anti-C5 antibodies described herein.

An antibody "retains its physical stability" in a pharmaceutical formulation if it shows little or no evidence of aggregation, precipitation, and/or denaturation when visually examined for color and/or clarity, or measured by UV light scattering or size exclusion chromatography.

The term "aggregation" refers to the assembly of native folded proteins to form aggregates containing non-native structures. Aggregation can occur even under physiological, non-denaturing conditions and is often irreversible, resulting in the formation of non-native aggregates that are inactive and sometimes immunogenic and toxic.

The phrase "low to undetectable levels of aggregation" as used herein refers to samples containing no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, no more than than about 1% and not more than about 0.5% aggregates by weight of protein as measured by high performance gel permeation liquid chromatography (GP-HPLC), high performance size exclusion chromatography (HPSEC), or static light scattering (SLS) techniques.

An antibody "retains its chemical stability" in a pharmaceutical formulation if the chemical stability at a given point in time is such that the antibody is still considered to retain its biological activity, as defined below. Chemical stability can be assessed by identifying and quantifying chemically altered forms of the antibody. Chemical changes may include size modification (e.g. truncation) , deamidation, racemization, hydrolysis, oxidation, beta-elimination and disulfide exchange, which can be assessed using known techniques, for example, size exclusion chromatography, SDS-PAGE, matrix-assisted laser desorption - ionization/time-of-flight mass spectrometry (MALDI/TOF MS) and/or ion exchange chromatography.

An antibody "retains its biological activity" in a pharmaceutical formulation if the antibody in the pharmaceutical formulation is biologically active for its intended purpose. For example, biological activity is maintained if the biological activity of an antibody in a pharmaceutical formulation is within about 30%, about 20%, or about 10% (within assay error) of the biological activity exhibited at the time the pharmaceutical dosage form was prepared (e.g., as determined in the analysis antigen binding). As used herein, the "biological activity" of a monoclonal antibody refers to the ability of the antibody to bind to an antigen. It may further include the binding of an antibody to an antigen and result in a measurable biological response that can be measured in vitro or in vivo .

The "shelf life" of a pharmaceutical product, such as an aqueous solution containing an anti-C5 antibody, is the period of time the product is stored before degradation occurs. For example, expiration date can be defined as the time to degrade 0.1%, 0.5%, 1%, 5%, or 10% of a product.

As used herein, the term "antibody" describes polypeptides containing at least one antigen-binding site derived from an antibody (eg , VH/VL region, or Fv, or CDR). Antibodies include known forms of antibodies. For example, the antibody may be a human antibody, a humanized antibody, a bispecific antibody, or a chimeric antibody. The antibody can also be a Fab, Fab'2, ScFv, SMIP, Affibody®, nanobody or domain antibody. The antibody may also be of any of the following isotypes: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secretory IgA, IgD, and IgE. An antibody may be a naturally occurring antibody, or may be an antibody that has been altered by protein engineering technology (eg , mutation, deletion, substitution, conjugation to a moiety other than the antibody). For example, an antibody may contain one or more amino acid variants (compared to a naturally occurring antibody) that alter a property (eg , functional property) of the antibody. For example, numerous changes are known in the art that affect, for example , half-life, effector function, and/or immune responses to an antibody in a patient. The term "antibody" also includes artificial or engineered polypeptide constructs that contain at least one antigen-binding site derived from an antibody.

As used herein, the terms "specific binding", "selective binding", "selectively binds", and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen, but not to other antigens. Typically, the antibody (i) binds to an equilibrium dissociation constant (K _D ) of less than about 10 ^-7 M, such as less than about 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M, or even lower. , when determined, for example , using Surface Plasmon Resonance (SPR) technology using a BIACORE ^® 2000 instrument based on the principle of surface plasmon resonance, using a predetermined antigen, such as C5, as an analyte and an antibody as a ligand, or assay binding the antibody to antigen-positive cells by the Scatchard method, and (ii) binds to the predetermined antigen with an affinity that is at least twice its binding affinity to a non-specific antigen (e.g. BSA , casein) other than the predetermined antigen or a closely related antigen. Accordingly, unless otherwise indicated, an antibody that "specifically binds to human C5" refers to an antibody that binds to soluble or cell-bound C5 with a K _D of 10 ^-7 M or less, such as, for example, less than about 10 ^-8 M, 10 ^-9 M or 10 ^-10 M or even lower.

The term "surface plasmon resonance" in the context of this document refers to an optical phenomenon that provides a real-time analysis of biospecific interactions by detecting changes in protein concentrations in a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, New Jersey). For further description, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognize. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

The term "K _off " as used herein is intended to refer to the dissociation rate constant of an antibody from an antibody/antigen complex.

The term "K _d " in the context of this document is intended to refer to the dissociation constant for a particular antibody-antigen interaction.

As used herein, the terms "subject" or "patient" are used interchangeably herein and refer to a mammal such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig, etc. In one embodiment, the patient is a human patient (eg , a human patient with a condition associated with the complement system).

The terms "treat", "treatment" and "treatment" in the context of this document refer to the therapeutic measures described in this document. Methods of "treatment" use the administration to a subject of a combination disclosed herein to cure, delay, reduce the severity or lessen the intensity of one or more symptoms of a disease or disorder or a recurrent disease and disorder, or to prolong the subject's survival beyond that expected in the absence of such treatment.

In the context of this document, the term "effective treatment" refers to treatment that provides a beneficial effect, for example, reducing the intensity of at least one symptom of the disease or disorder. The beneficial effect may take the form of an improvement from baseline, for example, an improvement compared to the measurement or observation carried out before the start of therapy in accordance with the method. Effective treatment may refer to the alleviation of at least one symptom of a disease or condition.

The term "effective amount" refers to the amount of the agent, which provides the desired biological, therapeutic and/or prophylactic result. This may result in attenuation, reduction in intensity, temporary relief, reduction in severity, delay and/or mitigation of one or more signs, symptoms or causes of a disease or condition, or any other desired change in the biological system. In one example, an "effective amount" is the amount of a stable aqueous solution to ameliorate at least one symptom of a disease or condition. An effective amount can be administered in one or more administrations.

In the context of this document, the terms "induction" and "induction phase" are used interchangeably and refer to the first phase of treatment.

In the context of this document, the terms "maintenance" and "maintenance phase" are used interchangeably and refer to the second phase of treatment. In certain embodiments, treatment is continued as long as clinical benefit is observed or until uncontrolled toxicity or disease progression occurs.

II. Antibodies to C5

The anti-C5 antibodies described herein bind to the C5 component of the complement system (eg human C5) and inhibit the cleavage of C5 into C5a and C5b fragments. As described above, such antibodies are also characterized, for example, by improved pharmacokinetic properties compared to other anti-C5 antibodies (eg eculizumab ) used for therapeutic purposes.

Anti-C5 antibodies (or VH/VL domains derived therefrom) suitable for use in the present invention can be generated using methods well known in the art. Alternatively, anti-C5 antibodies known in the art may be used. You can also use antibodies that compete for binding to C5 with any of the antibodies known in the art.

An exemplary anti-C5 antibody is ravulizumab containing heavy and light chains having the sequences shown under SEQ ID NOs: 14 and 11, respectively, or antigen-binding fragments and variants thereof. Ravulizumab (also known as BNJ441 and ALXN1210) is described in PCT/US2015/019225 and US Pat. No. 9,079,949, the teachings of which are incorporated herein by reference. Throughout this document, the terms ravulizumab, BNJ441, and ALXN1210 may be used interchangeably. Ravulizumab selectively binds to the C5 protein of the human complement system, inhibiting its cleavage into C5a and C5b during complement system activation. This inhibition prevents the release of the pro-inflammatory mediator C5a and the formation of the cytolytic pore-forming membrane attack complex (MAC) C5b-9 while maintaining the proximal or early components of the complement activation pathway ( e.g. C3 and C3b) essential for opsonization of microorganisms and clearance of immune complexes.

In other embodiments, the antibody comprises the heavy and light chain CDRs or variable regions of ravulizumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of the ravulizumab VH region having the sequence given under SEQ ID NO: 12 and the CDR1, CDR2 and CDR3 domains of the ravulizumab VL region having the sequence given under SEQ ID NO: : 8. In another embodiment, the antibody comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown under SEQ ID NOs: 19, 18, and 3, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences given under SEQ ID NO: 4, 5 and 6, respectively. In another embodiment, the antibody contains VH and VL regions having the amino acid sequences shown under SEQ ID NO: 12 and SEQ ID NO: 8, respectively.

Another exemplary anti-C5 antibody is the BNJ421 antibody containing heavy and light chains having the sequences shown under SEQ ID NOs: 20 and 11, respectively, or antigen-binding fragments and variants thereof. BNJ421 (also known as ALXN1211) is described in PCT/US2015/019225 and US Pat. No. 9,079,949, the teachings of which are incorporated herein by reference.

In other embodiments, the antibody comprises the heavy and light chain CDRs or variable regions of BNJ421. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the BNJ421 VH region, having the sequence given under SEQ ID NO: 12, and the CDR1, CDR2, and CDR3 domains of the BNJ421 VL region, having the sequence given under SEQ ID NO: : 8. In another embodiment, the antibody comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown under SEQ ID NOs: 19, 18, and 3, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences given under SEQ ID NO: 4, 5 and 6, respectively. In another embodiment, the antibody contains VH and VL regions having the amino acid sequences shown under SEQ ID NO: 12 and SEQ ID NO: 8, respectively.

The precise boundaries of the CDR have been determined in various ways according to various methods. In some embodiments, the positions of CDRs or framework regions in a light chain or heavy chain variable domain can be determined according to Kabat et al . [(1991) "Sequences of Proteins of Immunological Interest". NIH Publication No. 91-3242, US Department of Health and Human Services, Bethesda, MD]. In such cases, the CDRs may be referred to as "CDRs according to Kabat" ( e.g. "LCDR2 according to Kabat" or "HCDR1 according to Kabat"). In some embodiments, the CDR positions of the light or heavy chain variable region can be determined according to Chothia et al. (1989) Nature 342 :877-883. Accordingly, these regions may be referred to as "CDRs according to Chothia" (eg "LCDR2 according to Chothia" or "HCDR3 according to Chothia"). In some embodiments, the positions of the variable region CDRs of the light and heavy chains can be determined using a combined determination of Kabat and Chothia. In such embodiments, these regions may be referred to as "CDRs according to the combined definition of Kabat and Chothia". Thomas et al. [(1996) Mol Immuno 33(17/18) :1389-1401] illustrates the identification of CDR boundaries as defined by Kabat and Chothia.

In some embodiments, an anti-C5 antibody described herein comprises a heavy chain CDR1 comprising or consisting of the following amino acid sequence: G H IFSNYWIQ (SEQ ID NO: 19). In some embodiments, an anti-C5 antibody described herein comprises a heavy chain CDR2 comprising or consisting of the following amino acid sequence: EILPGSG H TEYTENFKD (SEQ ID NO: 18). In some embodiments, an anti-C5 antibody described herein comprises a heavy chain variable region comprising the following amino acid sequence:

( SEQ ID NO : 12).

In some embodiments, an anti-C5 antibody described herein comprises a light chain variable region comprising the following amino acid sequence:

(SEQ ID NO: 8).

An anti-C5 antibody described herein may, in some embodiments, comprise a human Fc constant region variant that binds to the neonatal human Fc receptor (FcRn) with greater affinity than the native human Fc constant region from which the constant region variant was derived. Fc human. For example, the Fc constant region may contain one or more (e.g. , two, three, four, five, six, seven, or eight or more) amino acid substitutions compared to the native human Fc constant region from which the variant human Fc constant region was derived. Substitutions can increase the binding affinity of an IgG antibody containing an Fc constant region variant to FcRn at pH 6.0 while maintaining the pH dependence of the interaction. Methods for testing whether one or more substitutions in the Fc constant region of an antibody increase the affinity of the Fc constant region for FcRn at pH 6.0 (while maintaining the pH interaction dependence) are known in the art and are illustrated in the illustrative examples. See, for example , PCT/US2015/019225 and US Pat. No. 9,079,949, the disclosure of each of which is incorporated herein by reference in its entirety.

Substitutions that enhance the binding affinity of an antibody Fc constant region to FcRn are known in the art and include, for example , (1) the M252Y/S254T/T256E triple substitution described in Dall'Acqua et al. (2006) J Biol Chem 281 : 23514-23524; (2) the M428L or T250Q/M428L replacements described in Hinton et al. (2004) J Biol Chem 279 :6213-6216 and in Hinton et al. (2006) J Immunol 176 :346-356; and (3) the N434A or T307/E380A/N434A substitutions described in Petkova et al. (2006) Int Immunol 18(12) :1759-69. Additional substitution pairs P257I/Q311I, P257I/N434H, and D376V/N434H are described, for example , in Datta-Mannan et al. (2007) J Biol Chem 282(3) :1709-1717, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the constant region variant has a valine substitution at amino acid residue position 255 according to EU. In some embodiments, the constant region variant has an asparagine substitution at amino acid residue position 309 according to EU. In some embodiments, the constant region variant has an isoleucine substitution at EU amino acid residue position 312. In some embodiments, the constant region variant has a substitution at amino acid residue position 386 according to EU.

In some embodiments , the Fc constant region variant contains no more than 30 , 12, 11, 10, nine, eight, seven, six, five, four, three, or two) amino acid substitutions, insertions, or deletions compared to the native constant region from which it was derived. In some embodiments, the Fc constant region variant comprises one or more amino acid substitutions selected from the group consisting of M252Y, S254T, T256E, N434S, M428L, V259I, T250I, and V308F. In some embodiments, the human Fc constant region variant contains a methionine at position 428 and an asparagine at position 434, each of which is identified by EU numbering. In some embodiments, the Fc constant region variant contains a 428L/434S double substitution, as described, for example , in US Pat. No. 8,088,376.

In some embodiments, the precise location of these mutations may be biased relative to the location of the native human Fc constant region due to antibody engineering. For example, the 428L/434S double substitution when used in a chimeric IgG2/4 Fc fragment may correspond to 429L and 435S, as in the M429L and N435S variants found for ravulizumab (BNJ441) and described in US Pat. No. 9,079,949, the disclosure of which is incorporated herein. by reference in its entirety.

In some embodiments, the constant region variant comprises a substitution at amino acid position 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 or 436 ( EU numbering) compared to native human Fc constant region. In some embodiments, the replacement is selected from the group consisting of methionine instead of glycine at position 237; alanine instead of proline at position 238; lysine instead of serine at position 239; isoleucine instead of lysine at position 248; alanine, phenylalanine, isoleucine, methionine, glutamine, serine, valine, tryptophan, or tyrosine instead of threonine at position 250; phenylalanine, tryptophan or tyrosine instead of methionine at position 252; threonine instead of serine at position 254; glutamic acid instead of arginine at position 255; aspartic acid, glutamic acid, or glutamine instead of threonine at position 256; alanine, glycine, isoleucine, leucine, methionine, serine asparagine, threonine, or valine instead of proline at position 257; histidine instead of glutamic acid at position 258; alanine instead of aspartic acid at position 265; phenylalanine instead of aspartic acid at position 270; alanine or glutamic acid instead of asparagine at position 286; histidine instead of threonine at position 289; alanine instead of asparagine at position 297; glycine instead of serine at position 298; alanine instead of valine at position 303; alanine instead of valine at position 305; alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan or tyrosine instead of threonine at position 307; alanine, phenylalanine, isoleucine, leucine, methionine, proline, glutamine, or threonine instead of valine at position 308; alanine, aspartic acid, glutamic acid, proline or arginine instead of leucine or valine at position 309; alanine, histidine, or isoleucine instead of glutamine at position 311; alanine or histidine instead of aspartic acid at position 312; lysine or arginine instead of leucine at position 314; alanine or histidine instead of asparagine at position 315; alanine instead of lysine at position 317; glycine instead of asparagine at position 325; valine instead of isoleucine at position 332; leucine instead of lysine at position 334; histidine instead of lysine at position 360; alanine instead of aspartic acid at position 376; alanine instead of glutamic acid at position 380; alanine instead of glutamic acid at position 382; alanine instead of asparagine or serine at position 384; aspartic acid or histidine instead of glycine at position 385; proline instead of glutamine at position 386; glutamic acid instead of proline at position 387; alanine or serine instead of asparagine at position 389; alanine instead of serine at position 424; alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan or tyrosine instead of methionine at position 428; lysine instead of histidine at position 433; alanine, phenylalanine, histidine, serine, tryptophan or tyrosine in place of asparagine at position 434; and histidine in place of tyrosine or phenylalanine at position 436, each of which is listed according to EU numbering.

Suitable anti-C5 antibodies for use in the methods described herein, in some embodiments, comprise a heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14 and/or a light chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14. NO: 11. Alternatively, anti-C5 antibodies for use in the methods described herein, in some embodiments, comprise a heavy chain polypeptide containing the amino acid sequence listed under SEQ ID NO: 20 and/or a light chain polypeptide containing the amino acid sequence given under SEQ ID NO: 11.

In one embodiment, the antibody binds to C5 at pH 7.4 and 25°C (and otherwise under physiological conditions) with an affinity dissociation constant (K _D ) that is at least 0.1 (e.g. , at least 0 .15, 0.175, 0.2, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575 , 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95 or 0.975) nM . In some embodiments, the K _D of the anti-C5 antibody or antigen-binding fragment thereof is no more than 1 (e.g. , no more than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2) nM.

In other embodiments, [(K _D of anti-C5 antibody at pH 6.0 at C)/(K _D of anti-C5 antibody at pH 7.4 at 25°C)] is greater than 21 (e.g. , greater than 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 350 400 450 500 600 700 800 900 1000 1500 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500 or 8000).

Methods for determining whether an antibody binds to a protein antigen and/or the affinity of an antibody for a protein antigen are known in the art. For example, the binding of an antibody to a protein antigen can be detected and/or quantified using a variety of techniques such as, but not limited to, Western blot, dot blot, Surface Plasmon Resonance (SPR) method (e.g. BIAcore system ; Pharmacia Biosensor AB, Uppsala , Sweden and Piscataway, NJ) or enzyme-linked immunosorbent assay (ELISA). See, for example , Benny KC Lo (2004) "Antibody Engineering: Methods and Protocols", Humana Press (ISBN: 1588290921); Johne et al. (1993) J Immunol Meth 160 :191-198; Jonsson et al. (1993) Ann Biol Clin 51 :19-26; and Jonsson et al. (1991) Biotechniques 11 :620-627. In addition, methods for measuring affinity (eg dissociation and association constants) are set out in the demos.

In the context of this document, the term "ka" refers to the rate constant of association of _an antibody with an antigen. The term "k _d " refers to the dissociation rate constant of an antibody from an antibody/antigen complex. And the term “K _D ” refers to the equilibrium dissociation constant for an antibody-antigen interaction. The equilibrium dissociation constant is derived from the ratios of the kinetic rate constants K _D =k _a /k _d . Such definitions are preferably determined at 25°C or 37°C (see demonstration examples). For example, the binding kinetics of an antibody to human C5 can be determined at pH 8.0, 7.4, 7.0, 6.5, and 6.0 by surface plasmon resonance (SPR) on a BIAcore 3000 instrument using an anti-Fc antibody method. for antibody immobilization.

In one embodiment, the anti-C5 antibody, or antigen-binding fragment thereof, blocks the formation or activity of active C5a and/or C5b fragments of a C5 protein (eg , human C5 protein). Through this blocking effect, the antibodies inhibit, for example , the pro-inflammatory effects of C5a and the formation of the membrane attack complex (MAC) of C5b-9 on the cell surface.

Methods for determining whether a particular antibody described herein inhibits C5 cleavage are known in the art. Inhibition of the C5 component of the human complement system can reduce the ability of the complement system to cell lyse in body fluids of the subject. Such reductions in the ability of the complement system present in the biological(biological) body fluid(s) to cell lysis can be measured using methods well known in the art, such as using a conventional hemolysis assay, such as the hemolysis assay described by Kabat. and Mayer (eds.), "Experimental Immunochemistry, 2^nd Edition", 135-240, Springfield, IL, CC Thomas (1961), pages 135-139, or a conventional variant of such an assay, such as the method using hemolysis of chicken erythrocytes, described, for example, in Hillman et al. (2004)N Engl J Med 350(6):552. Methods for determining whether a candidate compound inhibits the cleavage of human C5 into C5a and C5b forms are known in the art and are described in Evans et al. (1995)Mol Immunol 32(16):1183-95. For example, the concentration and/or physiological activity of C5a and C5b in a biological fluid can be measured using methods well known in the art. For C5b, hemolysis assays or soluble C5b-9 assays can be used, as discussed herein. Other assays known in the art may also be used. Using assays of these or other suitable types, candidate agents capable of inhibiting the C5 component of the human complement system can be screened.

Immunological techniques, such as, but not limited to, ELISA, can be used to measure the concentration of C5 protein and/or its cleavage products to determine the ability of an anti-C5 antibody or antigen-binding fragment thereof to inhibit the conversion of C5 to biologically active products. In some embodiments, C5a production is measured. In some embodiments, antibodies specific for the C5b-9 neoepitope are used to detect the formation of terminal components of the complement system.

Hemolysis assays can be used to determine the inhibitory activity of an anti-C5 antibody, or antigen-binding fragment thereof, on activation of the complement system. To determine the effect of an anti-C5 antibody or antigen-binding fragment thereof on hemolysis mediated by the classical pathway of complement activation in an in vitro test serum solution, for example, hemolysin-coated sheep erythrocytes or chicken erythrocytes sensitized with antibodies to chicken erythrocytes. The percentage of lysis was normalized considering 100% lysis as equal to lysis occurring in the absence of inhibitor. In some embodiments, the complement system is classically activated with a human IgM antibody, such as that used in the Wieslab® Classical Pathway Activation Kit (Wieslab® COMPL CP310, Euro-Diagnostica, Sweden). Briefly, test serum is incubated with an anti-C5 antibody or antigen-binding fragment thereof in the presence of a human IgM antibody. The amount of C5b-9 formed is measured by contacting the mixture with an enzyme-conjugated anti-C5b-9 antibody and a fluorogenic substrate and measuring the absorbance at the appropriate wavelength. As a control, the test serum is incubated in the absence of an anti-C5 antibody or antigen-binding fragment thereof. In some embodiments, the test serum is C5 deficient serum reconstituted with a C5 polypeptide.

To determine the effect of an anti-C5 antibody or antigen-binding fragment thereof on alternative mediated hemolysis, non-sensitized rabbit or guinea pig erythrocytes can be used as target cells. In some embodiments, the test serum solution is C5 deficient serum reconstituted with a C5 polypeptide. The percentage of lysis was normalized considering 100% lysis as equal to lysis occurring in the absence of inhibitor. In some embodiments, the complement system is activated via an alternative pathway with lipopolysaccharide molecules, such as those used in the Wieslab® Alternative Complement Pathway Activation Kit (Wieslab® COMPL AP330, Euro-Diagnostica, Sweden). Briefly, the serum to be tested is incubated with an anti-C5 antibody or antigen-binding fragment thereof in the presence of lipopolysaccharide. The amount of C5b-9 formed is measured by contacting the mixture with an enzyme-conjugated anti-C5b-9 antibody and a fluorogenic substrate and measuring the fluorescence at the appropriate wavelength. As a control, the test serum is incubated in the absence of an anti-C5 antibody or antigen-binding fragment thereof.

In some embodiments, C5 activity or inhibition is quantified using a CH50eq assay. The CH50eq assay is a method for measuring the total activity of the classically activated complement system in serum. This test is a lysis assay that uses antibody-sensitized erythrocytes as an activator of the complement system in the classical pathway and various dilutions of test serum to determine the amount needed to achieve 50% lysis (CH50). The percentage degree of hemolysis can be determined, for example, using a spectrophotometer. The CH50eq assay provides an indirect measure of terminal complement complex (TCC) formation, since TCC itself is directly responsible for the measured degree of hemolysis.

The analysis is well known and commonly practiced by those skilled in the art. Briefly, to activate the complement system in the classical pathway, undiluted serum samples (e.g., reconstituted human serum samples) are added to microplate wells containing antibody-sensitized erythrocytes, thereby obtaining TCC. The activated serum samples are then diluted in microplate wells that are coated with a capture reagent (e.g. an antibody that binds to one or more components of the TCC). The TCC present in the activated samples binds to the monoclonal antibodies that coat the surface of the microplate wells. The wells are washed and a detection reagent is added to each well that has a detectable label and recognizes bound TCC. The detectable label may be, for example, fluorescent label or enzymatic label. The results of the analysis are expressed in units of CH50 equivalents per milliliter (CH50 equiv/mL).

inhibition, for example to the extent that it relates to the activity of the terminal components of the complement system, includes at least 5% (for example, at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% reduction in terminal complement activity, e.g. in a hemolysis assay or CH50eq assay compared to the effect of a control antibody (or antigen-binding fragment thereof) under similar conditions and at equimolar concentration. Significant inhibition in the context of this document refers to the inhibition of a specified activity (for example, activity of the terminal components of the complement system) by at least 40% (for example, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more). In some embodiments, an anti-C5 antibody described herein contains one or more amino acid substitutions compared to the CDR of eculizumab (i.e., SEQ ID NOs: 1-6) but retains at least 30% (e.g., at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70 , 75, 80, 85, 90, or 95%) of the inhibitory activity of eculizumab on the complement system in a hemolysis assay or CH50eq assay.

The anti-C5 antibody described herein has a human serum half-life of at least 20 (e.g., at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55) days. In another embodiment, an anti-C5 antibody described herein has a human serum half-life of at least 40 days. In another embodiment, an anti-C5 antibody described herein has a human serum half-life of about 43 days. In another embodiment, the anti-C5 antibody described herein has a human serum half-life of 39-48 days. Methods for measuring the half-life of an antibody in serum are known in the art. In some embodiments described herein, the anti-C5 antibody, or antigen-binding fragment thereof, has a serum half-life of at least 20% (e.g., 30% ) greater than the serum half-life of eculizumab, e.g. when measured in one of the mouse model systems described in the demos (for example, in a model system in mice deficient in C5/NOD/SCID or mice transgenic for hFcRn).

In one embodiment, the antibody competes for binding and/or binds to the same epitope on C5 as the antibodies described herein. The term "binds to the same epitope" in relation to two or more antibodies means that the antibodies bind to the same segment of amino acid residues determined using this method. Techniques for determining whether antibodies bind to the "same epitope on C5" as antibodies described herein include, for example, epitope mapping methods such as X-ray analyzes of crystals of antigen:antibody complexes that provide atomic resolution of the epitope, and mass spectrometry of hydrogen-deuterium exchange (HDX-MS). Other methods provide control of antibody binding to peptide fragments of an antigen or mutant variants of an antigen, where loss of binding due to modification of an amino acid residue within the antigen sequence is often considered to be indicative of an epitope component. In addition, computational combinatorics techniques for epitope mapping can also be applied. These methods are based on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. Antibodies having the same VH and VL sequences or the same CDR1, 2 and 3 sequences are expected to bind to the same epitope.

Antibodies that "compete with another antibody for binding to a target" refer to antibodies that inhibit (partially or completely) the binding of another antibody to a target. Whether two antibodies compete with each other for binding to the target, i.e. whether and to what extent one antibody inhibits the binding of another antibody to a target can be determined using known competition experiments. In certain embodiments, an antibody competes with another antibody and inhibits its binding to a target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition may differ depending on which antibody is the "blocking antibody" (i.e., the cold antibody that is first incubated with the target). Competing antibodies bind to the same epitope, an overlapping epitope, or adjacent epitopes (eg, as evidenced by steric mismatch).

The anti-C5 antibodies or antigen-binding fragments thereof described herein used in the methods described herein can be prepared using a variety of techniques known in the art. Monoclonal antibodies can be obtained using various techniques known to experts in this field. Briefly, spleen cells from an animal immunized with the desired antigen are immortalized, usually by fusion with a myeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6: 511-519 (1976)). Alternative methods of immortalization include transformation with Epstein-Barr virus, oncogenes or retroviruses, or other methods well known in the art. Colonies emerging from individual immortalized cells are screened for antibody production of the desired specificity and affinity for the antigen, and the yield of monoclonal antibodies produced by such cells can be enhanced by various techniques, including injection into the abdominal cavity of the vertebrate host. Alternatively, DNA sequences that encode a monoclonal antibody or binding fragment thereof can be isolated by screening a DNA library from human B cells according to the general protocol described in Huse, et al ., Science 246: 1275-1281 (1989) .

III. Highly concentrated anti-C5 antibody solutions

Provided herein are stable aqueous solutions containing anti-C5 antibody (e.g., ravulizumab). The aqueous solutions described herein may be sterile pharmaceutical grade compositions, for example, for administration to a subject for the treatment or prevention of a disorder associated with the complement system, such as PNH or aHUS. The solutions described herein can be formulated according to standard methods. The preparation of pharmaceutical formulations is a well-established art and is further described, for example, in Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20^th Edition, Lippincott, Williams & Wilkins (ISBN: 0683306472); Ansel et al. (1999) "Pharmaceutical Dosage Forms and Drug Delivery Systems", 7^th Edition, Lippincott Williams & Wilkins Publishers (ISBN: 0683305727); and Kibbe (2000) Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3^rd Edition (ISBN: 091733096X). Suitable methods for formulating the highly concentrated antibody solutions described herein are illustrated in the illustrative examples.

The aqueous solutions described herein have a high concentration of an antibody that binds to the C5 component of the human complement system, such as ravulizumab. Such solutions are sometimes referred to herein as "highly concentrated antibody solutions". In the context of this document, a "high concentration" of an anti-C5 antibody (for example, ravulizumab) in aqueous solution is an antibody concentration that is at least equal to or greater than 40 (e.g., at least equal to or greater than 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 , 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215 , 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300) mg/mL. In one embodiment, the anti-C5 antibody is present in solution at a concentration greater than 100 (e.g., more than 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190 or 195) mg / ml. In another embodiment, the anti-C5 antibody is present in solution at a concentration greater than 200 (e.g., more than 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290 or 295) mg / ml. In another embodiment, the anti-C5 antibody is present in solution at a concentration greater than 300 mg/mL. In another embodiment, the antibody is present in solution at a concentration of, for example, 40 mg/mL to 200 mg/mL, 50 mg/mL to 200 mg/mL, 60 mg/mL to 200 mg/mL, 70 mg/mL to 200 mg/mL, 80 mg/mL to 200 mg/ml, 90 mg/ml to 200 mg/ml, 100 mg/ml to 200 mg/ml, 110 mg/ml to 200 mg/ml, 120 mg/ml to 200 mg/ml, from 130 mg/mL to 200 mg/mL, 140 mg/mL to 200 mg/mL, 150 mg/mL to 200 mg/mL, 40 mg/mL to 100 mg/mL, 50 mg/mL to 100 mg/ml, 60 mg/ml to 100 mg/ml, 70 mg/ml to 100 mg/ml, 80 mg/ml to 100 mg/ml, 90 mg/ml to 100 mg/ml, from 40 mg/mL to 150 mg/mL, 50 mg/mL to 150 mg/mL, 60 mg/mL to 150 mg/mL, 70 mg/mL to 150 mg/mL, 80 mg/mL to 150 mg /ml, 90 mg/ml to 150 mg/ml, 100 mg/ml to 150 mg/ml, 110 mg/ml to 150 mg/ml, 120 mg/ml to 150 mg/ml, 40 mg or more /ml to 50 mg/ml, from 40 mg/ml to 250 mg/ml, from 50 mg/ml to 250 mg/ml, from 60 mg/ml to 250 mg/ml, from 70 mg/ml to 250 mg/ ml, 80 mg/ml to 250 mg/ml, 90 mg/ml to 250 mg/ml, 100 mg/ml to 250 mg/ml, 110 mg/ml to 250 mg/ml, 120 mg/ml ml to 250 mg/ml, from 130 mg/ml to 250 mg/ml, 140 mg/mL to 250 mg/mL, 150 mg/mL to 250 mg/mL, 160 mg/mL to 250 mg/mL, 170 mg/mL to 250 mg/mL, 180 mg/mL to 250 mg/mL, 190 mg/mL to 250 mg/mL, 200 mg/mL to 250 mg/mL, more than 200 mg/mL (eg, at least 201 mg/mL) to 250 mg/mL or more than 200 mg/ml (eg, 201 mg/ml or more) to 300 mg/ml.

As described herein and illustrated in the illustrative examples, the present aqueous solutions provide marked physical and chemical stability as well as functional stability of the anti-C5 antibody formulated herein. For example, the formulations described herein are capable of maintaining the structural integrity of an anti-C5 antibody (e.g., ravulizumab), present in solution in high concentrations. In one embodiment, the solution is suitable for storage at 2-8°C (e.g., at 4°C). In another embodiment, the solution is formulated to be stored below 0°C (e.g., at -20°C or -80°C). In another embodiment, the solution is formulated to be stored for up to three years (e.g., within one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 1 year, 1 ½ years, 2 years, 2 ½ years, or 3 years) at 2-8°C (e.g. at 4°C). In another embodiment, the solution is suitable for storage for a period of at least 1, 2, or 3 years at 2-8°C (e.g., at 4°C).

As illustrated in the demonstrations described herein, the solutions described herein are suitable for maintaining an anti-C5 antibody at about 100 mg/mL in predominantly monomeric form for up to two years at about 2°C-8°C. As used herein, an anti-C5 antibody formulated at a high concentration in a given aqueous solution is "predominantly monomeric" or is in "predominantly monomeric form" if the antibody present in the solution is at least 95% (e.g., at least 95.1, 95.2, 95.3, 95.4, 95.5, 95.6, 95.7, 95.8, 95.9, 96, 96.1, 96.2, 96.3, 96.4, 96.5, 96.6, 96.7, 96.8, 96.9, 97, 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.9, 98, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8 or 99.9% or more) monomeric, for example, at determination by high performance liquid chromatography in size exclusion chromatography (SEC-HPLC, such as gel permeation HPLC). In one embodiment, the anti-C5 antibody in the solutions described herein may remain predominantly monomeric after storage for at least one month (e.g., for at least two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or more) at about 2°C to 8°C (e.g. after storage at, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10°C).

In one embodiment of any of the solutions described herein, an anti-C5 antibody (e.g., ravulizumab) remains at least 95% (e.g., at least 96, 97, 98, or 99%) monomeric when stored at 2°C-8°C for at least six months as determined by SEC-HPLC (e.g., gel permeation HPLC). In another embodiment, the anti-C5 antibody remains at least 95% (e.g., at least 96, 97, 98 or 99%) monomeric during storage at 2°C-8°C for at least nine months as determined by SEC-HPLC. In another embodiment, the anti-C5 antibody remains at least 95% (e.g., at least 96, 97, 98 or 99%) monomeric during storage at 2°C-8°C for at least one year as determined by SEC-HPLC. In another embodiment, the anti-C5 antibody remains at least 95% (e.g., at least 96%, 97%, 98%, or 99%) monomeric during storage at 2°C-8°C for at least 18 months at determination by SEC-HPLC. In another embodiment, the anti-C5 antibody remains at least 95% (e.g., at least 96%, 97%, 98%, or 99%) monomeric during storage at 2°C-8°C for at least two years at determination by SEC-HPLC.

In another embodiment, less than 5% (for example, less than 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3 .7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5 , 2.4, 2.3, 2.2, 2.1, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2 , 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1%) antibodies in solution are oligomeric, aggregated and/or fragmented. As used herein, antibody fragmentation refers to improperly assembled constituents or degradation products of a whole antibody having a lower molecular weight than the whole antibody. Such forms of fragmentation include, without limitation, a free monomeric polypeptide heavy chain, a dimeric polypeptide heavy chain (e.g., a disulfide-stabilized polypeptide heavy chain), a dimeric polypeptide heavy chain linked to a single polypeptide light chain, a monomeric polypeptide heavy chain linked to a single polypeptide light chain. chain, or additional product(s) of destruction or fragment(s) of the polypeptide light chain or heavy chain. In some embodiments, less than 2% (for example, less than 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7 , 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1%) antibodies are aggregated after storage for at least one month (e.g., for at least two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or more) at 2°C-8°C. In some embodiments, less than 1% (for example, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1%) antibodies are fragmented after storage for at least one month ( for example, for at least two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or more) at 2°C-8°C. Methods for determining the amount of monomeric antibody as well as the amount of oligomeric, aggregated, or fragmented forms of anti-C5 antibody present in solution are described herein and illustrated in the illustrative examples. For example, one skilled in the art can determine the percentage of intact, fragmented, non-folded intermediates and/or aggregated antibody molecules present in a given solution using, for example, high performance liquid chromatography, size exclusion chromatography (SEC-HPLC, such as gel penetrating HPLC), static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding techniques, tryptophan intrinsic fluorescence, sodium dodecyl sulfate polyacrylamide gel electrophoresis under non-reducing conditions (SDS -PAGE) and differential scanning calorimetry (DSC).

In one embodiment of any of the solutions described herein, less than 5% of the anti-C5 antibodies (eg ravulizumab ) in the solution are aggregated when determined by SEC-HPLC (eg gel permeation HPLC). In another embodiment, less than 4% of the anti-C5 antibodies in solution are aggregated as determined by SEC-HPLC. In another embodiment, less than 3% of the anti-C5 antibodies in solution are aggregated as determined by SEC-HPLC. In another embodiment, less than 2% of the anti-C5 antibodies in solution are aggregated as determined by SEC-HPLC. In another embodiment, less than 1% of the anti-C5 antibodies in solution are aggregated as determined by SEC-HPLC.

As described herein and illustrated in the demos, the anti-C5 antibody solutions provided herein can retain at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or even 100%) of their biological/functional activity (for example, ability to bind to human C5) after storage for at least one month (e.g., for at least two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months , 34 months, 35 months, 36 months or more) at 2°C-8°C.

In another embodiment, an anti-C5 antibody (e.g., ravulizumab) present in the solution described herein can retain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%) of its hemolysis inhibition activity after storage for at least one month (e.g. for at least two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months , 34 months, 35 months, 36 months or more) at 2°C-8°C. Suitable hemolysis assay methods for determining whether an antibody retains its activity in a present solution are described herein and are known in the art, for example, hemolysis assay using avian or porcine erythrocytes.in vitro. Suitable methods for assessing the ability of an antibody preparation to bind to the C5 component of the human complement system are known in the art and are described herein.

In another embodiment of any of the solutions described herein, an anti-C5 antibody (e.g., ravulizumab) retains at least 80% (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of its C5 binding activity during storage at 2°C-8°C for at least six months compared to a reference anti-C5 antibody corresponding to an anti-C5 antibody prior to storage. In another embodiment, an anti-C5 antibody (e.g., ravulizumab) retains at least 80% (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of its C5 binding activity during storage at 2°C-8°C for at least nine months compared to a reference anti-C5 antibody corresponding to an anti-C5 antibody prior to storage. In another embodiment, an anti-C5 antibody (e.g., ravulizumab) retains at least 80% (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of its C5 binding activity during storage at 2°C-8°C for at least one year compared to a reference anti-C5 antibody corresponding to an anti-C5 antibody prior to storage. In another embodiment, an anti-C5 antibody (e.g., ravulizumab) retains at least 80% (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of its C5 binding activity during storage at 2°C-8°C for at least eighteen months compared to a reference anti-C5 antibody corresponding to an anti-C5 antibody prior to storage. In another embodiment, an anti-C5 antibody (e.g., ravulizumab) retains at least 80% (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of its C5 binding activity during storage at 2°C-8°C for at least two years compared to a reference anti-C5 antibody corresponding to an anti-C5 antibody prior to storage. In another embodiment, an anti-C5 antibody (e.g., ravulizumab) retains at least 80% (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of its C5 binding activity during storage at 2°C-8°C for at least three years compared to a reference anti-C5 antibody corresponding to an anti-C5 antibody prior to storage.

In another embodiment of any of the solutions described herein, an anti-C5 antibody (e.g., ravulizumab) retains at least 80% (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of its ability to inhibit hemolysis during storage at 2°C-8°C for at least nine months compared to a reference anti-C5 antibody corresponding to an anti-C5 antibody prior to storage. In another embodiment of any of the solutions described herein, an anti-C5 antibody (e.g., ravulizumab) retains at least 80% (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of its ability to inhibit hemolysis during storage at 2°C-8°C for at least six months compared to a C5 reference antibody corresponding to a C5 antibody prior to storage. In another embodiment of any of the solutions described herein, an anti-C5 antibody (e.g., ravulizumab) retains at least 80% (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of its ability to inhibit hemolysis during storage at 2° C.-8° C. for at least one year compared to a reference anti-C5 antibody corresponding to an anti-C5 antibody prior to storage. In another embodiment of any of the solutions described herein, an anti-C5 antibody (e.g., ravulizumab) retains at least 80% (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of its ability to inhibit hemolysis during storage at 2°C-8°C for at least 18 months compared to a C5 reference antibody corresponding to a C5 antibody prior to storage. In another embodiment of any of the solutions described herein, an anti-C5 antibody (e.g., ravulizumab) retains at least 80% (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of its ability to inhibit hemolysis during storage at 2° C.-8° C. for at least two years compared to a reference anti-C5 antibody corresponding to an anti-C5 antibody prior to storage. In another embodiment of any of the solutions described herein, an anti-C5 antibody (e.g., ravulizumab) retains at least 80% (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of its ability to inhibit hemolysis during storage at 2° C.-8° C. for at least three years compared to a reference anti-C5 antibody corresponding to an anti-C5 antibody prior to storage.

The aqueous solutions described herein may contain one or more conventional agents (for example, one or more excipients and/or additives such as buffering agents, sugars or saccharides, salts, surfactants, solubilizers, diluents, binders, stabilizers, salts, lipophilic diluents, amino acids, chelators and/or preservatives).

In one embodiment, the aqueous solution contains one or more buffer agents. As used herein, the term "buffer" refers to one or more components that, when added to an aqueous solution, are capable of protecting the solution from changes in pH when an acid or alkali is added or when diluted with a solvent. In one embodiment, the solution contains at least one or more buffer agents. Non-limiting examples of typical buffers that may be included in the wash(s) solution(s) include tris (tris(hydroxymethyl)methylamine), bis-tris, bis-tris-propane, histidine, triethanolamine, diethanolamine, formate, acetate, MES (2-(N-morpholino)ethanesulfonic acid), phosphate, HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), citrate, MOPS (3-(N-morpholino)propanesulfonic acid), TAPS (3{[tris( hydroxymethyl)methyl]amino}propanesulfonic acid), bicine (N,N-bis(2-hydroxyethyl)glycine), tricine (N-tris(hydroxymethyl)methylglycine), TES (2-{[tris(hydroxymethyl)methyl]amino} ethanesulfonic acid), PIPES (piperazine-N, N'-bis(2-ethanesulfonic acid), cacodylate (dimethylarsinic acid), SSC (sodium citrate saline), and sodium phosphate.

In another embodiment, the buffering agent is an amino acid. The amino acid can be, for example, an amino acid selected from the group consisting of histidine (for example, L-histidine), serine (for example, L-serine) and glycine (for example, L-glycine). In another embodiment, the solution contains two or more buffer agents. In a particular embodiment, the buffering agent is sodium phosphate. In one embodiment, the provided solutions do not contain the free amino acid as a buffering agent. In another embodiment, the solutions provided contain one free amino acid (e.g., histidine) as a buffer. In another embodiment, the solutions provided may contain two or more (e.g., two, three, four, five, six, or seven or more) different amino acids as buffering agents, e.g., serine and histidine.

The buffer concentration is sufficient to maintain the desired pH and may also be varied, for example to keep the formulation isotonic. Typical concentrations of conventional buffer agents used in parenteral formulations can be found in: Pharmaceutical Dosage Form: Parenteral Medications, Volume 1, 2^nd Edition, Chapter 5, p. 194, De Luca and Boylan, "Formulation of Small Volume Parenterals", Table 5: Commonly used additives in Parenteral Product. In one embodiment, the concentration of one or more buffering agents in the formulation is from about 10 mM to 300 mM, inclusive. In another embodiment, the solution contains at least one buffering agent at a concentration of 10 mM to 200 mM, inclusive. In another embodiment, the aqueous solution described herein contains a buffering agent at a concentration of at least 10 (for example, at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220 , 230, 240, 250, 260, 270, 280, 290 or 300 or more) mM. In another embodiment, the aqueous solution contains a buffering agent at a concentration of about 10 mM to 50 mM, 15 mM to 50 mM, 20 mM to 50 mM, 25 mM to 50 mM, 30 mM to 50 mM, 40 mM up to 50 mM, 10 mM to 100 mM, 15 mM to 100 mM, 20 mM to 100 mM, 25 mM to 100 mM, 30 mM to 100 mM, 40 mM to 100 mM, 10 mM to 150 mM, 15 mM to 150 mM, 20 mM to 150 mM, 25 mM to 150 mM, 30 mM to 150 mM, 40 mM to 150 mM, 50 mM to 100 mM, 60 mM to 100 mM, 70 mM to 100 mM, 80 mM to 100 mM, 50 mM to 150 mM, 60 mM to 150 mM, 70 mM to 150 mM, 80 mM to 150 mM, 90 mM to 150 mM , 100 mM to 150 mM, 10 mM to 200 mM, 15 mM to 200 mM, 20 mM to 200 mM, 25 mM to 200 mM, 30 mM to 200 mM, 40 mM to 200 mM, 50 mM to 200 mM, 60 mM to 200 mM, 70 mM to 200 mM, 80 mM to 200 mM, 90 mM to 200 mM, 100 mM to 200 mM, 150 mM to 200 mM, from 10 mM to 250 mM, 15 mM to 250 mM, 20 mM to 250 mM, 25 mM to 250 mM, 30 mM to 250 mM, 40 mM to 250 mM, 50 mM to 250 mM, 60 mM to 250 mM, 70 mM to 250 mM, 80 mM to 250 mM, 90 mM to 250 mM, from 100 mM to 250 mM, 150 mM to 250 mM, or 200 mM to 250 mM. In another embodiment, the buffer concentration in the formulation is approximately 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 90 mM, 95 mM or about 100 mM. In another embodiment, the buffering agent is present in solution at a concentration of at least or equal to 20 mM. In another embodiment, the buffering agent is present in solution at a concentration of at least or equal to 25 mM. In another embodiment, the buffering agent is present in solution at a concentration of at least or equal to 50 mM. In embodiments in which the present solution contains two or more different buffering agents (e.g., at least two, three, four, five, six, seven, eight, nine, or 10 or more), each of two or more buffer means may be independently present, for example, at one of the above concentrations.

In one embodiment, the aqueous solution has a neutral pH or can be adjusted to it. In the context of this document, the term "neutral pH" means a pH of 7 to 8 inclusive. Accordingly, in the context of this document, neutral pH includes specific pH values such as 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7 .9 and 8.0. In some embodiments, the neutral pH is at least pH 7 (e.g., at least pH 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9), but less than pH 8 (e.g., less than than pH 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2 or 7.1). In other words, in some embodiments, the neutral pH may be, for example, at least pH 7, but less than pH 7.5. In some embodiments, the implementation of the neutral pH may be from pH 7 to pH 7.5. In some embodiments, the neutral pH may be between pH 7 and pH 7.2. In another embodiment, the pH of the solution is from 7.0 to 7.4. In another embodiment, the pH of the solution is from 7.2 to 7.8. In another embodiment, the pH of the solution is from 7.2 to 7.6. In some embodiments, the neutral pH may be, for example, pH 7. One of ordinary skill in the art will also appreciate that human blood (such as human blood from a healthy patient) has a neutral pH as defined herein, for example, the pH of human blood is between about 7.35 and pH 7.45.Cm., for example, Boron and Boulpaep (2003) "Medical physiology: a cellular and molecular approach", W.B. Saunders, New York (ISBN: 0721632564). In some embodiments, the pH of the highly concentrated antibody solution described herein is between about 6.4 and 7.5, inclusive (e.g., about 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4 , 7.5, 7.6 or 7.7). In one embodiment, the pH of the solution is from 7.2 to 7.6. In a specific embodiment, the pH of the solution is 7.4.

In one embodiment, the solution contains one or more surfactants, such as an anionic, cationic, or nonionic surfactant. In the context of this document, the term "surfactant" refers to a surfactant molecule containing both a hydrophobic moiety (e.g., alkyl chain) and a hydrophilic part (for example, carboxyl and carboxylate groups). Surfactants suitable for use in the compositions of the present invention include, without limitation, fatty acid esters (for example, sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate), sorbitan trioleate, esters of glycerol and fatty acids (for example, glycerol monocaprylate, glycerol monomyristate, glycerol monostearate), polyglycerol fatty acid esters (e.g., decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate), polyoxyethylene esters of sorbitan fatty acids (for example, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate), polyoxyethylene sorbitan fatty acid esters (for example, polyoxyethylene sorbitan tetrastearate, polyoxyethylene sorbitan tetraoleate), polyoxyethylene esters of glycerol and fatty acids (for example, polyoxyethylene glyceryl monostearate), polyethylene glycol fatty acid esters (e.g. polyethylene glycol distearate), polyoxyethylene alkyl ethers (for example, polyoxyethylene lauryl ether), polyoxyethylene-polyoxypropylene alkyl ethers (for example, polyoxyethylene-polyoxypropylene glycol, polyoxyethylene-polyoxypropylene propyl ether, polyoxyethylene-polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ethers (for example, polyoxyethylene nonylphenyl ether), polyoxyethylene hydrogenated castor oils (for example, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylene wax derivatives (for example, polyoxyethylene sorbitol wax), polyoxyethylene derivatives of lanolin (for example, polyoxyethylenelanolin) and polyoxyethylene fatty acid amides (for example, polyoxyethylene stearic acid amide); C12-C18-alkyl sulfates (for example, sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate), polyoxyethylene-C10-C18-alkyl ether sulfate with the addition of an average of 2 to 4 moles of ethylene oxide units (for example, sodium polyoxyethylene lauryl sulfate) and salts of C10-C18 alkyl sulfosuccinate esters (e.g. sodium salt of lauryl sulfosuccinate ester); as well as natural surfactants such as lecithin, glycerophospholipid, sphingophospholipids (for example, sphingomyelin) and esters of sucrose and C12-C18 fatty acids.

In one embodiment, the surfactant in the formulation is a nonionic surfactant. In certain embodiments, the surfactant in the formulation is a polyoxyethylene sorbitan fatty acid ester, such as polysorbate 20, 40, 60, 80, or a combination of one or more of these. In one embodiment, the surfactant in the formulation is polysorbate 80 (Tween 80). In another embodiment, the surfactant in the formulation is Polysorbate 60. In another embodiment, the surfactant in the formulation is Polysorbate 40. In another embodiment, the surfactant in the formulation is Polysorbate 20 (Tween 20).

The amount of surfactant added to the formulation is sufficient to reduce aggregation of the formulated antibody and/or minimize particulate formation in the formulation. For example, the surfactant may be present in the composition in an amount of from about 0.001% to about 1%, or from about 0.001% to about 0.5%, or from about 0.01% to about 0.2%. In one embodiment, aqueous solutions contain a surfactant at a concentration of at least or about 0.001 (e.g., at least or about 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0 .08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2 , 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0 .33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45 , 0.46, 0.47, 0.48, 0.49 or 0.5 or more)%. In another embodiment, the aqueous solution contains no more than 0.2 (for example, not more than 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, 0.002, or 0.001) % pharmaceutically acceptable surface -active substance.

In another embodiment, the formulations contain polysorbate at a concentration of from about 0.001% to about 0.5%, from about 0.005% to about 0.2%, from about 0.01% to about 0.1%, or from about 0.02 % to about 0.06%, or from about 0.03% to about 0.05% (w/v). In certain embodiments, the composition contains polysorbate at a concentration of 0.01%, or 0.02%, or 0.03%, or 0.04%, or 0.05%, or 0.06%, or 0.07%, or 0.08% or 0.09% or 0.1% or 0.15% or 0.2% (w/v). In certain embodiments, the surfactant is present in the composition in an amount of 0.02% or about 0.04% (w/v). In one embodiment, the surfactant is present in the composition in an amount of 0.05% (w/v).

In one embodiment, the formulation contains at least about 0.01%, at least about 0.02%, at least about 0.05%, at least about 0.1%, at least about 0.2% , at least about 0.3%, at least about 0.4%, or at least about 0.5% polysorbate 80. In a certain embodiment, the composition contains polysorbate 80 at a concentration of from about 0.01% to about 0, 5%, about 0.01% to about 0.3%, about 0.001% to about 0.2%, about 0.02% to about 0.5%, about 0.02% to about 0, 3%, about 0.02% to about 0.2%, about 0.05% to about 0.5%, about 0.05% to about 0.3%, about 0.05% to about 0.2%, from about 0.075% to about 0.5%, from about 0.075% to about 0.3%, or from about 0, 075% to approximately 0.2%. In a further embodiment, the formulation contains about 0.01%, about 0.02%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, or about 0 .5% polysorbate 80. In one embodiment, the composition contains about 0.05% polysorbate 80. In one embodiment, the composition contains about 0.04% polysorbate 80. In one embodiment, the composition contains about 0.03% polysorbate 80. in an embodiment, the composition contains about 0.02% polysorbate 80. In one embodiment, the composition contains about 0.01% polysorbate 80.

In one embodiment, the aqueous solution contains one or more salts, for example, sodium chloride, potassium chloride or magnesium chloride. In some embodiments, the aqueous solution described herein contains salt at a concentration of at least 10 (e.g., at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or more) mM In some embodiments, the aqueous solution described herein may contain salt at a concentration of less than or about 200 (for example, less than or about 190, 180, 170, 160, 150, 140, 130, 120, 110, , 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, or 10) mM. In some embodiments, the aqueous solution described herein may contain a salt at a concentration of about 10 mM to 50 mM, 15 mM to 50 mM, 20 mM to 50 mM, 25 mM to 50 mM, 30 mM to 50 mM, 40 mM to 50 mM, 10 mM to 100 mM, 15 mM to 100 mM, 20 mM to 100 mM, 25 mM to 100 mM, 30 mM to 100 mM, 40 mM to 100 mM, 10 mM to 150 mM, 15 mM to 150 mM, 20 mM to 150 mM, 25 mM to 150 mM, 30 mM to 150 mM, 40 mM to 150 mM, 50 mM to 100 mM , 60 mM to 100 mM, 70 mM to 100 mM, 80 mM to 100 mM, 50 mM to 150 mM, 60 mM to 150 mM, 70 mM to 150 mM, 80 mM to 150 mM, 90 mM to 150 mM, 100 mM to 150 mM, 10 mM to 200 mM, 15 mM to 200 mM, 20 mM to 200 mM, 25 mM to 200 mM, 30 mM to 200 mM, from 40 mM to 200 mM, 50 mM to 200 mM, 60 mM to 200 mM, 70 mM to 200 mM, 80 mM to 200 mM, 90 mM to 200 mM, 100 mM to 200 mM, from 150 mM to 200 mM, from 10 mM to 250 mM, from 15 mM to 250 mM, from 20 mM d o 250 mM, 25 mM to 250 mM, 30 mM to 250 mM, 40 mM to 250 mM, 50 mM to 250 mM, 60 mM to 250 mM, 70 mM to 250 mM, 80 mM to 250 mM, 90 mM to 250 mM, 100 mM to 250 mM, 150 mM to 250 mM, or 200 mM to 250 mM. In embodiments in which the present solution contains two or more different salts (e.g., at least two, three, four, five, six, seven, eight, nine, or 10 or more), each of the two or more salts may be independently present, for example, at one of the concentrations described above.

In one embodiment, the aqueous solution contains one or more carbohydrate excipients. Suitable carbohydrate excipients are described, for example, in Katakam and Banga (1995)J Pharm Pharmacol 47(2):103-107; Andya et al. (2003)AAPS PharmSci 5(2): Article 10; and Shire (2009) "Current Trends in Monoclonal Antibody Development and Manufacturing", Volume 11, Springer, 354 pages. Carbohydrate excipients suitable for use in the solutions described herein include, without limitation, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose, trehalose and cellobiose; polysaccharides such as maltodextrins, dextrans and starches; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol and sorbitol. In one embodiment, the carbohydrate excipient is present in the solution provided herein at a concentration of at least or about 0.5 (e.g., at least or about 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 , 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3 .25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 , 9.5, 10 or more)%. In embodiments in which the present solution contains two or more different carbohydrate excipients (e.g., at least two, three, four, five, six, seven, eight, nine, or 10 or more), each excipient may be independently present at any of the above concentrations.

In another embodiment, the stable aqueous solution contains one or more stabilizing agents. Exemplary stabilizers include, without limitation, polyols, sugars (e.g., sucrose or trehalose), amino acids (for example, arginine), amines and salting out salts. In one embodiment, the solution contains at least one stabilizing agent at a concentration of 2-10% inclusive. In one embodiment, the solution contains 5% sucrose. In another embodiment, the solution contains at least one or more stabilizing agents at a concentration of 10 mM to 50 mM, inclusive. In another embodiment, the stabilizing agent is present in solution at a concentration of at least or equal to 20 mM. In another embodiment, the stabilizing agent is present in solution at a concentration of at least or equal to 25 mM. In another embodiment, the stabilizing agent is present in solution at a concentration of at least or equal to 50 mM. In another embodiment, the solution contains 25 mM arginine.

In one embodiment, the solutions described herein contain one or more preservatives.

In the context of this document, the term "preservative" refers to an agent that reduces the vital activity of bacteria and optionally can be added to the compositions described in this document. The addition of a preservative may, for example, facilitate the preparation of a multi-dose formulation. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long chain compounds) and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol.

In one embodiment, the stable aqueous solution contains no more than five agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than four agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than three agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than two agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than one agent in addition to the anti-C5 antibody.

In another embodiment, the stable aqueous solution comprises an anti-C5 antibody that comprises a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, a heavy chain CDR3, containing the amino acid sequence listed under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence listed under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence listed under SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence listed under SEQ ID NO: 6 at a concentration of 100 ± 20 (for example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120) mg/ml; 50 ± 15 (for example, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 mM phosphate buffer; 5 ± 3 (for example, 2, 3, 4, 5, 6, 7 or 8)% sucrose and 25 ± 10 (e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34 or 35) mM arginine; where the solution has a pH of 7.4 ± 0.5 (for example, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9).

In another embodiment, the stable aqueous solution consists of an anti-C5 antibody that contains a heavy chain CDR1 containing the amino acid sequence listed under SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence listed under SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence listed under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence listed under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence listed under SEQ ID NO: 5, and a light chain CDR3, containing the amino acid sequence listed under SEQ ID NO: 6, at a concentration of 100 ± 20 (for example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120) mg/ml; 50 ± 15 (for example, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 mM phosphate buffer; 5 ± 3 (for example, 2, 3, 4, 5, 6, 7 or 8)% sucrose and 25 ± 10 (e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34 or 35) mM arginine; where the solution has a pH of 7.4 ± 0.5 (for example, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9).

In another embodiment, the stable aqueous solution comprises an anti-C5 antibody comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, a heavy chain CDR3 comprising the amino acid sequence shown under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence shown under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence shown under SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence given under SEQ ID NO: 6 at a concentration of 100 ± 20 (for example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120) mg/ml; 50 ± 15 (for example, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 mM phosphate buffer; 5 ± 3 (for example, 2, 3, 4, 5, 6, 7 or 8)% sucrose; 25 ± 10 (e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) mM arginine ; as well as 0.05 ± 0.03 (for example, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 and 0.08)% polysorbate 80, where the solution has a pH of 7.4 ± 0.5 (for example, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9).

In another embodiment, the stable aqueous solution consists of an anti-C5 antibody that contains a heavy chain CDR1 containing the amino acid sequence listed under SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence listed under SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence listed under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence listed under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence listed under SEQ ID NO: 5, and a light chain CDR3, containing the amino acid sequence listed under SEQ ID NO: 6, at a concentration of 100 ± 20 (for example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120) mg/ml; 50 ± 15 (for example, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 mM phosphate buffer; 5 ± 3 (for example, 2, 3, 4, 5, 6, 7 or 8)% sucrose; 25 ± 10 (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) mM arginine; as well as 0.05 ± 0.03 (for example, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 and 0.08)% polysorbate 80, where the solution has a pH of 7, 4 ± 0.5 (for example, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9).

In another embodiment, a stable aqueous solution is provided (for example, sterile solution), where the solution contains (a) an anti-C5 antibody (for example, ravulizumab) at a concentration of approximately 100 mg/ml, (b) approximately 50 mm phosphate buffer; (c) about 5% sucrose; and (d) about 25 mM arginine. In another embodiment, the stable aqueous solution contains (a) an anti-C5 antibody (e.g., ravulizumab) at a concentration of 100 mg/ml, (b) 50 mm phosphate buffer; (c) 5% sucrose; and (d) 25 mM arginine.

In another embodiment, the stable aqueous solution contains (a) about 100 mg/ml anti-C5 antibody, (b) about 50 mM phosphate buffer, (c) about 5% sucrose, (d) about 0.05% polysorbate 80, and (e) approximately 25 mM arginine. In another embodiment, the stable aqueous solution contains (a) 100 mg/ml anti-C5 antibody, (b) 50 mM phosphate buffer, (c) 5% sucrose, (d) 0.05% polysorbate 80, and (e) 25 mM arginine.

In another embodiment, the stable aqueous solution contains no more than three additional agents. In another embodiment, the stable aqueous solution contains no more than two additional agents. In another embodiment, the stable aqueous solution contains no more than one additional agent.

In another embodiment, the stable aqueous solution consists of (a) about 100 mg/ml anti-C5 antibody, (b) about 50 mM phosphate buffer, (c) about 5% sucrose, and (d) about 25 mM arginine. In another embodiment, the stable aqueous solution consists of (a) 100 mg/ml anti-C5 antibody, (b) 50 mM phosphate buffer; (c) 5% sucrose; and (d) 25 mM arginine.

In another embodiment, the stable aqueous solution consists of (a) about 100 mg/ml anti-C5 antibody, (b) about 50 mM phosphate buffer, (c) about 5% sucrose; (d) about 0.05% polysorbate 80; and (e) about 25 mM arginine. In another embodiment, the stable aqueous solution consists of (a) 100 mg/ml anti-C5 antibody, (b) 50 mM phosphate buffer, (c) 5% sucrose, (d) 0.05% polysorbate 80, and (e) 25 mM arginine.

In one embodiment, the stable aqueous solution contains (a) an anti-C5 antibody at a concentration of approximately 100 mg/mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 comprising the amino acid sequence shown under SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence , shown under SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence shown under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence shown under SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 6, (b) approximately 50 mm phosphate buffer, (c) approximately 5% sucrose and (d) approximately 25 mm arginine.

In another embodiment, the stable aqueous solution contains (a) an anti-C5 antibody at a concentration of 100 mg/mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 comprising the amino acid sequence shown under SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence, listed under SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence listed under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence listed under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence listed under SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 6, (b) 50 mM phosphate buffer, (c) 5% sucrose, and (d) 25 mM arginine.

In another embodiment, the stable aqueous solution comprises (a) an anti-C5 antibody at a concentration of approximately 100 mg/mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 comprising the amino acid sequence shown under SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence , shown under SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence shown under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence shown under SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 6, (b) approximately 50 mm phosphate buffer, (c) approximately 5% sucrose, (d) approximately 0.05% polysorbate 80 and (e) approximately 25 mM arginine.

In another embodiment, the stable aqueous solution contains (a) an anti-C5 antibody at a concentration of 100 mg/mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 comprising the amino acid sequence shown under SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence, listed under SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence listed under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence listed under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence listed under SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 6, (b) 50 mM phosphate buffer, (c) 5% sucrose, (d) 0.05% polysorbate 80, and (e ) about 25 mM arginine.

In another embodiment, the stable aqueous solution consists of (a) an anti-C5 antibody at a concentration of approximately 100 mg/mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 comprising the amino acid sequence shown under SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid the sequence shown under SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence shown under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence, listed under SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence listed under SEQ ID NO: 6, (b) approximately 50 mm phosphate buffer, (c) approximately 5% sucrose, (d) approximately 0.05% polysorbate 80 and (e) about 25 mM arginine.

In another embodiment, the stable aqueous solution consists of (a) an anti-C5 antibody at a concentration of 100 mg/mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 containing the amino acid sequence shown under SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence , shown under SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence shown under SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence shown under SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence shown under SEQ ID NO: 6, (b) 50 mm phosphate buffer, (c) 5% sucrose, (d) 0.05% polysorbate 80 and ( e) 25 mM arginine.

IV. Methods for obtaining highly concentrated antibody solutions

Also provided herein are methods for preparing a highly concentrated anti-C5 antibody solution. In one embodiment, methods are provided for preparing a stable concentrated antibody solution containing a 100 mg/mL anti-C5 antibody, 50 mM phosphate buffer, 5% sucrose, and 25 mM arginine, the method comprising:

i) obtaining a first aqueous solution containing an antibody to C5, while the first aqueous solution has a first composition and contains no more than 10 mg/ml of antibody to C5;

ii) diafiltering the first aqueous solution to obtain a composition containing 50 mM phosphate buffer, 5% sucrose and 25 mM arginine at pH 7.4, thereby obtaining a second aqueous solution, while the second aqueous solution has a second composition as a result of diafiltration ; and

iii) concentration of the second aqueous solution to obtain a stable concentrated antibody solution containing 100 mg/ml anti-C5 antibody, 50 mM phosphate buffer, 5% sucrose and 25 mM arginine.

In another embodiment, a method is provided for preparing a stable concentrated antibody solution containing an antibody to C5 at a concentration of 100 mg/ml, 50 mm phosphate buffer, 5% sucrose; 25 mM arginine and 0.05% polysorbate 80, the method comprising:

i) obtaining a first aqueous solution containing an antibody to C5, while the first aqueous solution has a first composition and contains no more than 10 mg/ml of antibody to C5;

ii) diafiltration of the first aqueous solution to obtain a composition containing 50 mM phosphate buffer, 5% sucrose, 25 mM arginine and 0.05% polysorbate 80 at pH 7.4, thereby providing a second aqueous solution, while the second aqueous the solution has a second composition as a result of diafiltration; and

iii) concentration of the second aqueous solution to obtain a stable concentrated antibody solution containing 100 mg/ml anti-C5 antibody, 50 mM phosphate buffer, 5% sucrose, 25 mM arginine and 0.05% polysorbate 80.

v. Routes of administration

The solutions described herein can be administered to a patient in a number of ways, depending in part on the route of administration. The route of administration may be parenteral, for example, intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, intraocular injection, intra-articular injection or intramuscular injection (IM). The terms "parenteral", "parenterally administered" and other grammatically equivalent phrases as used herein refer to routes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial , intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid and intrasternal injection and infusion.

In a specific embodiment, the solution is administered by subcutaneous injection. Subcutaneous administration can be performed using a device. The device may be a syringe, a pre-filled syringe, a disposable or reusable auto-injector, a pen, a patch injector, a body injector, an ambulatory syringe infusion pump with a subcutaneous infusion set, or other device.

In one embodiment, the solution described herein is delivered to a subject via topical administration. In the context of this document, the term "topical administration" or "topical delivery" refers to delivery that is not based on the transport of the composition or active agent (for example, anti-C5 antibodies) to their putative target tissue or site via the vascular system. After topical administration near the target tissue or site, the solution or one or more of its components may diffuse into the intended target tissue or site.

For example, the solution may be delivered by injection or by implantation of a device containing the solution. The implant may be made from a porous, non-porous or gel-like material, including membranes such as silastic membranes, or fibers. The implant may be configured to release the solution continuously or intermittently into the subject's body. See, for example, US Patent Application Publication No. 20080241223; US patents No. 5501856; 4863457 and 3710795; EP488401 and EP 430539, the disclosure of each of which is incorporated herein by reference in its entirety. The solution described herein may be delivered to the subject via an implantable device based on, for example, diffusion, eroding, or convection systems, e.g., osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmotic systems, vapor pressure pumps, electrolytic pumps, gas generating pumps , piezoelectric pumps, eroded systems or electromechanical systems.

In one embodiment, the solution described herein can be topically injected into a joint (eg, a hinge joint). For example, in embodiments where the disorder is arthritis, a therapeutically suitable solution may be administered directly into the joint (eg, into the joint space) or near the joint. Examples of joints into which the composition described herein may be topically administered include, for example, the hip, knee, elbow, wrist, sternoclavicular, temporomandibular, carpal, tarsal, ankle, and any other joint subject to arthritic conditions. The composition described herein may also be administered into a bursa, such as, for example, acromio-acromion, biceps-radius, ulnar-radius, deltoid, subpatellar, ischial, and any other bursa known in the medical field.

In another embodiment, the solution described herein can be topically administered to the eye. Used in this document, the term "eye" refers to all, without exception, anatomical tissues and structures associated with the eye. In one embodiment, the solution described herein is injected into the posterior chamber of the eye. In another embodiment, the solution described herein is injected into the vitreous. In another embodiment, the solution described herein is administered through the sclera.

In some embodiments, for example, in embodiments for the treatment or prevention of a disorder such as COPD or asthma, the solution described herein may be administered to a subject through the lung. Pulmonary drug delivery may be by inhalation, and inhalation administration herein may be oral and/or intranasal. In one embodiment, the solution described herein can be administered to the lungs of a subject via an inhaler. Inhalers use compressed air to deliver the compound as a liquid aerosol or mist. The inhaler may be, for example, a jet inhaler (eg, an air jet or liquid jet inhaler) or an ultrasonic inhaler. Additional devices and methods for intrapulmonary administration are set forth, for example, in US Patent Application Publication Nos. 20050271660 and 20090110679, the disclosure of each of which is incorporated herein by reference in its entirety.

In other embodiments, the solutions described herein are present in a single dose dosage form, which may be particularly suitable for self-administration. The formulated product of the present invention may be included in a container, typically, for example, a vial, cartridge, pre-filled syringe, or disposable pen. A dispenser may also be used, such as the dispenser described in US Pat. No. 6,302,855. The injection system may include a delivery pen as described in US Pat. No. 5,308,341. well known in the art. Such devices may contain at least one injection needle (for example, a 31 gauge needle approximately 5-8 mm long) are typically pre-filled with one or more single therapeutic doses of solution and are useful for rapidly delivering solution to a subject with as little pain as possible.

VI. Methods of treatment

The described solutions can be used to treat various diseases and conditions in a human patient. In one embodiment, the solutions can be used to treat a disorder associated with the complement system, including, but not limited to, rheumatoid arthritis (RA); antiphospholipid antibody syndrome; lupus nephritis; ischemic/reperfusion injury; atypical hemolytic uremic syndrome (aHUS), typical or infectious hemolytic uremic syndrome (tHUS); dense sediment disease (DDD); paroxysmal nocturnal hemoglobinuria (PNH); neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); macular degeneration (for example, age-related macular degeneration (AMD)); syndrome of hemolysis, increased activity of liver enzymes and thrombocytopenia (HELLP); thrombotic thrombocytopenic purpura (TTP); spontaneous abortion; low-immune vasculitis; bullous epidermolysis; recurrent miscarriage and traumatic brain injury (see, for example, Holers (2008)Immunological Reviews 223:300-316, and Holers and Thurman (2004)Molecular Immunology 41:147-152).

In another embodiment, the complement system-associated disorder is a complement system-associated vascular disorder, such as, without limitation, a diabetes mellitus-associated vascular disorder (e.g., in the eye), central retinal vein occlusion, cardiovascular disorder, myocarditis, cerebrovascular disorder, peripheral (eg, musculoskeletal) vessels, renovascular disorder, mesenteric/enteric vascular disorder, graft and/or replant revascularization, vasculitis, Henoch-Schonlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis, immune complex vasculitis, disease Takayasu, dilated cardiomyopathy, diabetic angiopathy, Kawasaki disease (arteritis), venous gas embolism (VGE) and restenosis after stenting, rotational atherectomy and percutaneous transluminal coronary angioplasty (PTCA) (see, for example, U.S. Patent Application Publication No. 20070172483).

Additional disorders associated with the complement system include, but are not limited to, myasthenia gravis, cold agglutinin disease, dermatomyositis, Graves' disease, atherosclerosis, Alzheimer's disease, Guillain-Barré syndrome, Degos disease, tissue rejection (eg, transplant rejection), sepsis, burns (eg, severe burn), septic systemic inflammatory response, septic shock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis, type I diabetes mellitus, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), idiopathic thrombocytopenic purpura (ITP), Goodpasture's syndrome, antiphospholipid syndrome ( APS), catastrophic APS (CAPS), amyotrophic lateral sclerosis (ALS), Alzheimer's disease and chronic inflammatory demyelinating neuropathy.

In another embodiment, the solutions described herein can be used to treat thrombotic microangiopathy (TMA), for example, TMA associated with a disorder associated with the complement system, such as any of the disorders associated with the complement system described herein.

Complement-associated disorders also include pulmonary disorders associated with the complement system such as, but not limited to, asthma, bronchitis, chronic obstructive pulmonary disease (COPD), interstitial lung disease, α-1-antitrypsin deficiency, emphysema, bronchiectasis, bronchiolitis obliterans , alveolitis, sarcoidosis, pulmonary fibrosis, and collagenous vascular disorders.

In another embodiment, the solution described herein is administered to a subject to treat, prevent, or ameliorate at least one symptom of an inflammatory response associated with the complement system (e.g., aspect of a disorder associated with the complement system, which is an inflammatory response associated with the complement system) in a subject. For example, the composition may be used to treat, prevent, and/or ameliorate one or more symptoms associated with the complement system-associated inflammatory response, such as tissue rejection/graft-versus-host disease (GVHD), reperfusion injury (e.g., after cardiopulmonary bypass or tissue transplantation) and tissue damage following other forms of traumatic injury such as burns (eg, severe burns), blunt trauma, spinal injury, or frostbite. See, for example, Park et al. (1999)Anesth Analg 99(1):42-48; Tofukuji et al. (1998)J Thorac Cardiovasc Surg 116(6):1060-1068; Schmid et al. (1997)shock 8(2):119-124 and Bless et al. (1999)Am J Physiol 276(1):L57-L63.

In another embodiment, the complement system mediated disorder is a complement system mediated vascular disorder such as, but not limited to, cardiovascular disorder, myocarditis, cerebrovascular disorder, peripheral (e.g., musculoskeletal) vessels, renovascular disorder, mesenteric/enteric vascular disorder, graft and/or replant revascularization, vasculitis, Henoch-Schonlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis, immunocomplex vasculitis, transplantation organ or tissue disease, Takayasu disease, capillary leak syndrome, dilated cardiomyopathy, diabetic angiopathy, thoracic and abdominal aortic aneurysm, Kawasaki disease (arteritis), venous gas embolism (VGE) and restenosis after stenting, rotational atherectomy and percutaneous transluminal coronary angioplasty ( PTCA) (see, for example, U.S. Patent Application Publication No. 20070172483).

VII. Types of combined treatment

In one embodiment, the solutions described herein are administered to a patient as monotherapy. In another embodiment, they are administered in conjunction with one or more additional agents and/or other therapies (for example, which are suitable for the treatment of disorders associated with the complement system). For example, combination therapy may include administering to a human patient one or more additional agents (eg, anticoagulants, antihypertensives, or anti-inflammatory drugs (for example, steroids)), which provide a therapeutically beneficial effect on the patient. In one embodiment, the solutions described herein are administered in combination with an anti-inflammatory agent (e.g., NSAIDs, corticosteroids, methotrexate, hydroxychloroquine, anti-TNF antibodies such as etanercept and infliximab, a B-cell depleting agent such as rituximab, an interleukin-1 antagonist, or an agent that blocks T-cell costimulation such as abatacept) .

Additional agents for treating a complement disorder in a subject will differ depending on the particular disorder being treated, but may include, without limitation, one or more antihypertensive agents (e.g., angiotensin converting enzyme inhibitor, labetalol, hydralazine, nifedipine, calcium channel antagonists, nitroglycerin or sodium nitroprusside), anticoagulants, corticosteroids (eg, prednisone), immunosuppressants (eg. vincristine or cyclosporine A), anticoagulants (eg. warfarin (Coumadin), aspirin, heparin, phenindione, fondaparinux, idraparinux), thrombin inhibitors (eg, argatroban, lepirudin, bivalirudin, or dabigatran), fibrinolytic agents (eg, ancrod, α-aminocaproic acid, antiplasmin-a₁, prostacyclin, and defibrotide), antihypertensive agents (eg, labetalol, hydralazine, nifedipine, calcium channel antagonists, nitroglycerin or sodium nitroprusside), lipid-lowering agents (eg, hydroxymethylglutaryl-CoA reductase inhibitor), anticonvulsants (eg, magnesium sulfate), antithrombotic agents (eg, heparin, antithrombin, prostacyclin, or low-dose aspirin), sympathomimetics (eg, albuterol), antibiotics, deoxyribonucleases (eg. Pulmozyme®), anticholinergic drugs, IgE inhibitors (eg, anti-IgE antibodies), corticosteroids, or non-steroidal anti-inflammatory drugs (NSAIDs). Many different NSAIDS are available, some over the counter including ibuprofen (Advil®, Motrin®, Nuprin®) and naproxen (Alleve®), and many others available by prescription including meloxicam (Mobic®), etodolac (Lodine®) , nabumetone (Relafen®), sulindac (Clinoril®), tolmetin (Tolectin®), choline magnesium salicylate (Trilasate®), diclofenac (Cataflam®, Voltaren®, Arthrotec®), diflunisal (Dolobid®), indomethacin (Indocin® ), ketoprofen (Orudis®, Oruvail®), oxaprozin (Daypro®) and piroxicam (Feldene®) (see e.g. mihuet al. (2007)J Gastrointestin Liver Dis 16(4):419-424). In another embodiment, the solution described herein may be formulated for administration to a patient in conjunction with intravenous gamma globulin (IVIG) therapy, plasmapheresis, plasma exchange, or plasma exchange.

In one embodiment, the solution and one or more additional agents and/or therapies are administered simultaneously. In another embodiment, the solution is administered prior to the administration of one or more additional agents and/or therapies. In another embodiment, the solution is administered following the administration of one or more additional agents and/or therapies.

If the antibody solution described herein is used in combination with a second active agent, then the agents (e.g., anti-C5 antibody and second agent) can be formulated separately or together. For example, solution and agent can be mixed, for example, immediately before administration, and administered together or separately, for example, at the same time or at different times.

VIII. Single dose kits and dosage forms

Also provided herein are kits that contain a stable aqueous solution containing an anti-C5 antibody such as ravulizumab or BNJ421, or an antigen-binding fragment thereof, in a therapeutically effective amount suitable for administration to a human patient (e.g., a patient with a disorder associated with the complement system). Kits may optionally also contain instructions, for example, contain introduction schedules to enable the practitioner (for example, doctor, nurse or patient) to administer the composition contained therein to administer the solution to the patient.

The kits may also contain a suitable means for delivering one or more solutions to a patient in need, for example, a patient suffering from a disorder associated with the complement system, presumably having it or at risk of developing it. In one embodiment, the agent is suitable for invasive (eg, intravascular (eg. intravenous), subcutaneous, intraarticular, intraocular, intravitreal or intramuscular) solution delivery to the patient. In another embodiment, the agent is suitable for subcutaneous delivery of a solution to a patient. In another embodiment, the agent is suitable for intravenous delivery of a solution to a patient. For example, the agent may be a syringe or an osmotic pump. In another embodiment, the solution may be formulated as eye drops, wherein the agent is an eye dropper.

The kits optionally contain multiple single dose solution packs, each containing an effective single dose amount of the solution. Instruments or devices needed to administer the solution may also be included in kits. For example, one or more pre-filled syringes containing a solution may be provided in a kit.

The following examples are illustrative only and should not be construed as limiting the scope of the present invention in any way, as many changes and equivalents will become apparent to those skilled in the art upon reading the present invention.

The contents of all literature, Genbank entries, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

EXAMPLES

Example 1 Development of a highly concentrated formulation of ravulizumab (AXLN1210) for subcutaneous administration

This example summarizes the development of a highly concentrated formulation of ALXN1210 for subcutaneous administration (e.g., 50 mM phosphate buffer, 5% sucrose, 25 mM arginine, pH 7.4 at 100 mg/ml). Preliminary experiments were performed early in formulation development to provide screening data for candidate compounds and to assess the reduction in opalescence at higher concentrations of ALXN1210. The original composition of ALXN1210 (10 mM phosphate, 150 mM sodium chloride, pH 7.0, 0.02% Tween 80, at 10 mg/mL) was colorless and slightly opalescent. As the concentration of ALXN1210 increased, the degree of opalescence also increased. Taking into account the results of the screening of candidate compounds, a stability study was carried out to obtain data on the stability of the leader compounds. After the initial stability study, a stability study of prototypes was conducted to obtain the optimal composition of the bulk drug substance and drug product. Preliminary studies and stability studies are discussed in detail below.

1. Ways

Appearance

Appearance was determined by visual observation using normal laboratory lighting on both white and black backgrounds.

C5 binding

The C5 binding ELISA is an assay for ALXN1210 activity. This test procedure is a direct binding immunoassay with colorimetric detection used to test the ability of ALXN1210 to bind to its target, which is the protein C5 of the human complement system. A PolySorp microtiter plate was coated with human C5 protein and blocked with bovine serum albumin (BSA). The calibration curve was obtained from the reference material ALXN1210. Reference material and test samples were obtained in three dilutions designed to fall within the working range of the assay. After incubation with standards and samples, the plate was then washed and incubated with mouse anti-human IgG4 antibody conjugated with horseradish peroxidase (HRP). The plate was washed again and then developed using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) substrate (ABTS). The amount of reacted substrate was read spectrophotometrically on a plate reader at 405 nm. The absorbance was proportional to the concentration of ALXN1210 binding to C5 on the plate. A four-parameter curve fitting was applied to the calibration curve, and the results for the reference material and test samples were interpolated from the curve. The test results of the samples were compared with the results for the reference material, and the relative activity (%) was recorded.

Density

Density measurements were made with a DMA 4500 density meter, determined by the U-tube method. A hollow U-shaped glass tube was filled with the sample and then transferred to an electronically excited state at the lowest possible amplitude. Density was determined by the following equation:

ρ=A(τ ² )-B,

ρ=density;

τ=oscillation period.

A and B are meter constants determined by calibrating the meter with two substances of known density.

Differential Scanning Fluorimetry

By means of differential scanning fluorimetry, the change in thermal stability is measured by obtaining a thermal denaturation curve in the presence of a fluorescent dye such as Sypro orange. As the protein unfolds, the available hydrophobic surfaces bind to the dye, increasing the level of fluorescence and providing a stability curve with a characteristic midpoint value at the hydrophobic surface exposure temperature T _h .

Dynamic Light Scattering

Dynamic Light Scattering measures the size and interactions of proteins, nanoparticles and other macromolecules in situ in microwell plates using an illumination system that allows imaging of wells in a microplate using a built-in 3 megapixel camera. The deviations in the scattering of light due to Brownian motion give a diffusion coefficient which is related to the hydrodynamic radius of the particles present in the solution.

Gel permeation HPLC

Gel permeation (exclusion) HPLC was used to separate monomeric IgG from larger multimeric antibody molecules that can form as a result of monomer aggregation. The test samples were loaded onto a TSK G3000 SWXL gel column equilibrated with phosphate buffered saline, pH 7.0, followed by isocratic elution. Protein peaks were monitored at 214 nm and the percentage purity of monomeric IgG was expressed as a percentage of the total integrated peak area. Identification of multimers with greater mass was carried out by observing the peaks that appear when eluting to the peak of the monomer.

Imaging capillary electrophoresis (iCE)

In this method, the Protein Simple iCE280 or iCE3 system was used, which provides a solution for IEF without impurities in a capillary column, and detection of protein accumulation zones was carried out using a UV detector for the entire column. Samples were obtained by premixing ALXN1210, carrier ampholytes and pI markers. The sample was loaded into a capillary electrophoresis cartridge and the electrolyzers at each end of the capillary were filled with acid and base. A voltage was applied and the analytes formed clusters at their pI. The CCD camera visualizes the UV absorption of the entire capillary column every 30 seconds, providing real-time monitoring of the clumping stage. The resulting separation pattern was recorded and analyzed. The pI of the proteins present in the sample was interpolated from the position of the pI markers introduced into the sample.

Lab on a Chip (LoC)

This method was used to test the homogeneity and purity of the product. Denaturation of non-reduced samples was carried out by treatment with lithium dodecyl sulfate (LDS). The reduced samples were denatured by treatment with lithium dodecyl sulfate (LDS) and disulfide bonds were broken with dithiothreitol (DTT). The polypeptide chains were mixed with a fluorescent dye that binds to LDS and separated according to molecular size by microcapillary electrophoresis. The protein was detected and quantified by laser-induced fluorescence.

Osmolality

The osmolality of the samples was determined using an osmometer that measures the drop in freezing point. The osmometer was calibrated prior to use with commercially available certified osmolality standards at 50 mOsm/kg and 850 mOsm/kg, which cover the sample range. A 290 mOsm/kg reference solution was used to confirm successful calibration prior to testing samples. The samples were tested in triplicate, and the average of those determined for the samples was recorded.

pH

pH measurement was performed using a protein-resistant saturated silver-free KCl combination electrode and an appropriate meter and temperature control device. Prior to use, the meter was calibrated with commercially available solutions in the appropriate pH range (i.e. pH 4.0 - pH 7.0).

Determination of protein concentration using SoloVPE

Absorbance at 280 nm was used to determine the concentration of proteins in the test samples using a technology with different optical path lengths using a theoretically determined extinction coefficient of 1.479. In the method, absorbance values were determined in triplicate for each sample.

Viscosity

The measured viscosities were determined using an AMVn viscometer using the falling ball method. The hollow tube was filled with a sample and a solid ball of known density and then tilted at a known angle. The time required for the ball to move from one end of the tube to the other was determined and used to calculate the viscosity using the following equation:

η=K*(ρb-ρs)*tr,

η=dynamic viscosity (mPa*s);

K=proportionality constant;

ρb=bead density (g/ml);

ρs=sample density (g/ml);

tr=time the ball falls.

To calculate the viscosity, the density of the sample, determined using a DMA 4500 M density measuring instrument, was used as ρs.

Determination of Particles Invisible to the Naked Eye by Microflow Imaging (MGI)

The goal is to quantify all the particles in the formulation that are invisible to the naked eye through micro-flow imaging (MFI). A sample was taken from storage at a temperature of 2-8°C and directly tested by MFI using a BOT1 autosampler. The sample was inverted six times before loading the sample into BOT1 to ensure complete mixing of the particles. Samples were loaded into three successive wells, and one measurement was made for each of them, with a total of 3 replicates. Three mixing cycles were performed in BOT1 to further ensure mixing uniformity.

2. Development of the composition

Figures 1-5 and Tables 1-5 show the results of experiments that were carried out early in the development of highly concentrated ALXN1210 formulations.

In the first experiment, the effect of adding an amino acid to ALXN1210 in sodium phosphate buffer on opalescence was observed. The cause of opalescence was determined to be the absence of charge to allow charge repulsion between antibody molecules in solution at high concentration. Conducted a number of experiments, described below, in order to ensure the optimal choice of a particular amino acid and the concentration required to obtain a stable clear solution. Based on the results of these experiments, it was determined that the addition of a positively charged amino acid (L-arginine) provided a reduction in the degree of opalescence in a sample containing 50 mg/ml ALXN1210 in sodium phosphate buffer. The same conclusion was reached by visual inspection of the vials (data not shown).

In addition, subsequent experiments were performed by using formulation ALXN1210 IV at 10 mg/mL and by concentrating the antibody and performing buffer exchange to evaluate different initial buffer systems to use when finding a highly concentrated formulation of ALXN1210. As shown in Table 1 below, all pooled samples were subjected to a final buffer change at 1:1000 to obtain the desired pH. Pooled samples ranged from 35.3 to 54.0 mg/mL. The % recovery after buffer change ranges from 70.6% to 108%. The results of the appearance analysis showed that the contents of the 25 mM histidine pH 7 and 25 mM phosphate pH 7 buffer exchange vials were clear and colorless, comparable to eculizumab, and the solutions in all other buffer exchange vials were opalescent. Imaging capillary electrophoresis (iCE) results show that pI ranges from 5.98 to 6.54, main pI ranges from 6.19 to 6.24, and % peak area ranges from 63.1% up to 65.9%. The results of size exclusion chromatography (SEC) show that the % of monomers (purity) is from 98.48% to 98.98%.

Table 1. ALXN1210 with buffer exchange, 10 mg/mL to 50 mg/mL (Pooled Sample Groups 1-3)

Buffer buffer pH after replacement Measured concentration (mg/ml) % recovery after buffer replacement Appearance iCE SEC (% monomers) 25 mM, citrate buffer, pH 5 5.05 54.0 108.0 Opalescent pi range 6.01-6.53 98.48 25 mM, citrate buffer, pH 6 6.09 41.7 83.4 Opalescent pi range 6.01-6.53 98.70 25 mM, acetate buffer, pH 5, 4.98 42.1 84.2 Opalescent pi range 6.01-6.53 98.98 25 mM, acetate buffer, pH 6, 5.86 35.3 70.6 Opalescent pi range 6.01-6.53 98.79 25 mM, HEPES buffer, pH 7 6.93 49.7 99.4 Opalescent pi range 6.03-6.54 98.59 25 mM, histidine, pH 6 5.90 37.4 74.8 Opalescent pi range 6.02-6.53 98.93 25 mM, histidine, pH 7 6.94 47.2 94.4 *Transparent, colorless pi range 5.98-6.48 98.82 25 mM, phosphate buffer, pH 7 7.10 48.6 97.2 *Transparent, colorless pi range 6.01-6.53 98.50 25 mM, citrate buffer, pH 6.8 6.83 49.6 99.2 Opalescent 25 mM, phosphate-citrate buffer, pH 6.9 6.89 46.5 93.1 Opalescent 25 mM, potassium phosphate buffer, pH 7 7.05 49.0 97.9 Opalescent 25 mM, sodium phosphate buffer, pH 7.5 7.55 44.5 89.1 Opalescent

*Comparable to eculizumab.

As shown in Figure 1, the results obtained by salt titration of histidine-buffered samples at pH 7 using DLS show that self-association of ALXN1210 also increases with increasing salt concentration.

As shown in Figure 2, the results obtained by dynamic light scattering (DLS) titration with L-arginine show that 25 mM L-arginine is the minimum amount required to reduce opalescence in ALXN1210 at 50 mg/mL.

As shown in Figure 3, the results obtained from the phosphate-buffered salt titration at pH 7 using DLS show that no salt addition and 150 mM salt addition also provide the lowest self-association in ALXN1210. For comparison, see peaks labeled 2 and 5.

As shown in Figure 4, the results obtained for buffer exchange samples using DSF show that the hydrophobic pockets of ALXN1210 are not accessible. Citrate and acetate buffers at pH 5 and 6 have poor thermal stability with the lowest melting points (Tm), while histidine and phosphate buffers at pH 7 are the most stable with the highest Tm.

As the results of the appearance analyzes shown in Table 2 show, ALXN1210 at approximately 100 mg/ml is clear and colorless with the addition of 25 mM L-arginine in 25 mM phosphate buffer at pH 7.

Table 2 Appearance of ALXN1210 samples at 100 mg/mL

Sample ID Buffer Measured concentration (mg/ml) Appearance 10 mM sodium phosphate, pH 7 100.0 Opalescent SPPD-14-0042-8 25 mM histidine, pH 7.2 66.0 Opalescent SPPD-14-0042-9 10 mM phosphate, 25 mM L-arginine, pH 7 114.5 Opalescent SPPD-14-0042-composition C 25 mM phosphate, pH 7 112.0 Opalescent SPPD-14-0042-formulation C with added L-arginine 25 mM phosphate, 25 mM L-arginine (introduced), pH 7 112.0 Transparent, colorless

As shown in Figure 5, the results for ALXN1210 at 10 mg/mL and 114 mg/mL with and without L-arginine supplementation obtained using DLS show that the addition of 25 mM L-arginine to the sample at at least 100 mg /ml provides a result that most closely matches the reference solution at 10 mg/ml. In a sample with at least 100 mg/ml without the addition of L-arginine, the formation of higher order molecules occurs, indicating self-association.

The results of the osmolality analysis presented in Table 3 show that ALXN1210 in 25 mM histidine, pH 7.2, with 8% sucrose or 4.5% sorbitol is within the desired osmolality range of 275-320. The osmolality value of ALXN1210 in buffer with 25 mM phosphate, 25 mM L-arginine, pH 7, supplemented with 7% sucrose or 4% sorbitol is also within the desired osmolality range.

Table 3. Osmolality of ALXN1210 in various formulations

Sample Description applied buffer Concentration (mg/ml) % filler Osmolality (mOsm/kg) SPPD-14-0042-8 25 mM histidine, pH 7.2 66.0 55 SPPD-14-0042-8 25 mM histidine, pH 7.2 66.0 8% sucrose 299 SPPD-14-0042-8 25 mM histidine, pH 7.2 66.0 4.5% sorbitol 295 SPPD-14-0042-formulation C with L-arginine 25 mM phosphate, 25 mM L-arginine, pH 7 112.0 69 SPPD-14-0042-formulation C with L-arginine 25 mM phosphate, 25 mM L-arginine, pH 7 112.0 7% sucrose 302 SPPD-14-0042-formulation C with L-arginine 25 mM phosphate, 25 mM L-arginine, pH 7 112.0 4% sorbitol 317

The results of the viscosity analysis presented in Table 4 show that as the concentration of ALXN1210 increases, the viscosity of ALXN1210 solutions in histidine and phosphate buffers also increases. The results of the density analysis presented in Table 4 show no significant change in density for histidine and phosphate buffers with changes in concentration.

Table 4. Viscosity and density of ALXN1210 samples at various concentrations

Sample Description/Sample Matrix ALXN1210 concentration (mg/ml) Dynamic viscosity (mPa⋅s) Density (g/cm3) 25 mM histidine buffer, pH 7.2 0 1.014 0.999 ALXN1210, histidine, pH 7.2 66.0 3.368 1.018 10 mM sodium phosphate buffer, pH 7 0 1.014 0.999 10 mM sodium phosphate one hundred 8.788 1.027 25 mM phosphate buffer, 25 mM arginine, pH 7 0 1.030 1.002 ALXN1210, phosphate, 25 mM arginine, pH 7 114.5 10.387 1.034 ALXN1210, phosphate, 25 mM arginine, pH 7 100.3 6.605 1.002 ALXN1210, phosphate, 25 mM arginine, pH 7 95.1 5.714 1.002 ALXN1210, phosphate, 25 mM arginine, pH 7 90.0 4.906 1.002 ALXN1210, phosphate, 25 mM arginine, pH 7 75.7 3.476 1.002 ALXN1210, 25 mM phosphate buffer, 25 mM L-arginine, pH 7, 7% sucrose 0 1,250 1.028 ALXN1210, 25 mM phosphate, 25 mM L-arginine, pH 7, 7% sucrose (SPPD-14-0042-formulation C with L-arginine) 93.6 4.109 1.056 ALXN1210, 25 mM phosphate buffer, 25 mM L-arginine, pH 7, 4% sorbitol 1.153 1.016 ALXN1210, 25 mM phosphate, 25 mM L-arginine, pH 7, 4% sorbitol (SPPD-14-0042-formulation C with L-arginine) 80.0 2.877 1.038

As shown in table 5, the addition of L-arginine in the form of a base significantly increases the pH of the sample. Sufficient L-arginine with monobasic sodium phosphate added to the sample provided a pH increase of 1 pH unit. L-arginine⋅HCl added to the sample provided a pH decrease of approximately 0.25 pH units. However, in terms of appearance, the degree of opalescence decreased from the addition of L-arginine⋅HCl to the addition of L-arginine in sufficient quantity with sodium phosphate monobasic to the addition of L-arginine in base form.

Table 5. Effects of 25 mM L-arginine in L-arginine buffers on pH and appearance for ALXN1210

Sample Description pH Appearance 0.5 M solution of L-arginine in base form 11.47 1210-PD-14-0042-composition C 7.04 SPPD-14-0042-formulation C with 25 mM L-arginine 8.85 0.5 M sodium phosphate monobasic solution 4.23 0.5 M dibasic sodium phosphate solution 8.94 0.5 M solution of L-arginine in base form in sufficient quantity with 0.5 M solution of monobasic sodium phosphate 8.31 SPPD-14-0042-formulation C supplemented with 25 mM L-arginine solution in sufficient quantity with sodium phosphate monobasic solution 7.38 0.5 M L-arginine⋅HCl 5.48 25 mM sodium phosphate, 7% sucrose, pH 7, addition of 25 mM L-arginine⋅HCl 6.79 25 mM sodium phosphate, 4% sorbitol, pH 7, addition of 25 mM L-arginine⋅HCl 6.81 Bulk 1210-75-25P-7.0-5S (SPAS-14-007) supplemented with 25 mM L-arginine⋅HCl 7.07 Highly opalescent Bulk 1210-75-25P-7.0-3So (SPAS-14-007), 25 mM sufficient L-arginine with monobasic sodium phosphate 7.55 Moderately opalescent Bulk 1210-75-25P-7.0-3So (SPAS-14-007), 25 mM L-arginine base 9.17 Slightly opalescent

3. Storage stability

Figures 6-21 show the results of the initial stability study. These results show that histidine containing formulations are the least stable and phosphate containing formulations are most stable after 2 months at 2-8°C, 23-27°C and 37°C. Also, as evidenced by the results of size exclusion chromatography, the compositions containing phosphate, with sorbitol and sucrose were comparable after 2 months at 2-8°C and 23-27°C. However, the sorbitol formulations were slightly more stable at 37°C compared to the sucrose formulations after 2 months. The results of dynamic light scattering show no significant change in the samples in phosphate buffer with 25 mm L-arginine after 2 months at 2-8°C with the addition of sucrose or sorbitol. Dynamic light scattering results for samples containing histidine at T=2 months may not overlap due to high polydispersity between acquisitions, indicating a less stable composition compared to formulations containing phosphate. The results of the 5 day freeze-thaw cycle did not show any significant change between T=0 and 5 freeze-thaw cycles.

4. Composition of prototypes

Figures 22-46 show the results of the study of the stability of the prototypes.

These results show that all formulations containing phosphate at 75 mg/ml and 100 mg/ml (bulk drug substance (BDS) and drug product (DP)) are stable throughout the stability study at 2-8°C, - 20°C and -80°C. All 100 mg/mL bulk formulations after 5 freeze-thaw cycles at -20°C and -80°C were stable and did not show significant change.

5. Intermediate conclusions before short-term failure testing

Based on the results of these studies, the optimal composition for the highly concentrated ALXN1210 material was determined. In preliminary experiments, the addition of L-arginine to reduce opalescence was suggested for ALXN1210 at 100 mg/mL. The initial stability study resulted in the selection of the lead formulation with phosphate buffer and L-arginine at >50 mg/mL. Based on the results of the study of the stability of the prototypes, the initial optimal composition for ALXN1210 was determined, containing 50 mm phosphate buffer, 5% sucrose, 25 mm arginine, pH 7.4 at 100 mg/ml.

6. Development of the final optimal composition

In order to evaluate the suitability of the initial optimal formulation of ALXN1210 at 100 mg/mL in formulation buffer (50 mM sodium phosphate, 25 mM arginine and 5% sucrose at pH 7.4), it was subjected to short term degradation studies to determine whether polysorbate was necessary. 80 (PS80) or other surfactant to prevent degradation. The two grades of PS 80 were 0.05% (w/v) POLYSORBATE 80 (HX2)™ from NOF America Corporation, reported to contain >99% pure oleic acid, and Polysorbate 80 AVANTOR™ 4117 from J.T. Baker®, which is a widely used surfactant that consists of a mixture of fatty acids containing oleic acid and palmitic acid. Both products are often referred to as TWEEN 80® and are nonionic surfactants derived from polyethoxylated sorbitan and oleic acid with hydrophilic groups derived from ethylene oxide polymers.

Test methods used to evaluate the potential application of the PS80 Avantor J.T. Baker 4117 in ALXN1210 at 100mg/mL, are listed in Table 6 below. See Individual Testing Method for more details and description of the method.

Table 6. Test methods to determine the potential application of the PS80 Avantor J.T. Baker 4117

Testing Analysis type Purpose and rationale Appearance General characteristics A change in appearance may indicate deterioration of the product. Protein concentration Protein concentration A change in the results of a protein concentration assay may indicate changes in the solubility or stability of the product. Turbidity (UV-visible spectroscopy) General characteristics An increase in turbidity may indicate a change in the solubility or stability of the product. SE-UPLC Purity To ensure sample integrity and compliance with purity requirements. HIAC in low volume General characteristics To quantify changes in the number of particles invisible to the naked eye in a solution. An increase in the number of particles invisible to the naked eye may indicate changes in physical properties in the product profile that may affect safety. CE-SDS Purity A change in the CE-SDS pattern may indicate degradation of the product, such as cleavage of the polypeptide(s) chain(s) or aggregation of the product. iCE Identity An unsatisfactory result may indicate the destruction of the protein with the formation of uncharacteristic isoforms. Dynamic Light Scattering (DLS) Stability To assess the degree of aggregation/destruction. Content of polysorbate 80 General characteristics For quantitative determination of the concentration of polysorbate 80 in the composition. A decrease in the concentration of polysorbate 80 can lead to antibody aggregation. Destruction of polysorbate 80 General characteristics For quantitative determination of the degree of destruction of polysorbate 80 in the composition. Increasing the degree of destruction of polysorbate 80 can lead to aggregation of the antibody.

Vials containing 100 mg/mL ALXN1210 with PS80 Avantor J.T. Baker 4117 or PS80 HX2 NOF were visually inspected. All samples showed no visible particles or obvious color changes in all samples subjected to destruction during storage at 45°C and additional agitation for 5 days at 2-8°C. The results are shown in Table 7.

Table 7. Visual inspection of 100 mg/mL ALXN1210 in 5 ^cc vials containing either PS80 Avantor JT Baker 4117 or PS80 HX2 NOF

Sample ID Transparency Colour Intermediate particle content Final particle content 100 mg/ml ALXN1210+Avantor, T=0 Transparent Slightly yellowish color Virtually free of particles Virtually free of particles 100 mg/ml ALXN1210+NOF, T=0 Transparent Slightly yellowish color Virtually free of particles Virtually free of particles 100 mg/ml ALXN1210+Avantor, T=7 days at 45°C Transparent Slightly yellowish color Virtually free of particles Virtually free of particles 100mg/ml ALXN1210+NOF, T=7 days at 45°C Transparent Slightly yellowish color Virtually free of particles Virtually free of particles 100mg/ml ALXN1210, Avantor, T=7 days at 45°C+5 days with shaking at 2-8°C Transparent Slightly yellowish color Virtually free of particles Virtually free of particles 100mg/ml ALXN1210, NOF, T=7 days at 45°C+5 days with shaking at 2-8°C Transparent Slightly yellowish color Virtually free of particles Virtually free of particles 100 mg/ml ALXN1210+Avantor, T=4 days at 45°C Transparent Slightly yellowish color Virtually free of particles Virtually free of particles 100 mg/ml ALXN1210+NOF, T=14 days at 45°C Transparent Slightly yellowish color Virtually free of particles Virtually free of particles 100mg/ml ALXN1210, Avantor, T=14 days at 45°C+5 days with shaking at 2-8°C Transparent Slightly yellowish color Virtually free of particles Virtually free of particles 100 mg/ml ALXN1210, NOF, T=14 days at 45°C+5 days with shaking at 5°C Transparent Slightly yellowish color Virtually free of particles Virtually free of particles

For the concentration of ALXN1210 in mg/ml, a slight decrease was shown due to the condition of the destruction. All concentration measurements are shown in Table 8.

ALXN1210 formulations at 100 mg/mL containing 0.05% PS80 Avantor or PS80 HX2 NOF had no significant change in concentration when subjected to the same disruption condition as shown in Figure 2.

Table 8. Concentration measurements

Sample ID Concentration (mg/ml) Sample ID Concentration (mg/ml) 100 mg/ml ALXN1210+Avantor, T=0 98.6 100 mg/ml ALXN1210+NOF, T=0 100.5 100 mg/ml ALXN1210+Avantor, T=7 days at 45°C 88.9 100mg/ml ALXN1210+NOF, T=7 days at 45°C 91.3 100mg/ml ALXN1210, Avantor, T=7 days at 45°C+5 days with shaking at 2-8°C 97.1 100mg/ml ALXN1210, NOF, T=7 days at 45°C+5 days with shaking at 2-8°C 98.2 100 mg/ml ALXN1210+Avantor, T=4 days at 45°C 98.4 100mg/ml ALXN1210+NOF, T=14 days at 45°C 96.5 100mg/ml ALXN1210, Avantor, T=14 days at 45°C+5 days with shaking at 2-8°C 98.5 100 mg/ml ALXN1210, NOF, T=14 days at 45°C+5 days with shaking at 5°C 96.9

As shown in Table 8 above, 100 mg/mL ALXN1210 formulations containing 0.05% PS80 Avantor or PS80 HX2 NOF had no significant change in concentration when subjected to the same degradation condition. The concentration remained comparable in ALXN1210 formulations at 100 mg/mL containing 0.05% PS80 Avantor or PS80 HX2 NOF.

Haze was measured by monitoring absorbance at 650 nm. The measurements are shown in Table 9.

ALXN1210 formulations at 100 mg/mL containing 0.05% PS80 Avantor or 0.05% PS80 HX2 NOF did not show significant turbidity changes. Haze values remain stable at all times and under all breaking conditions in this study.

Table 9. Haze measured by monitoring absorbance at 650 nm

Sample ID Absorption (650 nm) Sample ID Absorption (650 nm) 100 mg/ml ALXN1210+Avantor, T=0 5.02E-03 100 mg/ml ALXN1210+NOF, T=0 -9.28E-04 100 mg/ml ALXN1210+Avantor, T=7 days at 45°C 9.10E-04 100mg/ml ALXN1210+NOF, T=7 days at 45°C 7.31E-03 100mg/ml ALXN1210, Avantor, T=7 days at 45°C+5 days with shaking at 2-8°C -6.80E-04 100mg/ml ALXN1210, NOF, T=7 days at 45°C+5 days with shaking at 2-8°C 4.02E-04 100 mg/ml ALXN1210+Avantor, T=4 days at 45°C -1.21E-03 100mg/ml ALXN1210+NOF, T=14 days at 45°C 1.12E-03 100mg/ml ALXN1210, Avantor, T=14 days at 45°C+5 days with shaking at 2-8°C 6.82E-04 100 mg/ml ALXN1210, NOF, T=14 days at 45°C+5 days with shaking at 5°C 2.12E-03

There was a decrease in the percentage of monomers for samples that were incubated at 45°C for 7 days or 14 days with additional shaking (200 rpm) at 2-8°C, which was expected due to the conditions of destruction. However, for the percentage of monomers, no significant difference was shown between both formulations of ALXN1210 at 100 mg/mL containing 0.05% PS80 Avantor or 0.05% PS80 HX2 NOF at the same time points and exposure to the same the same conditions.

The percentage monomer data is shown in Table 10. Figure 4 shows the % monomer reduction expected after exposure to degradation conditions and no significant difference between both formulations of 100 mg/mL ALXN1210 containing 0.05% PS80 Avantor or 0 .05% PS80 HX2 NOF, at the same times and under the same condition.

Table 10. Percentage of monomers for samples that were incubated at 45°C for 7 days or 14 days with additional shaking (200 rpm) at 2-8°C

Sample ID % monomers Sample ID % monomers 100 mg/ml ALXN1210+Avantor, T=0 98.2 100 mg/ml ALXN1210+NOF, T=0 98.2 100 mg/ml ALXN1210+Avantor, T=7 days at 45°C 93.0 100mg/ml ALXN1210+NOF, T=7 days at 45°C 93.0 100mg/ml ALXN1210, Avantor, T=7 days at 45°C+5 days with shaking at 2-8°C 92.8 100mg/ml ALXN1210, NOF, T=7 days at 45°C+5 days with shaking at 2-8°C 92.9 100 mg/ml ALXN1210+Avantor, T=4 days at 45°C 88.1 100mg/ml ALXN1210+NOF, T=14 days at 45°C 88.0 100mg/ml ALXN1210, Avantor, T=14 days at 45°C+5 days with shaking at 2-8°C 85.3 100 mg/ml ALXN1210, NOF, T=14 days at 45°C+5 days with shaking at 5°C 87.7

The shift towards acidic molecules was detected by isoelectric focusing after incubation of ALXN1210 with 0.05% polysorbate 80 Avantor J.T. Baker or with 0.05% polysorbate 80 HX2 NOF at 45°C for 7 days and 14 days as shown in Table 11. Additional shaking of the samples did not significantly affect the additional shift of the main peak towards acidic molecules.

Table 11. Isoelectric focusing by CE-SDS

Sample ID Home pi Area of the main peak in % Acid peak area in % Main peak area in % Avantor-T0 6.2 64.2 23.4 12.4 NOF-T0 6.2 65.0 21.4 13.6 Avantor - 7 days. 6.2 38.9 52.2 8.9 NOF - 7 days+5 days 6.2 41.7 50.4 8.0 Avantor - 7 days+5 days/shaking 6.2 41.6 49.2 9.2 NOF - 7 days + 5 days / shaking 6.2 42.3 48.7 9.1 T=14 days_Avantor_45C 6.2 21.4 74.8 3.8 T=14 days_NOF_45C 6.2 18.1 79.0 2.9 T=14 days_Avantor_45C+5 days/ shaking 6.2 19.3 75.8 4.9 T=14 days_NOF_45C+5 days/ shaking 6.2 19.3 77.8 2.9

Shaking load testing was performed with respect to original optimum formulation ALXN1210 at 100 mg/mL in formulation buffer containing 50 mM sodium phosphate, 25 mM arginine, and 5% sucrose at pH 7.4 in the presence of two grades of PS 80 formulated at 0.05% , and in their absence. The degree of formation of particles invisible to the naked eye was used to assess the destruction. The samples were shaken at 200 rpm at a temperature of 2-8°C. The time points for making measurements as to whether formation of particles invisible to the naked eye occurred were days 0, 1, 3, and 5. The results shown in Table 12 indicate that adding 0.05% PS80 to the formulation significantly reduced the degree of formation of particles invisible to the naked eye, if the highly concentrated composition was subjected to short-term stress, such as shaking at 200 rpm.

Table 12. Reducing the formation of particles invisible to the naked eye through the addition of PS80 and filtration

Sample ID Run number Total particle concentration (number/mL) Total number of particles (number) The number of particles with a size of 1-10.5 microns The number of particles with a size of 11.5-25.5 microns Number of particles >25.5 µm Analyzed volume Unfiltered, without PS80 1 869694 454725 419989 33722 1014 0.5229 ml After filtering, no PS80 1 221485 115805 96661 18111 1033 0.5229 ml Unfiltered - PS80 NOF 1 605402 316239 312238 3414 587 0.5224 ml After filtering, PS80 NOF 1 199 104 101 2 1 0.5321 ml After filtering, PS80 4500 1 185 97 95 1 1 0.5231 ml

7. Conclusion

In conclusion, the optimal composition of ALXN1210 in terms of the number of particles invisible to the naked eye at a concentration of 100 mg/ml contains buffer containing 50 mM sodium phosphate, 25 mM arginine and 5% sucrose, and 0.05% PS80 at pH 7.4.

Example 2 Phase 1 Study to Evaluate Single Dose of ALXN1210 SC versus IV in Healthy Subjects

A phase 1 study was performed to evaluate the safety, tolerability, efficacy, pharmacokinetic (PK)/pharmacodynamic (PD) characteristics, and immunogenicity of the BNJ441 antibody (also known as ALXN1210) administered subcutaneously (SC) versus intravenously (IV) in a healthy subject .

1. Goals

The primary objectives of this study were (1) to assess the safety and tolerability of a single dose of ALXN1210 administered subcutaneously versus ALXN1210 administered intravenously in healthy subjects as assessed by physical examination, vital signs measurements, immunogenicity data, laboratory data analysis and assessment of adverse events (AE), and (2) determination of the absolute bioavailability of ALXN1210 administered subcutaneously.

The secondary objective was to evaluate the PD effects of ALXN1210 administered subcutaneously versus ALXN1210 administered intravenously as measured by free C5 and hemolysis of chick erythrocytes (cRBC).

2. Research plan

The overall study design was performed as shown in the image in figure 47. This was a phase 1 study designed to evaluate the safety, tolerability, PK, PD and immunogenicity of a single dose of 400 mg of ALXN1210 administered subcutaneously, compared with a single dose of 400 mg of ALXN1210 administered intravenous or subcutaneous placebo in 42 healthy subjects. All subjects were screened for eligibility for inclusion in the study. Subjects who did not meet study inclusion criteria were not rescreened for study participation unless the condition leading to study non-eligibility was transient, self-limiting and easily treatable, and is expected to be eliminated by the time of dosing.

Six subjects were initially randomly assigned in a 2:1 ratio to cohort 1a in a blinded manner to receive a single dose of 400 mg of ALXN1210 administered subcutaneously or a single dose of placebo administered subcutaneously. Clinical safety data obtained within the first 48 hours post-dose were assessed for subjects in cohort 1a prior to study enrollment in cohorts 1b or 2. Thirty-six subjects were then randomly assigned in a 2:1 ratio to cohort 1b (N= 24) or to cohort 2 (N=12). In cohort 1b, 24 subjects were additionally randomized in a 5:1 ratio in a blinded manner to receive a single dose of 400 mg ALXN1210 administered subcutaneously (20 subjects) or a single dose of placebo administered subcutaneously (4 subjects), respectively. The 12 subjects in cohort 2 received a single dose of 400 mg of ALXN1210 administered intravenously in an unmasked regimen.

All subjects included in the study were included in the analyzes as appropriate. Subjects in cohorts 1a and 1b were pooled for analyses. Subjects participated in the study for up to 39 weeks, including a screening period of up to 70 days, followed by a 200-day follow-up period to evaluate safety, PK, PD, and immunogenicity after administration of study drug.

Forty-two subjects were evaluated for primary and secondary goals in this study: 6 (4 received ALXN1210 SC, 2 received placebo SC) subjects in cohort 1a, 24 (20 received ALXN1210 SC, 4 received placebo SC) subjects in cohort 1b, and 12 ( ALXN1210 IV) subjects in cohort 2.

3. Rationale for dose selection

A single dose of 400 mg, equivalent to 4 ml, was administered subcutaneously by 4 injections of 1 ml into the abdomen. Administration of a single dose of 400 mg ALXN1210 SC was expected to have an acceptable safety profile. It was expected that single doses of 400 mg of ALXN1210 SC and placebo SC administered as described in this protocol would provide data from which multiple dose simulations could be performed to plan dosing regimens needed to achieve therapeutic serum concentrations (> 50 mcg/ml) in patients.

Parallel randomization of 36 subjects in cohort 1b and cohort 2 was performed based on an assessment of clinical safety data obtained during the first 48 hours after dosing from 6 subjects in cohort 1a. Study enrollment in cohort 1b and cohort 2 was performed as described in Table 13.

The rules for determining toxicity in groups were as follows. Toxicity refers to a clinically significant (significant) adverse reaction (s) associated with a drug. “Continuation of cohort participation in the study” refers to continued participation in a sequential dosing/dosing regimen study following the study's continued dosing guidelines and minimum data requirements. “Clinical study suspension” means that no additional IMP was administered at the appropriate dose level/dosing regimen, and that further cohort participation in the study was suspended.

Table 13. Rules for determining toxicity

CTCAE degree Severity/
seriousness Number of subjects exposed Action Impact on continued cohort participation in the study I Light n. d. Continuation of the dosing regimen. The study continued according to the protocol of the clinical study. II Moderate ≤ 2 subjects with different SOCs 2 entities with the same SOC or 3 entities with different SOCs Continuation of the dosing regimen. The study was started for cohorts 1b and 2 if it had not already been started. ≥3 subjects with same SOC or ≥4 subjects with different SOC All dosing regimens were suspended unless toxicity was due to local tolerance or an injection/infusion site reaction, in which case dosing regimens were suspended for the exposed cohort only. A significant adjustment was required to continue the study (if it was stopped for both cohorts). A significant adjustment was required to continue the study for the exposed cohort (for reactions due to local tolerability or reactions at the injection/infusion site). III heavy 1 subject Continuation of the dosing regimen. The study was started for cohorts 1b and 2 if it had not already been started. ≥2 subjects All dosing regimens were suspended unless toxicity was due to local tolerance or an injection/infusion site reaction, in which case dosing regimens were suspended for the exposed cohort only. A significant adjustment was required to continue the study (if it was stopped for both cohorts). A significant adjustment was required to continue the study for the exposed cohort (for reactions due to local tolerability or reactions at the injection/infusion site). IV life threatening ≥1 subject Study stop. A significant amendment was required to continue the study. V deadly SAE serious ≥1 subject Study stop. A significant amendment was required to continue the study.

Abbreviations: CTCAE=common terminological criteria for adverse events; SAE=serious adverse event; SOC=system organ class.

4. Schedule of assessments

The time frames of the applied research procedures are presented in tables 14-15.

Table 14. Schedule of evaluations: from screening to visit 1

Visit 1 Research Day Screening Day 1 Day 1 Day 2 Day 3 Day 5 Ratings ¹ Day -70 - day -2 Day -1, entry into the study Before Dosing 0 h (SOI) EOI ² 15 min after EOI 30 min after EOI 2 hours after SOI 4 hours after SOI 8 hours after SOI 24 hours 48 h 96 h Status (OP or CRU) OP Entry into the study CRU CRU CRU CRU CRU CRU CRU CRU CRU CRU CRU ³ Informed Consent ⁴ X Immunization MCV4 (Day -56) ⁵ X Immunization with serogroup B meningococcal vaccine (day -56 and day -28) ⁵ X Serum bactericidal antibody (for meningococcal serogroups A, C, W135 and Y) X History and demographics X Physical examination X X X Height, weight and BMI X Quanti-FERON®-TB Testing X Biochemical indicators X X X X Hematological indicators X X X X Coagulation indicators X X X X Screening for hepatitis B and C X Screening for HIV, types I and II X Complement system activity ⁶ X CH50 ⁷ X Serum testing for pregnancy ⁸ X X Breath alcohol testing X X General analysis of urine and biochemical analysis of urine X X X X Urine screening for drugs X X Vital signs measurements X X ^x9 X ^{x9 x9 x9 x9} X X X ECG X X ¹⁰ X X X X Teleelectrocardiography ¹¹ X X (X) (X) (X) (X) Randomization X Investigational drug administration X Samples for PK X X X X X X X X X PD panel (serum C5, cRBC hemolysis) X X X X X X X X X X Infusion site score ¹² X X X X X X X Immunogenicity (ADA) X Considering Potential Safety Risks ALXN1210 ¹³ X X X Concomitant medicinal products ←Continuous monitoring (after ICF signing at screening)→ Adverse events ¹⁴ ←Continuous monitoring (after ICF signing at screening)→ Prophylactic antibiotic treatment ¹⁵ ←Prevention with antibiotics→

¹ Tolerances for study estimates are described in the Study Procedures Manual.

² End of infusion (EOI) occurred approximately 15 minutes after start of infusion (SOI).

³ Subjects were withdrawn from the clinical study unit upon completion of all evaluations on day 5. Subjects were given a "study patient record" with information to healthcare professionals and the study participant about the symptoms of infectious meningitis.

⁴ Signed and dated informed consent forms were obtained prior to any study-related screening procedures.

⁵ For subjects without appropriate documentation of prior MCV4 or serogroup B vaccination, MCV4 immunization was administered at least 56 days prior to the first dose on Day 1 and serogroup B meningococcal vaccine was administered at least at least 56 days before the dose on day 1 with a booster injection at least 28 days before the dose on day 1.

⁶ Determination of the activity of the complement system, confirmed by a suitable assay, such as an ELISA for the alternative pathway of activation of the complement system (CAP)/C5 inhibition (hemolysis) assay, was performed at screening to confirm that subjects do not have complement deficiency.

⁷ The sample taken on day -1 was stored for future analysis, which will need to be done in the event that the sample taken after dosing shows that the functioning of the complement system is not normal.

⁸ Serum pregnancy testing was performed on all female subjects to confirm that the female subjects were not pregnant.

⁹ Pre-dose vital signs measurements (within 15 minutes before SOI) and at the end of the infusion, 30 minutes after the end of the infusion, 2 hours after the start of the infusion, 4 hours after the start of the infusion, and 8 hours after the infusion were assessed on Day 1 start of infusion.

¹⁰ On day 1, a 12-lead ECG was performed in triplicate before dosing and approximately 15 minutes after completion of the infusion.

¹¹ Conducted continuous recording of cardiac activity from the moment before dosing during the IV infusion period (cohort 2) and up to 3 hours after SC injection (cohort 1a and 1b).

¹² Assessment at the site of infusion or injection was performed within 15 minutes of the start of the infusion/injection and ±15 minutes of other scheduled times on Day 1. Nodules or reactions <1 cm in size were not reported as an adverse event unless they persisted for more than 24 hours Pain at the infusion or injection site was assessed using a visual analogue scale (0-10). Pain was not assessed prior to dosing.

¹³ The investigator or designee met with the subject at each visit to discuss potential safety risks with ALXN1210 and to respond to any safety concerns from the subject.

¹⁴ Data collection for adverse events (AEs) and serious adverse events (SAEs) began with the signing of the informed consent form.

¹⁵ For prophylactic antibiotic treatment, subjects were administered penicillin V 500 mg orally twice daily (equivalent to 1 x 10 ⁶ units) starting on the evening of day -1, until the activity of the complement system returned to normal, as determined by CH50 analysis.

Abbreviations: ADA=antibody to drug; BMI=body mass index; cRBC=chicken erythrocyte; CRU=Clinical Research Unit; ECG=electrocardiogram; EOI=completion of infusion/injection; HIV=human immunodeficiency virus; ICF=informed consent form; MCV4 = tetravalent meningococcal conjugate vaccine; OP=outpatient; SOI=start of infusion/injection; TB=tuberculosis.

Table 15. Schedule of assessments: visit 2 to visit 14

Visit 2 Visit 3 Visit 4 Visit 5 Visit 6 Visit 7 Visit 8 Visit 9 Visit 10 Visit 11 Visit 12 Visit 13 Visit 14 Procedures Day 8 Day 15 Day 22 Day 29 Day 36 Day 43 Day 50 Day 57 Day 71 Day 90 Day 120 Day 150 Day 200 Status (OP or CRU) OP OP OP OP OP OP OP OP OP OP OP OP OP Physical examination X X X X X X Vital signs measurements X X X X X X X X X X X X X ECG X X Biochemical indicators X X X X X X X X X Hematological indicators X X X X X X X X X Coagulation indicators X X X X X X X X X General analysis of urine and biochemical analysis of urine X X X X X X Serum testing for pregnancy X X CH50 testing X ^x1 Samples for determination of pharmacokinetic characteristics X X X X X X X X X X X X X Pharmacodynamic performance panel (serum C5, cRBC hemolysis) X X X X X X X X X X X X X Immunogenicity (ADA) X X X X X X X Considering Potential Safety Risks ALXN1210 ² ←--- Discussion of potential safety risks of ALXN1210 --→ Concomitant medicinal products ←---Continuous monitoring (after ICF signing at screening)-- → Adverse events ³ ←---Continuous monitoring (after ICF signing at screening)-- → Prophylactic treatment ⁴ antibiotics ⁴ ←---Prevention with antibiotics--→

¹ Additional samples were taken after day 57.

² The investigator or designee met with the subject at each visit to discuss potential safety risks with ALXN1210 and to respond to any safety concerns from the subject.

³ Data collection on adverse events began with the signing of the informed consent form.

⁴ For prophylactic antibiotic treatment, subjects were administered oral penicillin V 500 mg twice daily (equivalent to 1 x 10 ⁶ units) until complement system activity returned to normal, as determined by CH50 analysis.

Abbreviations: ADA=antibody to drug; cRBC=chicken erythrocyte; CRU=Clinical Research Unit; ECG=electrocardiogram; ICF=informed consent form; OP=outpatient.

5. Selection and Termination of Subjects

To qualify for study inclusion, subjects must meet all of the following criteria.

1. Healthy subjects aged 25 to 55 years inclusive at the time of dosing.

2. A body mass index (BMI) of 18 to 29.9 kg/m ² inclusive and a weight of 50 to 100 kg inclusive.

3. Friedrichia-corrected QT interval (QTcF) of ≤450 ms for male subjects and ≤470 ms for female subjects at screening and before dosing on day 1.

4. Consent and ability to provide written informed consent and adhere to study visit schedules.

5. Documented vaccination with MCV4 at least 56 days and no more than 3 years prior to dosing. Documentation should include a positive antibody titer to confirm an immune response prior to administration of the investigational medicinal product.

6. Vaccination with serogroup B meningococcal vaccine at least 56 days prior to Day 1 dose with a booster at least 28 days prior to Day 1 dose at least 28 days apart , between the first and second injections.

7. Female subjects of childbearing potential, if they are heterosexually active, should use highly effective or acceptable contraceptives, as defined below, starting at screening and continuing for at least 6 months after study drug administration. This study required antibiotic prophylaxis, which can adversely affect the effectiveness of hormonal contraceptives. Therefore, it is recommended that subjects using hormonal contraceptives also use barrier contraceptives (eg, a condom or diaphragm with spermicide) during the entire period of antibiotic prophylaxis. Male subjects, if they are heterosexually active and have a fertile female spouse or partner, or a spouse or partner who is pregnant or breastfeeding, must agree to use barrier contraceptives (male condom) during the period treatment and for at least 6 months after study drug administration. Barrier contraceptives were required even in the case of a documented medical report of a successful surgical vasectomy. Male subjects' spouses or partners of childbearing potential must use highly effective contraceptives as defined above or acceptable contraceptives as defined below, beginning at screening and continuing for at least 6 months after study drug administration. Male subjects should not be sperm donors during the screening and treatment periods and for at least 6 months after study drug administration.

Subjects meeting any of the following exclusion criteria were not eligible to participate in the study.

1. Subjects who have been in close and prolonged contact (defined as living under the same roof or providing personal care) with individuals younger than 2 years of age or older than 65 years of age, or who have been immunocompromised or one of the following primary medical conditions: anatomical or functional asplenia (including sickle cell anemia); congenital deficiencies of the complement system, properdin, factor D, or primary antibodies; acquired complement system deficiencies (eg, patients receiving eculizumab) or human immunodeficiency virus (HIV).

2. Subjects who belonged to one of the following groups: professionals who were exposed to environmental conditions with an increased risk of meningococcal disease; scientific, industrial and clinical-laboratory personnel who are regularly exposed to N. meningitidis; military personnel during recruit training (military personnel may be at increased risk of meningococcal disease when housed in cramped conditions); employees of preschool institutions; subjects residing in a college or university dormitory; and subjects who plan to travel during the study or have traveled to areas endemic for meningococcal meningitis (eg, India, sub-Saharan Africa, pilgrimage to Saudi Arabia for the Hajj) within 6 months prior to dosing.

3. Any history of Neisseria infection.

4. Recurrent infection of unknown etiology or infection requiring treatment with systemic antibiotics within 90 days prior to dose history.

5. HIV infection (as evidenced by the titer of antibodies to HIV-1 or HIV-2).

6. Acute or chronic hepatitis B virus (HBV) infection. All subjects were required to be tested for hepatitis B surface antigen (HBsAg) before enrollment in the study. Subjects who tested positive for HBsAg were not included in the study. For HBsAg-negative subjects, the following testing algorithm was required. If the test result for antibodies to hepatitis B core antigen (HBcAb) was negative, then the subject was eligible for inclusion in the study. If the result of the HBcAb test was positive, then tested for antibodies to the surface antigen of hepatitis B virus (HBsAb). If both HBcAb and HBsAb were positive, then the subject was eligible for inclusion in the study. If the HBcAb test result was positive and the HBsAb test result was negative, then the subject was not included in the study.

7. Acute or chronic hepatitis C virus infection (indicated by antibody titer).

8. Active systemic viral or fungal infection within 14 days prior to dosing.

9. Positive or indeterminate QuantiFERON®-TB test result, indicating the possible presence of tuberculosis (TB) infection.

10. History of latent or active TB or exposure to endemic areas within 8 weeks prior to screening visit.

11. Female subjects who are breastfeeding or heterosexual and do not agree to use contraceptives and are not postmenopausal. Postmenopausal was defined as the absence of menses for ≥12 consecutive months with no other cause and a documented serum follicle-stimulating hormone level of ≥40 mIU/mL and an estradiol concentration of ≤110 pmol/L within 6 months prior to study drug administration. medicinal product.

12. Positive serum pregnancy test result at screening or day -1.

13. Serum creatinine above upper limit of normal (ULN) within reference range in lab tests at Screening or Day -1.

14. Alanine aminotransferase (ALT) or aspartate aminotransferase (AST) level > ULN in laboratory test reference range at screening or > 1.5 × ULN in laboratory test reference range on day -1.

15. Any of the following hematological findings: hemoglobin level <130 g/L for male subjects and <115 g/L for female subjects, hematocrit <0.37 L/L for male subjects and <0.33 L /l for female subjects, white blood cell count (WBC) <3.0×10 ³ /μl, absolute neutrophil count <2.0×10 ³ /μl and platelet count <150 or >400×10 ³ /μl at screening or day -1. Clinical laboratory complete blood count (CBC) results that the Investigator considers clinically important and unacceptable on Day -1.

16. History of complement deficiency or complement system activity below normal reference range as assessed by CAP ELISA at screening.

17. Malignant neoplasms excluding non-melanoma skin cancer or carcinoma of the cervix in situ, which were treated without signs of recurrence, in the anamnesis.

18. Participation in a clinical study within 30 days prior to the start of the Day 1 dose or use of any investigational small molecule therapeutic agent within 30 days prior to the Day 1 dose.

19. Participation in more than one mAb clinical study or participation in a mAb clinical study within 12 months prior to screening during which the subject was exposed to active investigational medicinal product. Subjects who participated in only one mAb study could be considered eligible for inclusion in this study if they completed their participation in the previous study more than 12 months prior to screening.

20. Prior exposure to ALXN1210.

21. Major surgery or hospitalization within 90 days prior to dosing.

22. Allergy to excipients for ALXN1210 (for example, polysorbate 80) in history.

23. Documented history of allergy to penicillin or cephalosporin.

24. Significant allergic reaction (for example, anaphylaxis or angioedema) to any product (food, pharmaceutical, etc.) in history.

25. Current smokers >10 cigarettes per day (ex-smokers may be allowed to be included in the study at the discretion of the investigator).

26. History of illicit drug abuse, severe alcohol abuse in the 1 year prior to the Screening Visit, or regular alcohol intake in the 6 months prior to the Screening Visit (more than 14 drinks of alcohol per week [1 unit = 150 ml of wine, 360 ml of beer, or 45 ml of 40% alcohol]) in history.

27. Positive urine toxicological screening for drugs at screening or day -1.

28. Drinking alcohol within 48 hours prior to study drug administration or a positive breath alcohol test result on day -1.

29. Plasma donation within 7 days prior to dosing. Donation or loss (excluding screening volume) of more than 50 ml of blood within 30 days prior to dosing or more than 499 ml of blood within 56 days prior to dosing.

30. History of continuous topical, inhaled, or systemic steroid use for >28 days, or history of inhaled or topical use of any immunosuppressive therapeutic agent within 90 days prior to study drug administration.

31. Use of prescription drugs (excluding oral contraceptives) within 14 days prior to study drug administration, unless prior approval by the clinical trial sponsor.

32. Regular use of over-the-counter, over-the-counter medicinal products, including herbal preparations and dietary supplements, within 14 days prior to study drug administration. Multivitamins, acetaminophen ≤2 g daily, and topical skin preparations may be used without significant systemic absorption.

33. Any clinically diagnosed autoimmune or rheumatic disease (for example, systemic lupus erythematosus, rheumatoid arthritis).

34. Immunization with a live attenuated vaccine 28 days prior to dosing or routine vaccination during the study (except for vaccination scheduled according to the study protocol). Immunization with inactivated or recombinant influenza vaccine was allowed.

35. Presence of fever (confirmed body temperature >37.6°C) (eg, fever associated with symptomatic viral or bacterial infection) within 14 days prior to dosing.

36. Subjects with any history, condition, or risk that, in the opinion of the Investigator, might preclude the subject's full participation in the study or adherence to the protocol, or might expose the subject to any additional risks or misrepresent the subject's assessment or study outcome.

6. Infection

To reduce the risk of infection associated with inhibition of terminal components of the complement system, subjects in this study were administered the following.

1. Vaccination with MCV4 at least 56 days prior to ALXN1210 dose on Day 1 (if subjects have not been vaccinated with MCV4 within the last 3 years or if subjects have been previously vaccinated but lacked appropriate documentation to support prior vaccination).

2. Two injections of serogroup B meningococcal vaccine. at least 28 days between the first and second injections.

3. For prophylactic antibiotic treatment, penicillin V 500 mg twice daily (equivalent to 1 x 10 ⁶ units) was administered orally until the activity of the complement system returned to normal (determined by CH50 analysis).

The first antibiotic dose was administered orally on day -1 in the evening prior to day 1 of study drug dosing. For the outpatient portion of the study, subjects were instructed to take the antibiotic at approximately the same time (twice daily) on each scheduled day. An appropriate system (such as text messaging) was used to monitor subjects' adherence to an antibiotic prophylaxis regimen on a daily basis.

The following observations support the rationale for administering prophylactic antibiotics in this single dose study.

1. Penicillin was the drug of choice for the eradication of N. meningitidis in carriers.

2. Complement-deficient patients who received once-monthly injections of benzathinepenicillin G as a means of preventing recurrent meningococcal disease over a 2- to 4-year period experienced significantly fewer episodes of Neisseria infections than complement-deficient individuals not receiving prophylaxis. (Figueroa JE, et al ., Clin. Microbiol. Rev. 1991 Jul;4(3):359-95).

3. High levels of penicillin resistance due to plasmid-encoded β-lactamases are rare in meningococcal strains (Yazdankhah SP, et al., J. Med. Microbiol . 2004 Sep;53(Pt 9):821-32).

4. Some physicians have suggested antibiotic prophylaxis with oral penicillin V 500 mg twice daily in the treatment of patients with PNH and aHUS with eculizumab (Kelly RJ, et al., Blood 2011 Jun 23;117(25):6786 -92 and Leeds Teaching Hospitals NHS Trust, Kings College Hospital NHS Foundation Trust, National Specialized Commissioning Team (NSCT) Service Specification Paroxysmal Nocturnal Haemoglobinuria (PNH) 2013).

5. Uncertainty about vaccine efficacy in immunocompromised patients has led several countries, such as France, to recommend continuous antibiotic prophylaxis throughout eculizumab treatment in patients with PNH and aHUS (Zuber J, Fakhouri F, Roumenina LT, Loirat C, Fremeaux -Bacchi V. Use of eculizumab for atypical haemolytic uraemic syndrome and C3 glomerulopathies Nat.Rev.Nephrol.2012 Nov;8(11):643-57).

7. Previously prescribed and concomitant drugs and procedures

Previously prescribed medications (any drug or substance taken by the subject within 14 days prior to the time the subject signed the ICF prior to study drug administration) and concomitant medications (any drug or substance taken by the subject after study drug administration until completion of the last visit during the course of the study) were recorded in the electronic individual subject registration card (eCRF). Previously prescribed procedures (any therapeutic intervention [eg, surgery/biopsy, physiotherapy] performed within 14 days prior to the time the subject signed the informed consent prior to study drug administration) and concomitant procedures (any therapeutic intervention [eg, surgery/biopsy, physical therapy] after administration of study drug until the end of the last study visit) were recorded in the subject's eCRF.

A concomitant therapeutic agent was any drug or substance administered from the time the subject was screened for the study until the end of the last visit during the study. Subjects were instructed not to initiate any new medications, including over-the-counter drugs and herbal preparations, unless authorized by the investigator, during the entire study period. During the study, occasional use of over-the-counter fever or pain relievers (eg, acetaminophen).

An accompanying procedure was any therapeutic intervention (e.g., surgery/biopsy, physical therapy) or diagnostic evaluation outside the study (eg, blood gas measurement, microflora analysis) performed from the moment the subject signed the informed consent until the last visit during the study. Concomitant procedures were not allowed unless they were prescribed for medical reasons.

8. Randomization and masking

Eligible subjects who met the inclusion and exclusion criteria were assigned individual numbers for inclusion in the study and randomization.

This study was the following partially blind study.

Cohort 1a. Dosing (single dose of 400 mg ALXN1210 or placebo SC) was done in double blind mode. Subjects in cohort 1a were randomized in a 2:1 ratio (4 ALXN1210 SC, 2 placebo SC; N=6).

Cohort 1b. Dosing (single dose of 400 mg ALXN1210 or placebo SC) was done in double blind mode. Subjects in cohort 1b were randomized in a 5:1 ratio (20 ALXN1210 SC, 4 placebo SC; N=24).

Cohort 2 . Dosing (single dose of 400 mg ALXN1210 IV) occurred in non-masked mode (N=12).

At the time of cohort 2 dosing, the drug/dose to be administered was known to both the subjects and the on-site medical/nursing staff.

At the time of dosing cohorts 1a and 1b, both subjects and on-site medical/nursing personnel at the study site were masked with study drug prescribing data. Pharmaceutical staff who prepared SC injections and staff administering study drug were not masked, while all other study site staff involved in the safety assessment were masked for study drug administration. Clinical study sponsor staff were subjected to data demasking as necessary (eg, to control preparation of SC injections appropriately, to determine whether SAE data could be reported), and were prevented from providing any study drug prescribing information to study site staff.

9. Description of the investigational medicinal product

Study drug is described in Table 16.

Table 16 Study drug

Investigational drug The product's name ALXN1210IV ALXN1210SC Placebo SC Dosage form Sterile solution for infusion Sterile solution for injection Sterile solution for injection single dose 150 mg/vial ¹⁾ 100 mg/vial ²⁾ n.a. Route of administration Intravenous subcutaneous injection subcutaneous injection Description of physical properties Sterile, preservative-free solution Sterile, preservative-free solution Sterile, preservative-free 0.9% sodium chloride solution for injection according to the European or British Pharmacopoeia Manufacturer Alexion Pharmaceuticals Inc. Alexion Pharmaceuticals Inc. Salt solution introduced to the UK market

¹ Each ALXN1210 IV drug vial was slightly overfilled to allow 15 ml (150 mg ALXN1210) to be withdrawn for administration.

² Each ALXN1210 SC drug vial had a slight overflow to allow 1 ml (100 mg ALXN1210) to be withdrawn for administration.

Abbreviations: BP=British Pharmacopoeia; IV=intravenous; n.a.=no data; Ph Eur=European Pharmacopoeia; SC=subcutaneous.

10. ALXN1210 and placebo

Each vial of ALXN1210 SC contained 100 mg ALXN1210 (100 mg/mL) in 50 mM sodium phosphate, 25 mM arginine, 5% sucrose, and 0.05% polysorbate 80. ALXN1210 SC was formulated at pH 7.4 and provided as a fully formulated , a sterile, preservative-free aqueous solution of ALXN1210 at a concentration of 100 mg/ml, supplied in 2 ml disposable vials. Each ALXN1210 SC vial had a slight overflow to allow 1 ml (100 mg ALXN1210) to be withdrawn for administration.

Each placebo SC dose contained 0.9% sodium chloride for injection according to the European or British Pharmacopoeia in the same volume as indicated for cohorts 1a and 1b.

Each vial of ALXN1210 IV contains 150 mg of ALXN1210 in 10 mM sodium phosphate, 150 mM sodium chloride, 0.02% polysorbate 80 and water for injection. ALXN1210 IV was formulated at pH 7.0 and provided as a fully formulated, sterile, preservative-free aqueous solution of ALXN1210 at 10 mg/mL supplied in 20 mL disposable vials. ALXN1210 IV was diluted in 0.9% sodium chloride for injection according to the European Pharmacopoeia or British Pharmacopoeia and was administered by IV infusion at a maximum rate of 333 ml/h, excluding safety or technical interruptions.

Vials with ALXN1210 were stored refrigerated at 2°C-8°C (36°F-46°F) and protected from light. Vials with ALXN1210 were not frozen or shaken.

ALXN1210 SC and placebo SC were blinded in an SC injection syringe. There was no dilution of ALXN1210 SC or placebo SC. ALXN1210 SC and placebo SC were placed directly into the syringe.

ALXN1210 IV was designed to be infused by dilution in commercially available saline (0.9% sodium chloride for injection according to the European or British Pharmacopoeia) for IV infusion at a maximum rate of 333 ml/h, excluding interruptions for safety or technical reasons.

ALXN1210 IV was diluted with 0.9% sodium chloride for injection according to the European or British Pharmacopoeia before administration (dosing solution). The shelf life of the dosing solution after opening the package was 4 hours at room temperature from 15°C to 25°C (from 59°F to 77°F). The expiry date and time of the dosing solution was calculated from the moment the first vial was opened. The dose was administered before the expiration date and time. The drawn contents of each 1 ml ALXN1210 SC or placebo SC syringe (4 syringes per subject) were injected within 1 hour after the contents of the vial were drawn into the syringe.

11. Introduction

All doses of ALXN1210 SC or placebo SC were administered by four injections of 100 mg SC in a volume of 1 ml each (Table 17) in the abdomen. All four 1 ml injections were administered over a 15 minute period, and at least 15 minutes must elapse between the completion of an injection in one subject and the start of an injection in the next subject.

Table 17. Reference dosing regimen for ALXN1210 SC and placebo SC preparations

Cohort Investigational drug and dose Number of prepared 1 ml syringes Total injected volume 1a 1 dose 400 mg ALXN1210 SC or placebo SC four 4 ml 1b 1 dose 400 mg ALXN1210 SC or placebo SC four 4 ml

All doses of ALXN1210 IV were administered by IV infusion using IV kits with built-in filters at a maximum rate of 333 ml/h, excluding safety or technical interruptions. At least 15 minutes must elapse between the completion of an injection in one subject and the start of an injection in the next subject.

Table 18. Reference Dosing Schedule for ALXN1210 IV

Cohort Investigational drug and dose Volume of ALXN1210 per dose (ml) Volume of diluent per dose (ml) Volume of infusion (ml) Maximum infusion rate (ml/h) Minimum duration of infusion, ¹⁾ minutes (hours) 2 1 dose 400 mg ALXN1210 IV 40 40 80 333 15 (0.25)

¹ Duration of infusion was approximate.

12. Control of potential adverse events during study drug administration

Some subjects who have been treated with IV infusions of monoclonal antibodies have experienced concomitant infusion reactions with signs or symptoms that can be classified as acute allergic/hypersensitivity reactions or cytokine release syndrome.

Subjects were closely monitored during and after study drug administration for any symptoms of anaphylaxis and other hypersensitivity reactions, including changes and circulatory and/or respiratory arrest or urticaria, arthralgia, myalgia, or other signs of associated reactions. Appropriate treatments were available for immediate use. Adverse events associated with the infusion may occur, and depending on their type and severity, it may be necessary to stop the infusion. Subjects were informed of the early symptoms and signs of hypersensitivity reactions, including hives, swelling of the face, eyelids, lips, or tongue, or breathing problems. To control infusion reactions, an algorithm of actions was used in case of an acute reaction to infusion. In this study, regular evaluations were performed to monitor infusion reactions and reactions at the infusion site. To ensure prompt resolution of these reactions, at least 15 minutes were available between the completion of the infusion/injection in one subject and the start of the infusion/injection in the next subject. Doses were administered to no more than 6 subjects per day. Any reactions were subjected to treatment and taken into account in the rules of continuation of administration / increase in dose and determination of toxicity. In the event of an anaphylactic reaction, the current UK Treatment Guideline for Anaphylactic Reactions of the UK Resuscitation Council was followed.

13. Evaluations of pharmacokinetic (PK) and pharmacodynamic (PD) characteristics

After the drug administration study, serum samples for determination of serum ALXN1210 concentrations and for analyzes of total and free C5 concentrations, degree of cRBC hemolysis, and other possible indicators of C5 activation were collected at the following time points, recording the actual dates and time points of serum sampling and using them in PK and PD calculations.

Serum ALXN1210 concentrations were analyzed at the following sampling times: pre-dose (within 15 minutes prior to infusion/injection [SOI]); on day 1 at the end of infusion/infection (EOI), 30 minutes after EOI and at the following time points after SOI: 2 h, 4 h and 8 h; day 2 (24 h); day 3 (48 hours); day 5 (96 h); day 8 (168 h); day 15 (336 h); day 22 (504 h); day 29 (672 h); day 36 (840 h); day 43 (1008 h; day 50 (1176 h); day 57 (1344 h); day 71 (1680 h); day 90 (2136 h); day 120 (2856 h); day 150 (3576 h); and day 200 (4776 hours).

All subjects who provided sufficient serum samples for PK analysis to characterize the concentration-time profile were included in the PK analysis population. All subjects who provided samples for PD analysis were included in the PD analysis population.

14. Immunogenicity scores

Serum samples were collected at the following time points: before dosing (within 15 minutes before SOI) and on days 15 (336 h), 29 (672 h), 57 (1344 h), 90 (2136 h), 120 (2856 h). h), 150 (3576 h) and 200 (4776 h) and analyzed for the production of ADA against ALXN1210. Additional characterization of the humoral response was performed as appropriate based on PK/PD and ALXN1210 safety data.

All subjects who provided pre-dose and post-dose sample for ADA analysis were included in the immunogenicity assay population.

Anti-drug antibody (ADA) production against ALXN1210 was assessed by immunogenicity assay. Detailed instructions for the procedure for collecting, handling, storing and transporting serum samples for immunogenicity testing are provided in the laboratory manual.

15. Safety assessment

Safety assessments included TB testing, physical examination findings, vital signs measurements, immunogenicity (ADA) testing, laboratory evaluations, ECG, infusion site and injection site evaluations (e.g., bleeding, bruising, redness, swelling, induration and pain) and control of adverse events. Adverse events were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.03 (CTCAE v4.03), published June 14, 2010. Laboratory assessments include haematological, biochemical, and coagulation panels; CBC with leukocyte count; urinalysis and serum pregnancy testing for female subjects.

Clinical and laboratory evaluations were performed to assess the safety of ALXN1210. The timing of evaluations is described in the evaluation schedule. Abnormal results were monitored until they were resolved or stabilized.

Consideration of demographic parameters, including age, gender, race and ethnicity, was carried out as described in the assessment schedule. A complete history was taken.

Vital signs measurements were taken after the subject was in the supine or semi-recumbent position for at least 5 minutes and included body temperature (°C; oral), respiratory rate, blood pressure in the supine position. back and heart rate. The timing of the measurements of vital signs is described in the assessment schedule. Blood pressure or pulse rate measurements that were out of range were repeated at the investigator's discretion. Any confirmed clinically significant measurements of vital signs were recorded as adverse events.

Weight, height and BMI were recorded as described in the evaluation schedule. Each examination included the following assessments: general appearance; leather; head, ears, eyes, nose and throat; neck; lymph nodes; rib cage; heart; abdomen; limbs; central nervous system and musculoskeletal system.

ECG in triplicate in 12 leads was obtained after the subject was at rest for at least 5 minutes. The timing of the ECG is described in the Evaluation Schedule. In addition, continuous recording of cardiac activity was performed at each dose from pre-dose to completion of IV infusion in cohort 2 and from pre-dose to 3 hours after completion of SC injection in cohorts 1a and 1b. Heart rate, PR, QRS, RR and QT were measured and corrected QTcF intervals were calculated.

Blood samples for haematological, clinical chemistry, coagulation, and serological testing for viruses, as well as urine samples for urinalysis, urinalysis, and drug and alcohol screening were collected as described in the assessment schedule.

Blood samples were analyzed for the following hematological parameters: platelet count, red blood cell (RBC) count, and WBC count; leukocyte formula with automated counting (neutrophils, lymphocytes, monocytes, eosinophils, basophils); hemoglobin level; hematocrit and RBC indices (mean erythrocyte volume, mean erythrocyte hemoglobin, and mean erythrocyte hemoglobin concentration). The timing of hematological evaluations is described in the evaluation schedule.

Blood samples were analyzed for the following clinical and biochemical parameters: blood urea nitrogen level; creatinine; glucose; sodium; phosphorus; potassium; chloride; total carbon dioxide; total calcium; magnesium; AST; ALT; gamma-glutamyl transpeptidase; alkaline phosphatase; lactate; total, direct and indirect bilirubin; uric acid; albumin and total protein. Given that indirect bilirubin was calculated from total and direct bilirubin values, results for indirect bilirubin were not available if direct bilirubin was below the limit of quantitation.

FSH levels and serum estradiol concentrations were measured at screening for postmenopausal female subjects to confirm their postmenopausal status.

The timing of biochemical assessments is described in the Assessment Schedule.

Blood samples were analyzed for prothrombin time, international normalization index and partial thromboplastin time. The timing of coagulation assessments is described in the Assessment Schedule.

A urinalysis includes tests for specific gravity, pH, glucose, protein, blood, and ketones. Microscopic examination of urine samples was performed only if abnormalities were detected. Urine samples were also sent to a pathology laboratory for protein and creatinine measurements to calculate the urinary protein:creatinine ratio. The timing of evaluations in the urinalysis and urinalysis is described in the evaluation schedule.

Blood samples collected at screening were analyzed for antibody titers to HIV-1, HIV-2, HBsAg and HCV. All subjects were required to be tested for HBV surface antigen before enrollment. Subjects who tested positive for HBsAg were not included in the study. For HBsAg-negative subjects, the following testing algorithm was required.

1. If the result of the HBcAb test was negative, then the subject met the requirements for inclusion in the study.

2. If the result of the HBcAb test was positive, then testing for antibodies to the hepatitis B surface antigen (HBsAb) was performed.

If both HBcAb and HBsAb were positive, then the subject was eligible for inclusion in the study.

If the HBcAb test result was positive and the HBsAb test result was negative, then the subject was not included in the study.

The drug screening urine sample was analyzed for the following compounds: amphetamines, barbiturates, benzodiazepines, cocaine, methadone, opiates, phencyclidine, methamphetamine, 3,4-methylenedioxymethamphetamine, and tetrahydrocannabinol (cannabinoids). Breath alcohol testing was carried out. If a positive result was observed prior to dosing, dosing was not continued. The timing of urine drug testing and breath alcohol testing is described in the evaluation schedule.

Pregnancy testing (beta human chorionic gonadotropin) was performed on all female subjects. The timing of pregnancy testing is described in the evaluation schedule.

Serum samples for QuantiFERON-TB testing were collected as described in the evaluation schedule.

A suitable assay for determining the activity of the complement system, such as a CAP ELISA/C5 inhibition (hemolysis) assay, was performed at screening to confirm that subjects were not complement deficient. Subjects found to be complement deficient were excluded from participation in the study.

Serum samples were collected at baseline and during follow-up to measure CH50 activity using LIA in vitro to confirm normalization of complement system activity. If a normal CH50 result was obtained for the first CH50 sample from the subject collected during follow-up, then antibiotic prophylaxis could be stopped and a second scheduled CH50 sample was not required. If the first and second CH50 samples were not normal, a baseline sample could be analyzed and additional CH50 samples collected until the activity of the complement system was restored.

The titer of serum bactericidal antibodies (SBA) to meningococci from serogroups A, C, W135 and Y was performed at screening. Titer measurements were used to exclude subjects without an immune response from dosing.

Evaluations were made at the subcutaneous injection site or IV infusion site. Pain at the site of the SC injection or IV infusion was assessed using a visual analog scale (0-10). Pain was not assessed prior to dosing. Seals or reactions ≤1 cm in size were not reported as an adverse event unless they persisted for more than 24 hours.

Serum samples were analyzed for anti-drug antibodies (ADA). The timing of the collection of serum samples for ADA analysis is described in the assessment schedule.

16. Control of adverse events

The investigator was responsible for identifying, evaluating, documenting and reporting all adverse events (AEs). All AEs were enrolled from the moment of signing the informed consent form until the end of the study. There was no time limit for SAEs that were considered to be causal.

All observed or suspected AEs, regardless of causation, were reported and all recorded in the data collection system. Adverse events reported by the subject and/or their parent or legal guardian, and/or identified in response to an open-ended question from the study team, or identified as a result of observation, physical examination, or other study procedures, were collected and recorded.

AE was defined as any adverse and unintended symptom (eg, including abnormal laboratory findings), a symptom or disease commensurate with the use of a medicinal product or procedure, whether or not it is thought to be associated with the use of a medicinal product or procedure that occurs during a clinical trial.

All exacerbations of a chronic or intermittent pre-existing condition, including also an increase in the frequency and/or intensity of the condition, were considered AE.

Abnormal test results were considered AE. When abnormal laboratory values were identified, investigators were strongly encouraged to report the diagnosis or sign or symptom rather than a single abnormal test value. An abnormal test result was documented as AE if it met any of the following conditions: was associated with a sign or symptom; required additional diagnostic testing (retests were not considered additional testing); required medical or surgical intervention; led to a change in the dose of study drug outside the doses specified in the protocol, or led to termination of participation in the study; required significant additional treatment; did not meet any of the conditions listed above.

This definition also includes signs or symptoms due to: drug overdose, drug withdrawal, drug abuse, drug interactions, extravasation, exposure during pregnancy, exposure through breastfeeding, drug misuse, and occupational exposure .

AE does not necessarily include the following.

Medical or surgical procedures ( e.g. surgery, endoscopy, tooth extraction, blood transfusion); the state that leads to such a procedure was AE ( e.g., laparoscopic cholecystectomy was a procedure or treatment for SAE in the form of a necrotic process in the gallbladder)

Pre-existing diseases or conditions present at or prior to the screening evaluation that are not getting worse

Situations where an adverse medical event has not occurred (for example, hospitalization for non-emergency surgery, if scheduled prior to study entry; hospital admission for social reasons and/or convenience purposes)

Any AE that meets any 1 of the criteria listed below must be registered as an SAE.

SAE has been described as any adverse medical event that, at any dose:

1. Leads to death

2. Life threatening ^a

3. Requires hospitalization or prolongation of hospitalization ^b . Hospitalization does not necessarily include the following:

Medical rehabilitation / placement in a palliative care facility / placement in a nursing facility

Visit to the emergency department for a period of less than 24 hours

Non-Emergency or Pre-Scheduled Hospital Admission/Surgery/Surgical Day Hospital Admission

The admission to the hospital indicated in the protocol

Hospital admission for a pre-existing condition associated with neither new AE nor exacerbation of pre-existing AE

4. Leads to permanent or significant disability/incapacity

5. Represents a congenital anomaly/birth defect

6. Is an important medical event ^c

^a The term "life-threatening" in the sense of "serious" refers to an event in which the subject had a risk of death at the time of the event; it does not refer to a phenomenon that could hypothetically cause death if it were more severe.

^b Hospitalization requires admission of the patient to a hospital or extension of an ongoing hospitalization. AEs that were associated with hospitalization or prolongation of hospitalization were considered SAEs.

^c An important medical event. Medical and scientific judgment should be used in deciding whether to report urgently in other situations, such as important medical events that may not be directly life threatening or result in death or hospitalization, but may put the subject at risk or may require intervention to prevent one of the other the outcomes listed in the definition above. They should also usually be considered serious. Examples of such events were intensive care in the emergency department or at home for allergic bronchospasm; immediate-type hypersensitivity or convulsions that do not lead to hospitalization; or developing drug dependence or drug abuse.

It is necessary to differentiate severity and seriousness. Severity describes the intensity of the AE, while the term "severity" refers to an AE that meets the criteria for SAE described above.

All AEs were graded according to the following criteria from CTCAE v4.03 published June 14, 2010.

Grade 1: mild (awareness of the sign or symptom but mild tolerance)

Grade 2: moderate (discomfort sufficient to interfere with normal activities)

Grade 3: severe (limited ability, with inability to perform normal activities)

Grade 4: life threatening

Degree 5: fatal.

Changes in AE severity were documented to allow estimation of AE duration at each intensity level to be assessed. Adverse events characterized as intermittent require documentation of the onset and duration of each episode if the severity of the intermittent episode has changed.

Investigator assessment of causality was provided for all AEs (both non-serious and serious). This assessment was recorded in the data collection system and in any additional forms, as appropriate. The definitions for causal studies were as follows:

Not related (unrelated). This relationship suggests that there is no association between the study drug and the reported event.

Incredibly related. This association suggests that the clinical presentation was highly consistent with a cause other than study drug use, but such an assignment cannot be made with absolute certainty, and an association between study drug and AE cannot be ruled out with certainty.

Possibly related. This relationship suggests that treatment with study drug may cause or contribute to AE; for example, the event is consistent with a reasonable time sequence from the time of study drug administration and/or is consistent with a known pattern of response to study drug, but could also be caused by other factors.

Probably related. This relationship suggests that there is a reasonable temporal sequence of the event and administration of the study drug, as well as a likely association of the event with the study drug. This is based on the known pharmacological effects of the investigational drug, known or previously reported adverse reactions to the investigational drug or drug class, or judgment based on the investigator's clinical experience.

Definitely related. Relationship in time with study drug. Other conditions (comorbidity, concomitant drug reaction, or progression/manifestation of a disease state) do not appear to explain the phenomenon; the phenomenon corresponds to a known pharmaceutical profile; improvement upon completion of treatment; reappearance with repeated administration of the drug.

If the subject had undergone a fatal SAE, the following procedures were performed. The SAE that resulted in death has an outcome documented as death/death, with the end date being the date of death. If the subject had additional AE/SAE that were ongoing at the time of death, these events were documented as ongoing with no end date. Only 1 event is fatal/fatal unless otherwise noted in the autopsy report or investigator's report.

17. Statistical analysis

A formal statistical analysis plan (SAP) was developed and finalized before the database was locked.

The safety analysis population consists of all subjects who received at least 1 dose of study drug. Subjects in this population were used for safety analysis.

The PK population consists of all subjects for whom sufficient serum concentration data were available to allow calculation of PK parameters. The PK population was used to obtain summary PK analysis data.

The population for PD consists of all subjects for whom there were sufficient data on the concentration of total and free C5 and data on the degree of hemolysis of cRBC. The PD population was used to obtain summary data from the PD analysis.

The immunogenicity assay population consisted of all subjects from which pre-dose and post-dose ADA samples were collected.

The total estimated sample size of 36 subjects - 24 subjects with ALXN1210 SC from cohort 1 and 12 subjects with ALXN1210 IV from cohort 2 provided > 80% power to conclude that the lower bound of the 90% confidence interval for the bioavailability ratio of ALXN1210 SC and IV was >0.4 assuming an absolute bioavailability of 0.6 and a coefficient of variation of 0.35. In addition, 6 subjects received placebo SC: 2 in cohort 1a and 4 in cohort 1b. Cohort 1a was randomized 2:1 and cohort 1b 5:1 to receive ALXN1210 SC or placebo SC. This brought the total planned number of subjects to N=42.

In general, descriptive statistics options for continuous variables include the number of non-missing values, arithmetic mean, standard deviation, median, minimum value, and maximum value. Descriptive statistics parameters for PK parameters included number of observations, arithmetic mean, standard deviation, arithmetic coefficient of variation (% CV), median, minimum value, maximum value, geometric mean, and geometric % CV. Categorical variables were summarized using percentages and frequency counts across cohorts and time points.

All subjects were included in a summary of the subject distribution, which summarized the frequency and percentage of screened and treated subjects who completed or discontinued participation in the study, as well as the reason for termination by cohort. Demographics and baseline characteristics were summarized for all subjects for each cohort and overall.

Safety analyzes were performed on the safety analysis population and the results reported for each group. Safety analyzes included analysis of all AEs, ECGs, clinical laboratory data, physical examinations, and measurements of vital signs and were presented using descriptive statistics parameters. No inferential statistical analyzes were planned for the safety parameters of this study. The incidence of AE and SAE occurring during treatment was summarized by system organ class and preferred terms for each cohort and overall by relationship to study drug. AEs that occurred during treatment were also summarized by cohort and overall by severity. Serious AEs and AEs leading to withdrawal from the study were listed. Subjects with multiple AEs within the same category ( for example, in general, in the system-organ class, under the preferred term) were counted once in this category. For severity tables, the most severe event in a subject within a category was taken into account.

Changes in vital signs measurements and laboratory assessments (eg, in biochemistry, CBC with WBC count, and urinalysis) from baseline were summarized for each cohort. Values of laboratory parameters were classified into degrees according to CTCAE. Tables of shifts by cohort were obtained for these laboratory parameters. These tables summarize the number of subjects with each grade at baseline relative to the range of reference values and change to the worst highest grade assessed post-dose during the study.

ECG parameters were measured at the indicated time points, including heart rate, PR, RR, QRS, QT intervals and corrected QTcF. The mean of triplicate ECG scores at collection time points was calculated and changes from pre-treatment baseline values were assessed for each cohort.

An outlier analysis was performed that summarized the frequency and percentage of subjects that met any of the following outlier criteria at each visit by cohort:

QT, QTcF interval >450 ms

QT, QTcF interval >480 ms

QT, QTcF interval >500 ms

QT, QTcF interval increases from baseline >30 ms

QT, QTcF interval increases from baseline > 0 ms.

All concomitant medications were coded using the WHO Drug Dictionary and the frequency and percentage of concomitant medication use were summarized.

Individual serum concentration data for ALXN1210 treated subjects with actual sampling dates and times were used to derive PK parameters using non-compartmental assays using Phoenix WinNonlin 6.3 or later.

The following PK parameters were obtained: maximum observed serum concentration (C _max ), time to reach maximum observed serum concentration (T _max ), area under the curve of serum concentration versus time from zero to the last quantifiable concentration (AUC _t ), area under the curve from time zero to infinity (AUC _∞ ), terminal elimination rate constant (λ _z ), terminal half-life (T _½ ), total clearance (CL or CL/F), and volume of distribution (V _d or _Vd /F).

The geometric mean ratio (ALXN1210 SC/ALXN1210 IV) and its 90% CI were calculated for C _max , AUC _t and AUC _∞ and presented in tabular form. CI was calculated using intersubject variability. Estimates of changes in concentration over time were presented.

The PD effects of ALXN1210 SC and IV were assessed by assessing changes in serum total and free C5 concentrations, degree of cRBC hemolysis, and other indicators of C5 activation over time. Analyzes were performed on samples collected as described in the evaluation schedule.

Immunogenicity as measured by ADA was summarized in tabular form by cohort and by list of subjects.

Example 3 Results of a Phase 1 Study to Evaluate a Single Dose of ALXN1210 Administered Subcutaneously vs. IV Administration in Healthy Subjects

The following is a summary of the data from the Phase 1 single dose study, which was conducted essentially as described in Example 2 above. In particular, the study was designed to evaluate safety, tolerability, pharmacokinetic characteristics (PK), pharmacodynamic characteristics (PD) and immunogenicity of a single dose of 400 mg ALXN1210 administered subcutaneously versus a single dose of 400 mg ALXN1210 administered intravenously or placebo administered subcutaneously in 42 healthy subjects.

1. Distribution of subjects

Of 161 screened subjects, 42 (26.09%) subjects were randomly assigned to receive study drug: placebo SC (n=6), ALXN1210 SC (n=24), and ALXN1210 IV (n=12) (Figure 48). None of the randomized subjects terminated early from the study.

2. Deviations from the protocol

At least one protocol deviation was reported in 36 subjects (placebo SC: n=6; ALXN1210 SC: n=20 and ALXN1210 IV: n=10). Categories of protocol deviations included: time window deviation, subject compliance, assessment failure, exclusion criteria, and dose administration.

In two subjects in the ALXN1210 IV group, protocol deviations were assessed as significant. In one subject on day 29, ADA, PK, PD and laboratory evaluations were not performed because the subject did not attend a follow-up visit. The other subject was not sampled for PK and PD analysis on day 71 because the subject did not attend a follow-up visit. Although these deviations were judged to be significant due to the nature of the clinical study design (primary endpoint associated with PK), they were not considered to have an impact on the interpretation of the results. None of the other protocol deviations were considered to affect the interpretation of the results or the safety of the subjects. Serum pregnancy test results were negative in all subjects during the study.

3. Evaluation of pharmacokinetic characteristics, pharmacodynamic characteristics and immunogenicity

All 42 randomized subjects received study drug and were included in the safety analysis group (Table 19). All of these subjects were also included in the PD analysis group and the immunogenicity analysis group based on the definitions. For 36 subjects in the safety analysis group who received ALXN1210 SC or ALXN1210 IV, there were sufficient serum concentration data to allow calculation of PK parameters and were included in the PK analysis group (Table 19).

Table 19. Population analysis (all randomized subjects)

Placebo SC (N=6), n (%) ALXN1210 SC (N=24), n (%) ALXN1210 IV (N=12), n (%) Security Analysis Group 6 (100.0) 24 (100.0) 12 (100.0) Group for the analysis of pharmacokinetic characteristics 0 24 (100.0) 12 (100.0) Group for the analysis of pharmacodynamic characteristics 6 (100.0) 24 (100.0) 12 (100.0) Group for immunogenicity analysis 6 (100.0) 24 (100.0) 12 (100.0)

Note: Percentage (%) equals n/N × 100.

Abbreviations: IV=intravenous; N=total number of subjects; n=number of subjects; SC=subcutaneous.

4. Demographic and other background characteristics

Among the treatment groups, the majority of subjects were Caucasian males (66.7%) (69.0%) with a mean age (± SD) of 35.0 (± 7.65) years. The mean (± SD) BMI for the general population was 24.035 (± 3.1582). Overall, demographics were well balanced across treatment groups (Table 20).

Table 20. Demographic data - parameters of descriptive statistics by type of treatment (group for safety analysis)

Demographic parameter Placebo SC (N=6) ALXN1210SC (N=24) ALXN1210 IV (N=12) Total (N=42) Gender, n (%) Male 4 (66.7) 16 (66.7) 8 (66.7) 28 (66.7) Female 2 (33.3) 8 (33.3) 4 (33.3) 14 (33.3) Age (years) N 6 24 12 42 Mean value (±SD) 34.2 (± 6.46) 36.2 (± 7.73) 33.2 (± 8.20) 35.0 (± 7.65) Weight, kg) N 6 24 12 42 Mean value (±SD) 71.30 (± 6.727) 72.69 (± 12.892) 72.45 (± 11.882) 72.42 (± 11.698) BMI (kg/ ^m2 ) N 6 24 12 42 Mean value (±SD) 23.220 (± 2.4948) 23.846 (± 3.3895) 24.820 (± 3.0353) 24.035 (± 3.1582) Ethnicity, n (%) Hispanic or Latino 0 1 (4.2) 1 (8.3) 2 (4.8) Not Hispanic or Hispanic 6 (100.0) 23 (95.8) 11 (91.7) 40 (95.2) Race, n (%) Mongoloids 1 (16.7) 2 (8.3) 2 (16.7) 5 (11.9) Negroids or African Americans 1 (16.7) 5 (20.8) 0 6 (14.3) Caucasoids 2 (33.3) 17 (70.8) 10 (83.3) 29 (69.0) Other 2 (33.3) 0 0 2 (4.8)

Note: Percentage (%) equals n/N × 100.

Abbreviations: BMI=body mass index; IV=intravenous; Max. = maximum; min. = minimum; N=total number of subjects; n=number of subjects; SC=subcutaneous; SD=standard deviation.

The use of previously prescribed drugs was reported by 5 (20.8%) subjects in the ALXN1210 SC group. There were no reports of the use of previously prescribed drugs in the SC and ALXN1210 IV placebo groups. Concomitant drug use was reported by 3 (50.0%), 13 (54.2%) and 8 (66.7%) subjects in the placebo SC, ALXN1210 SC and ALXN1210 IV groups, respectively. The most commonly used concomitant medications were anilides such as acetaminophen/paracetamol (15 subjects) for the treatment of AE, followed by progestogens and estrogens, a fixed dose combination (7 subjects) for preventing conception. None of the reported concomitant medications affected the results of the study.

None of the subjects received any non-pharmacological therapies and procedures. All doses of ALXN1210 SC or placebo SC were administered by four SC injections of 100 mg in a volume of 1 ml each in the abdomen. All doses of ALXN1210 IV were administered by IV infusion using IV kits with built-in filters. All subjects received their prescribed doses.

5. Results of analysis of pharmacokinetic characteristics, pharmacodynamic characteristics and immunogenicity and data tables for individual subjects

PK analyzes were performed on the PK analysis group, which consisted of all subjects from the safety analysis group who received either ALXN1210 SC or ALXN1210 IV and for whom sufficient serum concentration data were available to allow calculation of PK parameters.

Figures 49-50 illustrate mean (± SD) serum concentration versus time profiles for healthy subjects after SC and IV administration of ALXN1210 (linear and linear-logarithmic scales). Graphs of individual concentrations of ALXN1210 in serum depending on the nominal time are presented using a linear scale (figure 49) and a linear-logarithmic scale (figure 50), respectively.

Pharmacokinetic parameters of ALXN1210 after SC and IV administration are summarized in Table 21. A total of 24 subjects received ALXN1210 SC administration; the median value (range) t _max was 169.8 (96.0-508.1) hours after SC injection. The geometric mean (CV%) t _½ was similar, being 31.3 (13.6) days and 29.9 (15.4) days for the introduction of ALXN1210 SC and IV, respectively. Elimination of ALXN1210 was similar for the IV and SC pathways (Figure 49).

Table 21 Summary of ALXN1210 Pharmacokinetic Parameters (PK Analysis Group)

_{Cmax tmax AUCt AUC∞ λz t½} CL or CL/F _Vd or _Vd /F Treatment Statistical parameter [µg/ml] [h] [h*mcg/ml] [h*mcg/ml] [/h] [h] [l/h] [l] ALXN1210SC (n=24) geometric mean 35.1 ^169.8a 46734.5 47653.4 0.001 751.6 0.009 9.1 Geometric CV (%) 32.9 n. d. 28.4 28.5 0.00 13.6 27.7 24.7 Range (min., max.) 19.5, 65.2 96.0, 508.1 26294.3, 99354.7 26834.0, 103195.2 0.001, 0.001 614.4, 1012.2 0.004, 0.015 5.7, 14.3 ALXN1210 IV (n=12) geometric mean 134.6 0.8a 77892.8 78902.9 0.001 718.4 0.005 5.3 Geometric CV (%) 19.0 n. d. 24.9 25.4 0.00 15.4 22.6 24.8 Range (min., max.) 99.6, 170.0 0.27, 2.0 57748.3, 151090.9 58146.6, 155019.7 0.001, 0.001 601.5, 990.1 0.003, 0.007 3.1, 7.1

^a Median value is given for t _max .

Note: For ALXN1210 SC treatment, columns CL and V _d represent CL/F and V _d /F, respectively.

Abbreviations: AUC _t = area under the serum concentration versus time curve from time 0 to the last quantifiable concentration; AUC _∞ = area under the serum concentration versus time curve from time 0, extrapolated to infinity; CL or CL/F = total drug clearance from blood serum; C _max =maximum observed serum concentration; CV=coefficient of variation; h=hour; IV=intravenous; l=liter; max=maximum; min=minimum; n=number of subjects; n.a.=no data; SC=subcutaneous; t _½ = terminal half-life; t _max =time to reach maximum observed serum concentration; V _d or V _d /F=volume of distribution; λ _z =terminal elimination rate constant.

Table 22 summarizes the absolute bioavailability of ALXN1210 SC. The PK parameters for ALXN1210 SC (C _max , AUC _t , and AUC _∞ ) were compared to a reference (ALXN1210 IV) by mixed model statistical analysis after log transformation of the data. GMR C _max for ALXN1210 (SC/IV) was 26.1% (95% CI: 21.3, 32.0). The absolute bioavailability of ALXN1210 SC, based on GMR estimated AUC _∞ (SC/IV), was 60.4% (95% CI: 49.7, 73.3).

Table 22. Statistical Analysis of Subcutaneous Absolute Bioavailability of ALXN1210 (PK Analysis Group)

geometric mean Pharmacokinetic parameter ALXN1210SC (n=24) ALXN1210 IV (n=12) GMR (%) 90%CI 95%CI C _max [µg/ml] 35.1 134.6 26.1 22.0-30.9 21.3-32.0 AUC _t [h.*mcg/ml] 46734.5 77892.8 60.0 51.1-70.4 49.5-72.8 AUC _∞ [h.*mcg/ml] 47653.4 78902.9 60.4 51.4-71.0 49.7-73.3

Note: The ratio was defined as the geometric mean of the ALXN1210 SC group divided by the geometric mean of the ALXN1210 IV x 100 group. A linear mixed model with fixed and random effects for the subject was used.

Abbreviations: AUC _t = area under the serum concentration versus time curve from time 0 to the last quantifiable concentration; AUC _∞ = area under the serum concentration versus time curve from time 0, extrapolated to infinity; CI=confidence interval; C _max =maximum observed serum concentration; GMR=geometric mean ratio; h=hour; IV=intravenous; n=number of subjects; SC=subcutaneous.

PD analyzes were performed in the PD analysis group, which consisted of all subjects from the safety analysis group for whom there were sufficient data on free and total C5 concentration and data on the degree of hemolysis of cRBC.

Figure 51 depicts the percent change in mean (±SD) serum free C5 concentration from baseline over time for subjects treated with placebo SC, ALXN1210 SC and ALXN1210 IV. The mean free C5 concentration remained relatively constant after placebo SC administration. The introduction of a single dose of 400 mg ALXN1210 IV resulted in an immediate and almost complete inhibition of free C5 (≥99%) for a period up to and including day 8 after IV administration. Administration of a single dose of 400 mg of ALXN1210 SC also resulted in a decrease in free C5, but not to the same extent or with the same rapid onset as observed after IV administration. After administration of ALXN1210 SC, the maximum mean free C5 inhibition (77%) was observed 1 week post-dose.

The duration and extent of the decrease in the mean concentration of free C5 depended on the exposure. Figure 52 depicts the percentage change in mean (± SD) serum total C5 concentrations from baseline over time for subjects treated with placebo SC, ALXN1210 SC and ALXN1210 IV. Mean concentrations of total C5 concentrations remained relatively constant after placebo SC administration. However, administration of a single dose of 400 mg of ALXN1210 resulted in a maximum mean increase in total C5 concentration of 82% and 107% from baseline after SC and IV dosing, respectively.

Figure 53 depicts the percent change in mean (± SD) hemolysis rate of chicken red blood cells (cRBC) from baseline over time for subjects treated with placebo SC, ALXN1210 SC and ALXN1210 IV. The mean degree of cRBC hemolysis remained relatively constant after placebo SC administration. Administration of a single dose of 400 mg ALXN1210 IV resulted in immediate inhibition of moderate cRBC hemolysis with a maximum mean reduction of 88%. Administration of a single dose of 400 mg of ALXN1210 SC also resulted in inhibition of cRBC hemolysis, but not to the same extent or with the same rapid onset compared to IV administration. The maximum mean inhibition of cRBC hemolysis of 29% was observed approximately 1 week after dosing with ALXN1210 SC. The duration and degree of cRBC inhibition depended on exposure.

The immunogenicity assay was performed on an immunogenicity assay group that consisted of all subjects in the safety assay group from which pre-dose and post-dose ADA samples were collected. Anti-drug antibody testing was performed pre-dose and post-dose on days 15, 29, 57, 90, 120, 150 and 200.

One subject (Subject 0344-185) in the ALXN1210 SC treatment group had a confirmed ADA-positive sample at baseline (pre-dose) and in all post-dose samples. All post-dose antibody titers in this subject were below pre-dose titers. A positive anti-drug antibody response in this subject was not considered clinically significant or associated with ALXN1210. Therefore, this subject was not included in the immunogenicity summary below.

A total of 4 subjects (ALXN1210 SC group: 3/23 [13%] subjects and ALXN1210 IV group: 1/12 [8.3%] subjects) developed ADA during treatment. In the ALXN1210 SC group, the first subject tested positive for ADA on days 57, 90, 120, 150, and 200. All ADA positives were positive for eculizumab cross-reactivity. The second subject tested positive for ADA on days 29, 57, 90, 120, 150 and 200. All positive ADA values were positive for eculizumab cross-reactivity, except for day 90 when they were negative. A third subject tested positive for ADA on days 90, 120, 150, and 200. All ADA positives were positive for eculizumab cross-reactivity.

In the ALXN1210 IV arm, one subject tested positive for ADA on days 15, 29, 90, 120, 150, and 200. All ADA positives were negative for eculizumab cross-reactivity.

The earliest post-dose ADA-positive responses were observed on day 29 and day 15 for SC and IV dosing, respectively. ADA titers for ADA-positive specimens were low and ranged from <1.0 to 27. Most ADA-positive specimens showed cross-reactivity with eculizumab after SC administration of ADA. Following IV administration, ADA did not cross-react with eculizumab. All ADA-positive subjects remained positive until the end of the follow-up period. A formal assessment of the effect of ADA on PK and PD could not be performed due to the small number of subjects who tested positive for ADA. Examination of the limited individual results of PK and PD analysis in these subjects suggests that there is no clear effect of immunogenicity on the PK or PD of ALXN1210.

6. Conclusions regarding pharmacokinetic characteristics, pharmacodynamic characteristics and immunogenicity

The median value (range) t _max was 169.8 (96.0-508.1 hours) after SC injection. The geometric mean terminal half-life was similar, 31.3 days and 29.9 days after administration of ALXN1210 SC and IV, respectively.

The GMR of the estimated C _max (SC/IV) was 26.1% (95% CI: 21.3, 32.0). The absolute bioavailability of ALXN1210 SC based on GMR estimated AUC _∞ (SC/IV) was 60.4% (95% CI: 49.7, 73.3).

The extent and duration of the PD response, as assessed by serum concentrations of free and total C5 and the extent of cRBC hemolysis, were exposure dependent. Administration of a single dose of 400 mg ALXN1210 IV resulted in immediate and nearly complete inhibition of free C5 (≥99%) up to and including day 8 after study drug administration. Administration of a single dose of 400 mg of ALXN1210 SC given as four 100 mg SC injections also resulted in a reduction in free C5, but not to the same extent or with the same rapid onset as IV administration. The maximum mean inhibition of free C5 was 77%, which occurred about 1 week after SC administration. Administration of 400 mg of ALXN1210 resulted in a maximum mean increase in total C5 of 82% and 107% from baseline after SC and IV dosing, respectively. Administration of a single dose of 400 mg ALXN1210 IV resulted in immediate inhibition of moderate cRBC hemolysis with a maximum mean reduction of 87%. Administration of a single dose of 400 mg of ALXN1210 SC also resulted in inhibition of cRBC hemolysis, but not to the same extent or with the same rapid onset compared to IV administration. The maximum mean inhibition of cRBC hemolysis of 29% was observed around day 8 after SC administration.

Treatment-induced ADA was reported in 3/23 (13%) of subjects and 1/12 (8.3%) of subjects in the ALXN1210 SC and ALXN1210 IV arms, respectively, with low ADA titer values ranging from <1.0 to 27. The earliest post-dose ADA response was observed at day 29 and day 15 for SC and IV dosing, respectively. Following SC administration, ADA was cross-reactive with eculizumab in most ADA-positive samples. Following IV administration, ADA did not cross-react with eculizumab. All ADA-positive subjects remained positive until the end of the follow-up period. No clear effect of immunogenicity on the PK or PD of ALXN1210 was observed.

One additional subject in the ALXN1210 SC treatment group had a confirmed ADA-positive sample at baseline (pre-dose) and in all post-dose samples. All post-dose antibody titers in this subject were below pre-dose titers. The anti-drug antibody response in this subject was not associated with ALXN1210.

7. Degree of impact

All subjects who received a single dose of study drug were included in the safety analysis arm (N=42): placebo SC (n=6); ALXN1210 SC (n=24) and ALXN1210 IV (n=12). The total infusion volume (80 ml) of study drug was administered to each subject assigned to receive ALXN1210 IV. In one subject, the infusion was interrupted for a minute because insufficient time was programmed into the pump for a complete infusion. Each subject who received ALXN1210 SC or placebo SC was administered a total volume of study drug (4 ml).

8. Adverse events

In all three treatment groups, 35/42 (83.3%) subjects experienced 75 TEAEs (all grade 1). The proportion of subjects experiencing at least 1 TEAE was 91.7%, 83.3%, and 79.2%, respectively, in the ALXN1210 IV group, placebo SC group, and ALXN1210 SC group. No deaths or SAEs were reported during the study. None of the subjects discontinued study medication or discontinued study participation due to TEAE (Table 23) . All TEAEs were eliminated during the course of the study. Most TEAEs did not require any drug, and none of the subjects required non-pharmacological interventions at any time.

Table 22 Treatment-Emerged Adverse Events (TEAE) - Overall Summary (Safety Analysis Group)

Placebo SC (N=6) ALXN1210SC (N=24) ALXN1210 IV (N=12) Total (N=42) E n(%) E n(%) E n(%) E n(%) Subjects with at least 1 TEAE 15 5 (83.3) 38 19 (79.2) 22 11 (91.7) 75 35 (83.3) Related TEAE 0 0 2 2 (8.3) 1 1 (8.3) 3 3 (7.1) Unrelated TEAE 15 5 (83.3) 36 18 (75.0) 21 11 (91.7) 72 34 (81.0) 1 degree 15 5 (83.3) 38 19 (79.2) 22 11 (91.7) 75 35 (83.3) 2 degree 0 0 0 0 0 0 0 0 3 degree 0 0 0 0 0 0 0 0 4 degree 0 0 0 0 0 0 0 0 5 degree 0 0 0 0 0 0 0 0 Subjects with at least 1 SAE 0 0 0 0 0 0 0 0 Subjects with TEAE leading to treatment discontinuation 0 0 0 0 0 0 0 0 Subjects with TEAE during administration of study drug ^a 0 0 0 0 0 0 0 0 Deaths 0 0 0 0 0 0 0 0

Note: Percentage (%) equals n/N × 100.

1 degree = mild; Grade 2=moderate; 3 degree = strong; Grade 4 = life-threatening or disabling; Grade 5=death associated with TEAE.

Associated TEAE = possibly associated, likely associated, or definitely associated TEAE; unrelated TEAE = unrelated or unlikely related TEAE.

For ALXN1210 IV, TEAE was considered to have occurred during study drug administration if TEAE occurred during infusion; for placebo SC and ALXN1210 SC, an adverse event was considered to have occurred during study drug administration if the adverse event occurred between the first and last injections.

Abbreviations: E=number of events; IV=intravenous; N=total number of subjects at risk; n=number of subjects having AE; SC=subcutaneous; SAE=serious adverse event; TEAE=adverse event occurring during treatment.

A total of 75 TEAEs were reported in 35 subjects. Among the TEAE treatment groups that were most frequently reported were nasopharyngitis (23/42 subjects, 54.8%) and headache (7/42 subjects, 16.7%). All TEAEs were summarized by system organ class (SOC) and preferred terms by treatment in Table 23.

Table 23 Adverse Events Occurring During Treatment - Frequency Table by System Organ Class and Preferred Terms (Safety Analysis Group)

Main system organ class
Preferred Term Placebo SC (N=6) ALXN1210SC (N=24) ALXN1210 IV (N=12) Total (N=42) E n(%) E n(%) E n(%) E n(%) Subjects with TEAE 15 5 (83.3) 38 19 (79.2) 22 11 (91.7) 75 35 (83.3) Infectious and parasitic diseases 10 5 (83.3) eighteen 14 (58.3) nine 8 (66.7) 37 27 (64.3) Nasopharyngitis 7 5 (83.3) 13 11 (45.8) 8 7 (58.3) 28 23 (54.8) lower respiratory tract infection 1 1 (16.7) 1 1 (4.2) 0 0 2 2 (4.8) Candida balanitis 0 0 1 1 (4.2) 0 0 1 1 (2.4) Inflammation of the subcutaneous tissue 0 0 0 0 1 1 (8.3) 1 1 (2.4) Gastroenteritis 1 1 (16.7) 0 0 0 0 1 1 (2.4) Dermatophytosis 0 0 1 1 (4.2) 0 0 1 1 (2.4) Upper respiratory tract infection 0 0 1 1 (4.2) 0 0 1 1 (2.4) Viral infection of the upper respiratory tract 0 0 1 1 (4.2) 0 0 1 1 (2.4) Vulvovaginal candidiasis 1 1 (16.7) 0 0 0 0 1 1 (2.4) Nervous System Disorders 3 3 (50.0) five 3 (12.5) four 4 (33.3) 12 10 (23.8) Headache 3 3 (50.0) 2 1 (4.2) 3 3 (25.0) 8 7 (16.7) Migraine 0 0 2 2 (8.3) 1 1 (8.3) 3 3 (7.1) migraine with aura 0 0 1 1 (4.2) 0 0 1 1 (2.4) Gastrointestinal disorders 2 1 (16.7) four 3 (12.5) 3 3 (25.0) nine 7 (16.7) Diarrhea 1 1 (16.7) 2 1 (4.2) 1 1 (8.3) four 3 (7.1) Nausea 1 1 (16.7) 0 0 1 1 (8.3) 2 2 (4.8) Dyspepsia 0 0 1 1 (4.2) 0 0 1 1 (2.4) Toothache 0 0 0 0 1 1 (8.3) 1 1 (2.4) Vomit 0 0 1 1 (4.2) 0 0 1 1 (2.4) Musculoskeletal and connective tissue disorders 0 0 3 3 (12.5) 3 3 (25.0) 6 6 (14.3) Musculoskeletal pain 0 0 1 1 (4.2) 1 1 (8.3) 2 2 (4.8) Myalgia 0 0 1 1 (4.2) 1 1 (8.3) 2 2 (4.8) Backache 0 0 1 1 (4.2) 0 0 1 1 (2.4) Pain in the limb 0 0 0 0 1 1 (8.3) 1 1 (2.4) Respiratory, thoracic and mediastinal disorders 0 0 3 3 (12.5) 3 3 (25.0) 6 6 (14.3) Cough 0 0 0 0 2 2 (16.7) 2 2 (4.8) Nasal congestion 0 0 1 1 (4.2) 1 1 (8.3) 2 2 (4.8) allergic sinusitis 0 0 1 1 (4.2) 0 0 1 1 (2.4) Pain in the oropharynx 0 0 1 1 (4.2) 0 0 1 1 (2.4) Injury, poisoning and procedural complications 0 0 2 2 (8.3) 0 0 2 2 (4.8) Arthropod bites 0 0 1 1 (4.2) 0 0 1 1 (2.4) joint dislocation 0 0 1 1 (4.2) 0 0 1 1 (2.4) General disorders and disorders at the injection site 0 0 1 1 (4.2) 0 0 1 1 (2.4) Bruising at puncture sites 0 0 1 1 (4.2) 0 0 1 1 (2.4) Immune System Disorders 0 0 1 1 (4.2) 0 0 1 1 (2.4) seasonal allergies 0 0 1 1 (4.2) 0 0 1 1 (2.4) Skin and subcutaneous tissue disorders 0 0 1 1 (4.2) 0 0 1 1 (2.4) Papular rash 0 0 1 1 (4.2) 0 0 1 1 (2.4)

Note: Percentage (%) equals n/N × 100.

Each subject was counted only once for a given SOC and preferred term, regardless of the actual number of adverse events that occurred.

The SOC classification and preferred term are in accordance with MedDRA v20.0.

Abbreviations: E=number of events; IV=intravenous; MedDRA=Medical Dictionary for Regulatory Activities; N=total number of subjects at risk; n=number of subjects having an adverse event; SC=subcutaneous; SOC=system organ class; TEAE=adverse event occurring during treatment.

The majority of TEAEs (72/75 TEAE, 96%) were considered unrelated to ALXN1210 treatment. Among the treatment groups, 3/42 (7.1%) subjects reported 3 TEAEs that the investigator assessed as related ("possibly related") to treatment with ALXN1210 and grade 1 (mild): (1) upper respiratory infections in one subject in the ALXN1210 SC group (2), migraine in one subject in the ALXN1210 SC group, and (3) headache in one subject in the ALXN1210 IV group. All 75 TEAEs were classified as grade 1 (mild). No subject died, experienced SAE, or discontinued study drug or study participation due to TEAE.

In general, mean values in hematology analysis, coagulation analysis, blood chemistry, urinalysis and urinalysis were within the range of reference values, and there were no clear trends in the mean value from baseline.

The majority of subjects entered the study with normal values (ie, within the respective reference ranges) of parameters for hematology analysis, urinalysis, coagulation analysis, blood chemistry, and urinalysis. There were no obvious trends for shifts among the treatment groups. For some laboratory parameters, a shift from normal values at baseline to abnormal values (grade 1 [mild] or grade 2 [moderate]) was observed during the study, which, however, was not considered clinically significant. Most of the shifts were temporary and corrected during the study.

During the study, 3 subjects in the ALXN1210 SC group reported a shift to grade 3 abnormal values. None of the shifts to grade 3 abnormal values were reported as AE.

First, a decrease in the number of neutrophils was reported in one subject. The number of neutrophils in this subject at baseline was 3.77×10 ⁹ /L. The estimated neutrophil count on day 43 was 0.95×10 ⁹ /l (normal range: 2.0-7.5×10 ⁹ /l). The neutrophil count was in the normal range at day 57.

Elevated potassium levels were reported in two subjects (normal range: 3.5-5.1 mmol/l). In 1 subject with a baseline potassium level of 4.5 mmol/l, the estimated potassium level on day 150 was 6.1 mmol/l. Re-measured potassium levels were within the normal range on the same day (unscheduled visit). In another subject with a baseline potassium level of 4.6 mmol/l, the estimated potassium level on day 90 was 6.4 mmol/l. This subject had abnormal potassium levels at screening (ranging in the range of 5.2-6.2 mmol/L at various screening visits) and at most study visits. The increase in potassium levels was temporary; the recorded values were within the range of normal values at day 150 and day 200.

No notable changes in vital signs measurements from baseline and no clinically significant anomalies of vital signs consistently observed in individual subjects were detected.

No subject had clinically significant physical examination findings other than those reported as AE. Significant changes in the mean values from baseline in the results of ECG or telemetry control were not observed.

Changes in QT intervals were corrected using the Friedrichia formula (QTcF). One subject in the placebo SC group had a mean QT interval of >500 ms at screening (510.0 ms) and on day 2 (508.7 ms), day 150 (516.6 ms) and day 200 (612.9 ms) . The mean QTcF interval in the same subject was 449.7 ms, 443.7 ms, 451.9 ms, and 501.3 ms at screening and on days 2, 150, and 200, respectively. The increase in the QT interval and QTcF was not considered clinically significant in this female subject in the placebo group. Such changes were also not reported as AE. There were no significant changes in mean QT and QTcF intervals from baseline during the study.

Assessments at the site of infusion or injection were performed within 15 minutes after SOI and ± 15 minutes at time points of 30 minutes, 2 hours, 4 hours, 8 hours, and on Day 2 (48 hours), Day 3 (72 hours, total 6 ratings). Lesions or reactions <1 cm in size were not considered AE unless they persisted for more than 24 hours. At 30 minutes post-EOI, 5/24 subjects in the ALXN1210 SC group had redness, and 1 subject had minimal (3 mm) redness at 2/4 injection sites at 2 hours post-injection and none at the last time points. Minimal induration or swelling (10 mm) was recorded 30 minutes after EOI in 1/24 of the subjects in the ALXN1210 SC group, which was not observed in recent evaluations. However, none of them met the criteria defined in the protocol to be considered AE. Pain at the infusion or injection site was assessed by subjects using a VAS (0-100 mm). For most infusions and injections, pain at the infusion site was rated as 0 mm for all assessments. Two subjects in the SC group reported transient pain corresponding to 3-5 mm on day 1, and three subjects in group IV reported minimal (1-5 mm) pain after infusion.

9. Safety Conclusions

All subjects who received a single dose of study drug (placebo SC, ALXN1210 SC or ALXN1210 IV) were included in the safety analysis group (N=42). Among the 3 treatment groups, 35/42 subjects (83.3%) experienced 75 TEAEs. Only 3/75 TEAEs (4%) were considered related to ALXN1210, while 72/75 (96%) were considered not related to ALXN1210 treatment. All TEAEs were mild (grade 1) and were eliminated during the course of the study. Most TEAEs did not require any drug, and none of the subjects required non-pharmacological interventions at any time. The most frequently reported TEAEs were nasopharyngitis (23/42 subjects, 54.8%) and headache (7/42 subjects, 16.7%).

No deaths or SAEs were observed during the study. None of the reported TEAEs resulted in discontinuation of the study drug or withdrawal of the subject from the study. In general, no clinically significant changes in laboratory parameters, vital signs, physical examination parameters, ECG, or telemetry were observed during the study or follow-up. No clinical manifestations of hypersensitivity were observed during or after administration of any single dose by SC injection or IV infusion. ADA-positive subjects have no clinical signs or symptoms associated with an allergic reaction or hypersensitivity.

10. Discussion and general conclusions

The aim of this phase 1 study was to evaluate the safety, tolerability, PK, PD and immunogenicity of a single dose of 400 mg ALXN1210 SC versus a single dose of 400 mg ALXN1210 IV or placebo SC given by injection in healthy subjects. A total of 42 subjects were randomized to receive study drug: placebo SC (n=6); ALXN1210 SC (n=24) and ALXN1210 IV (n=12).

The introduction of ALXN1210 at a dose of 400 mg was well tolerated by the SC route of administration in healthy subjects. The absolute bioavailability of ALXN1210 SC based on GMR estimated AUC _∞ (SC/IV) was 60.4% (95% CI: 49.7, 73.3). Estimated geometric mean _t½ was 31.3 days and 29.9 days after administration of ALXN1210 SC and IV. The extent and duration of the PD response, as assessed by serum concentrations of free and total C5 and the extent of cRBC hemolysis, were exposure dependent.

Anti-drug antibodies were reported in 3/23 (13%) subjects and 1/12 (8.3%) subjects in the ALXN1210 SC and ALXN1210 IV arms, respectively, with positive ADA titer values ranging from <1.0 to 27. Most an early post-dose response was observed on day 29 and day 15 for SC and IV dosing, respectively. In the majority of ADA-positive samples following SC administration, ADA was cross-reactive with eculizumab. ADA-positive subjects showed no clinical signs or symptoms consistent with an allergic reaction or hypersensitivity (including anaphylaxis). In addition, no clear effect of ALXN1210 on PK or PD could be identified.

No unexpected safety issues were observed in any of the treatment groups during the study. No deaths or SAEs occurred during the course of the study, and no subject experienced any TEAE leading to discontinuation of study drug or withdrawal from the study.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1
eculizumab heavy chain CDR1 amino acid sequence (as determined according to the combined definition of Kabat and Chothia)
GYIFSNYWIQ SEQ ID NO: 2
eculizumab heavy chain CDR2 amino acid sequence (as determined by Kabat definition)
EILPGSGSTEYTENFKD SEQ ID NO: 3
eculizumab heavy chain CDR3 amino acid sequence (as determined according to Kabat's combined definition)
YFFGSSPNWYFDV SEQ ID NO: 4
eculizumab CDR1 light chain amino acid sequence (as determined according to Kabat definition)
GASENIYGALN SEQ ID NO: 5
amino acid sequence of eculizumab light chain CDR2
(as defined by Kabat definition)
GATNLAD SEQ ID NO: 6
eculizumab light chain CDR3 amino acid sequence (as determined by Kabat definition)
QNVLNTPLT SEQ ID NO: 7
amino acid sequence of the variable region of the heavy chain of eculizumab
QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWM
GEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARY
FFGSSPNWYFDVWGQGTLVTVSS SEQ ID NO: 8
amino acid sequence of the light chain variable region of eculizumab, ravulizumab and BNJ421 antibody
DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGA
TNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTK
VEIK SEQ ID NO: 9
amino acid sequence of the heavy chain constant region of eculizumab and antibody BNJ421
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKC
CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF
NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS
VMHEALHNHYTQKSLSLSLGK SEQ ID NO: 10
amino acid sequence of the entire heavy chain of eculizumab
QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWM
GEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
YFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYT
CNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV
DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 11
amino acid sequence of the entire light chain of eculizumab, ravulizumab and BNJ421 antibody
DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYG
ATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATFATCQNVLNTPLTFGQ
GTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN
ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKSFNRGEC SEQ ID NO: 12
amino acid sequence of the heavy chain variable region of ravulizumab and antibody BNJ421
QVQLVQSGAEVKKPGASVKVSCKASG H IFSNYWIQWVRQAPGQGLEW
MGEILPGSG H TEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYC
ARYFFGSSPNWYFDVWGQGTLVTVSS SEQ ID NO: 13
amino acid sequence of ravulizumab heavy chain constant region
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER
KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE
VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN
VFSCSV L HEALH S HYTQKSLSLSLGK SEQ ID NO: 14
amino acid sequence of the entire heavy chain of ravulizumab
QVQLVQSGAEVKKPGASVKVSCKASG H IFSNYWIQWVRQAPGQGLEWM
GEILPGSG H TEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
YFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYT
CNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV
DKSRWQEGNVFSCSV L HEALH S HYTQKSLSLSLGK SEQ ID NO: 15
amino acid sequence of an IgG2 heavy chain constant region variant containing YTE substitutions
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKC
CVECPPCPAPPVAGPSVFLFPPKPKDTL Y I T R E PEVTCVVVDVSHEDPEVQF
NWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 16
the amino acid sequence of the entire heavy chain of the eculizumab variant containing the heavy chain constant region shown under SEQ ID NO: 15 (above)
QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWM
GEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
YFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNF
GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKP
KDTL Y I T R E PEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQ
FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGK SEQ ID NO: 17
amino acid sequence of eculizumab light chain CDR1 (as determined by Kabat definition) with a glycine to histidine change at position 8 compared to SEQ ID NO: 4
GASENIY H ALN SEQ ID NO: 18
shows the amino acid sequence of the heavy chain CDR2 of eculizumab, in which the serine at position 8 compared to SEQ ID NO: 2 is replaced by histidine.
EILPGSG H TEYTENFKD SEQ ID NO: 19
the amino acid sequence of the CDR1 of the heavy chain of eculizumab, in which the tyrosine at position 2 (compared to SEQ ID NO: 1) is replaced by histidine
G H IFSNYWIQ SEQ ID NO: 20
amino acid sequence of the entire heavy chain of the BNJ421 antibody
QVQLVQSGAEVKKPGASVKVSCKASG H IFSNYWIQWVRQAPGQGLEW
MGEILPGSG H TEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYC
ARYFFGSSPNWYFDVWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQT
YTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV
DKSRWQEGNVFSCSV M HEALH N HYTQKSLSLSLGK

--->

SEQUENCE LIST

<110> ALEXION PHARMACEUTICALS, INC.

<120> HIGHLY CONCENTRATED C5 ANTIBODY COMPOSITIONS

<130> AXJ-226PC

<140>

<141>

<150> 62/537741

<151> 2017-07-27

<160> 20

<170> PatentIn version 3.5

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Gly Ala Ser Glu Asn Ile Tyr Gly Ala Leu Asn

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Gly Ala Thr Asn Leu Ala Asp

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Gln Asn Val Leu Asn Thr Pro Leu Thr

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ser Asn Tyr

20 25 30

Trp Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Glu Tyr Thr Glu Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Phe Phe Gly Ser Ser Pro Asn Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val Leu Asn Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Leu Gly Lys

325

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ser Asn Tyr

20 25 30

Trp Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Glu Tyr Thr Glu Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Phe Phe Gly Ser Ser Pro Asn Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys

210 215 220

Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val Leu Asn Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 12

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly His Ile Phe Ser Asn Tyr

20 25 30

Trp Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly His Thr Glu Tyr Thr Glu Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Phe Phe Gly Ser Ser Pro Asn Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 13

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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Leu Gly Lys

325

<210> 14

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 14

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly His Ile Phe Ser Asn Tyr

20 25 30

Trp Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly His Thr Glu Tyr Thr Glu Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Phe Phe Gly Ser Ser Pro Asn Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys

210 215 220

Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala

420 425 430

Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 15

<211> 326

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 15

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Thr Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Met Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 16

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 16

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ser Asn Tyr

20 25 30

Trp Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Glu Tyr Thr Glu Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Phe Phe Gly Ser Ser Pro Asn Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Thr Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys

210 215 220

Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg

245 250 255

Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Met Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val

290 295 300

Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 17

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 17

Gly Ala Ser Glu Asn Ile Tyr His Ala Leu Asn

1 5 10

<210> 18

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 18

Glu Ile Leu Pro Gly Ser Gly His Thr Glu Tyr Thr Glu Asn Phe Lys

1 5 10 15

asp

<210> 19

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 19

Gly His Ile Phe Ser Asn Tyr Trp Ile Gln

1 5 10

<210> 20

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 20

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly His Ile Phe Ser Asn Tyr

20 25 30

Trp Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly His Thr Glu Tyr Thr Glu Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Phe Phe Gly Ser Ser Pro Asn Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys

210 215 220

Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<---

Claims (39)

1. A stable aqueous solution for the treatment of a human patient with a condition associated with the C5 protein of the complement system, containing: (a) an anti-C5 antibody at a concentration of 100 mg/ml ± 5%, wherein the anti-C5 antibody comprises a heavy chain polypeptide containing the amino acid sequence shown under SEQ ID NO: 14 and a light chain polypeptide containing the amino acid sequence given under SEQ ID NO: 11; (b) 50 mM ± 5% phosphate buffer; (c) 5% ± 5% sucrose (w/v); and (d) 25 mM ± 5% arginine. 2. A stable aqueous solution for the treatment of a human patient with a condition associated with the C5 protein of the complement system, containing: (a) an anti-C5 antibody, wherein the anti-C5 antibody comprises a heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14 and a light chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11; (b) 50 mM ± 5% phosphate buffer; (c) 5% ± 5% sucrose (w/v); (d) 0.05%±5% polysorbate 80 and (f) 25 mM ± 5% arginine. 3. Stable aqueous solution according to claim 1, additionally containing a surfactant. 4. Stable aqueous solution according to claim 3, wherein the surfactant is 0.05% ± 5% polysorbate 80. 5. Stable aqueous solution according to claim 2, wherein the anti-C5 antibody is present at a concentration of 100 mg/ml ± 5%. 6. A stable aqueous solution according to any one of paragraphs. 1-5 wherein the anti-C5 antibody is ALXN1210 (ravulizumab). 7. Stable aqueous solution according to any one of paragraphs. 1-6, where the pH of the solution is from 7.2 to 7.6. 8. Stable aqueous solution according to claim 7, wherein the pH of the solution is 7.4. 9. A stable aqueous solution according to any one of paragraphs. 1-8, where the solution is sterile. 10. A stable aqueous solution according to any one of paragraphs. 1-9 wherein the anti-C5 antibody remains at least 97% monomeric during storage at 2-8°C for at least six months as determined by SEC-HPLC. 11. A stable aqueous solution according to any one of paragraphs. 1-10 wherein the anti-C5 antibody remains at least 97% monomeric during storage at 2-8° C. for at least one year as determined by SEC-HPLC. 12. A stable aqueous solution according to any one of paragraphs. 1-11, wherein less than 3% of the anti-C5 antibodies in solution are aggregated as determined by SEC-HPLC. 13. A stable aqueous solution according to any one of paragraphs. 1-12, wherein less than 2% of anti-C5 antibodies in solution are aggregated as determined by SEC-HPLC. 14. A stable aqueous solution according to any one of paragraphs. 1-13, wherein less than 1% of anti-C5 antibodies in solution are aggregated as determined by SEC-HPLC. 15. A stable aqueous solution according to any one of paragraphs. 1-14, wherein during storage at 2-8° C. for at least six months, the anti-C5 antibody retains at least 90% of its C5 binding activity compared to the anti-C5 antibody prior to storage. 16. A stable aqueous solution according to any one of paragraphs. 1-15, wherein during storage at 2-8° C. for at least six months, the anti-C5 antibody retains at least 95% of its ability to inhibit hemolysis compared to the anti-C5 antibody prior to storage. 17. A stable aqueous solution according to any one of paragraphs. 1-16 where the solution is suitable for subcutaneous administration. 18. A stable aqueous solution according to any one of paragraphs. 1-17, where the solution is suitable for intravenous administration. 19. A method for preparing a stable concentrated solution of an antibody containing an anti-C5 antibody containing a polypeptide heavy chain containing the amino acid sequence listed under SEQ ID NO: 14 and a polypeptide light chain containing the amino acid sequence listed under SEQ ID NO: 11, at a concentration 100 mg/ml, 50 mM phosphate buffer, 5% sucrose (w/v) and 25 mM arginine, including: i) obtaining a first aqueous solution containing not more than 10 mg/ml of anti-C5 antibody; ii) diafiltration of the first aqueous solution to obtain a composition containing 50 mm phosphate buffer, 5% sucrose and 25 mm arginine, at pH 7.4, thereby providing a second aqueous solution; and iii) concentration of the second aqueous solution to obtain a stable concentrated antibody solution containing 100 mg/ml anti-C5 antibody, 50 mM phosphate buffer, 5% sucrose and 25 mM arginine. 20. A method of treating a human patient with a condition associated with the complement protein C5 system, comprising administering to the patient a stable aqueous solution according to any one of paragraphs. 1-17 in an amount effective to treat the condition. 21. The method of claim 20, wherein the condition associated with the complement C5 protein system is selected from the group consisting of rheumatoid arthritis, antiphospholipid antibody syndrome, lupus nephritis, ischemic/reperfusion injury, atypical hemolytic uremic syndrome (aHUS), typical hemolytic - uremic syndrome, paroxysmal nocturnal hemoglobinuria (PNH), dense sediment disease, neuromyelitis optica, multifocal motor neuropathy, multiple sclerosis, macular degeneration, HELLP syndrome, spontaneous abortion, thrombotic thrombocytopenic purpura, low immune vasculitis, epidermolysis bullosa, repeated miscarriage, traumatic brain injury, myocarditis, cerebrovascular disorder, peripheral vascular disorder, renovascular disorder, mesenteric/enteric vascular disorder, vasculitis, Henoch-Schonlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis arthritis, immune complex vasculitis, Takayasu's disease, dilated cardiomyopathy, diabetic angiopathy, Kawasaki disease, venous gas embolism, restenosis after stenting, rotational atherectomy, percutaneous transluminal coronary angioplasty, myasthenia gravis, cold agglutinin disease, dermatomyositis, paroxysmal cold haemoglobinuria, antiphospholipid Graves' disease, atherosclerosis, Alzheimer's disease, septic systemic inflammatory response, septic shock, spinal cord injury, glomerulonephritis, transplant rejection, Hashimoto's thyroiditis, type I diabetes mellitus, psoriasis, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpasture's syndrome, Degos disease and catastrophic antiphospholipid syndrome. 22. The method of claim 20 wherein the condition associated with the C5 complement system protein is atypical hemolytic uremic syndrome (aHUS). 23. The method of claim 20 wherein the condition associated with the C5 complement system protein is paroxysmal nocturnal hemoglobinuria (PNH). 24. The method according to any one of paragraphs. 20-22 where the stable aqueous solution is administered subcutaneously to the patient. 25. The method according to any one of paragraphs. 20-22, where the stable aqueous solution is administered intravenously to the patient. 26. A therapeutic kit for the treatment of a human patient with a condition associated with the C5 protein of the complement system, containing (i) a stable aqueous solution according to any one of paragraphs. 1-17 and (ii) a means for delivering a stable aqueous solution to a human. 27. The therapeutic kit of claim 26, wherein the agent is a syringe.

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