RU2847524C2 - Highly concentrated anti-c5 antibody formulations - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to stable aqueous solutions containing anti-C5 antibody. Stable aqueous solution contains anti-C5 antibody at concentration of 70 mg/ml ± 5 %, 50 mM ± 5 % of phosphate buffer, 5 % ± 5 % of sucrose, 25 mM ± 5 % of arginine. Antibody to C5 contains a heavy chain with SEQ ID NO: 14 and a light chain with SEQ ID NO: 11. Stable aqueous solution is used in a method of treating a human with a condition associated with the complement system C5 protein. Also presented is a therapeutic kit containing said solution and a means for delivering the stable aqueous solution to a human.

EFFECT: invention provides a stable, concentrated aqueous solution of an anti-C5 antibody that remains at least 97 % monomeric and retains at least 90 % of its C5-binding activity during storage at 2–8 °C for at least six months; less than 3 % of the anti-C5 antibody in the solution aggregates.

15 cl, 53 dwg, 24 tbl, 3 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Ser. 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, which was filed electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on July 26, 2018, is named AXJ-226PC_SL.txt and is 32,987 bytes in size.

LEVEL OF TECHNOLOGY

The complement system works in concert with other immune systems to protect against cellular and viral pathogens. At least 25 complement proteins are known, which are found as a complex network of plasma proteins and membrane cofactors. Plasma proteins constitute approximately 10% of the globulins in vertebrate serum. Components of the complement system exert their immune defense function by interacting in a series of complex but precise enzymatic cleavage and membrane binding events. The resulting complement cascade leads to the formation of products with opsonic, immunoregulatory, and lytic functions. A brief description of the biological activities associated with complement activation is presented, for example, in The Merck Manual, ^16th Edition.

Although a properly functioning complement system provides robust protection against microbial challenge, inappropriate regulation or activation of complement 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 complement-associated disorders, such as PNH or aHUS, are at risk for significant morbidity and mortality. Accordingly, the aim of the present invention is to provide improved compositions and methods for treating patients with complement-associated disorders.

BRIEF DESCRIPTION OF THE INVENTION

Provided herein are stable, highly concentrated aqueous solutions of anti-C5 antibodies, as well as methods for preparing and using the formulations. The present invention provides, among other aspects, formulation conditions suitable for maintaining the physical and functional stability of the anti-C5 antibody (e.g., ravulizumab, also known as "antibody BNJ441" and "ALXN1210") for significant periods of time in highly concentrated solutions. For example, the present invention provides formulation conditions capable of maintaining an anti-C5 antibody in a 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. Furthermore, as described herein and illustrated in the demonstrative examples, such formulations also minimize aggregation, fragmentation, or degradation of the anti-C5 antibody (e.g., ravulizumab) in highly concentrated solutions. For example, the present invention provides formulation conditions capable of maintaining an anti-C5 antibody in a highly concentrated form for two years without detectable fragmentation or degradation products of the antibody (as determined by a size-exclusion high-performance liquid chromatography (SEC-HPLC) technique, such as gel permeation HPLC) and with no more than 2% aggregates. Conditions suitable for formulating solutions of an anti-C5 antibody, such as ravulizumab, at a concentration greater than 200 mg/mL are also provided herein.

The advantages of stable, highly concentrated aqueous solutions of anti-C5 antibodies are numerous. First, for therapeutic routes that require administration of the 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. Without the ability to formulate anti-C5 antibodies at high concentrations, the use of, for example, subcutaneous, intravitreal, and/or intra-articular delivery routes will often be precluded. Similarly, highly concentrated antibody formulations provide the patient with greater choice of administration routes. For 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, such as by subcutaneous or intravenous injection. Thus, the ability to formulate antibodies in high concentrations may improve compliance by providing a simple alternative for at-home administration for patients with complement-related disorders.

Furthermore, the methods for producing the aqueous solutions described herein do not require a lyophilization step, nor do the disclosed highly concentrated aqueous solutions require reconstitution from lyophilized material. The highly concentrated antibody solutions disclosed herein offer several advantages over reconstituted lyophilized antibody formulations. First, healthcare professionals must reconstitute lyophilized antibody solutions on-site under sterile conditions, increasing the potential for microbial contamination of the solution prior to administration. Furthermore, reconstitution requires considerable care to ensure that all solids contained in the reconstitution vessel are properly dissolved in the solution. The highly concentrated aqueous solutions disclosed herein thus provide a healthcare professional, caregiver, and/or patient with a rapid, simple, safe, and effective means of delivering a therapeutic antibody to a patient in need thereof.

Other benefits of highly concentrated formulations include, for example, Reducing production costs by reducing bulk storage space and/or product waste. Furthermore, 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), comprising heavy and light chains having the sequences shown at SEQ ID NOs: 14 and 11, respectively, or antigen-binding fragments and variants thereof. In other embodiments, the antibody comprises the complementarity determining regions (CDRs) of the heavy and light chains or the variable regions (VRs) of ravulizumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the variable region of the heavy chain (VH) of ravulizumab having the sequence shown under SEQ ID NO: 12, and the CDR1, CDR2, and CDR3 domains of the variable region of the light chain (VL) of ravulizumab having the sequence shown under SEQ ID NO: 8. In another embodiment, the antibody comprises the heavy chain CDR1, CDR2, and CDR3 sequences set forth under SEQ ID NOs: 19, 18, and 3, respectively, and the light chain CDR1, CDR2, and CDR3 sequences set forth under SEQ ID NOs: 4, 5, and 6, respectively.

In another embodiment, the antibody comprises VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 8, respectively.

In another embodiment, the antibody comprises a heavy chain constant region as set forth in SEQ ID NO: 13.

In another embodiment, the antibody comprises a variant human Fc constant region that binds to the human neonatal Fc receptor (FcRn), wherein the variant human CH3 Fc constant region comprises 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 indicated according to EU numbering.

In another embodiment, the antibody comprises the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 19, 18, and 3, respectively, and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, and a variant human Fc constant region that binds to the human neonatal Fc receptor (FcRn), wherein the variant human Fc CH3 constant region comprises 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 indicated according to EU numbering.

In another embodiment, the antibody competes for binding to and/or binds to the same epitope on C5 as the aforementioned antibodies. In another embodiment, the antibody has at least about 90% amino acid sequence identity of the variable region with the aforementioned antibodies (e.g., at least about 90%, 95% or 99% identity of the variable region 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 of 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, the [(K _D of the antibody or antigen-binding fragment thereof to human C5 at pH 6.0 and at 25°C)/(K _{D of} the antibody or antigen-binding fragment thereof to human C5 at pH 7.4 and at 25°C)] of the antibody is greater than 25.

In one aspect, a stable aqueous solution is provided (e.g., sterile solution), wherein the solution comprises an anti-C5 antibody at a concentration of about 100 mg/mL, wherein the anti-C5 antibody 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 comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In another embodiment, the solution comprises an anti-C5 antibody (e.g., ravulizumab) at a concentration of or about 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 another embodiment, the stable aqueous solution contains one or more additional agents (e.g., stabilizing agents, buffering agents, surfactants, and/or preservatives). For example, in one embodiment, the stable aqueous solution comprises a stabilizer. Illustrative stabilizers include, but are not limited to, polyols, sugars (e.g., sucrose or trehalose), amino acids (eg, arginine), amines and salting-out salts. In one embodiment, the solution comprises at least one stabilizing agent at a concentration of 2-10%, inclusive. In one embodiment, the solution comprises 5% sucrose. In another embodiment, the solution comprises 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 the solution at a concentration of at least or equal to 20 mM. In another embodiment, the stabilizing agent is present in the solution at a concentration of at least or equal to 25 mM. In another embodiment, the stabilizing agent is present in the solution at a concentration of at least or equal to 50 mM. In another embodiment, the solution comprises 25 mM arginine.

In another embodiment, the solution comprises at least one or more buffering agents. Non-limiting examples of typical buffers that may be contained in the wash 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, M°PS (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 (e.g., L-histidine), serine (e.g. L-serine) and glycine (eg. L-glycine). In another embodiment, the solution contains two or more buffering agents. In a specific embodiment, the buffering agent is sodium phosphate.

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

In another embodiment, the solution comprises a carbohydrate filler at a concentration of 0.1 to 5%. In one embodiment, the carbohydrate filler is present in the solution at a concentration of at least or equal to 1.5%. In another embodiment, the carbohydrate filler is present in the 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 comprises a surfactant. Surfactants suitable for use in the compositions of the present invention include, but are not limited to, fatty acid esters (e.g., sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate), sorbitan trioleate, glycerol esters of fatty acids (eg, glycerol monocaprylate, glycerol monomyristate, glycerol monostearate), polyglycerol esters of fatty acids (eg, decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate), polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate), polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan tetrastearate, polyoxyethylene sorbitan tetraoleate), polyoxyethylene glycerol esters of fatty acids (e.g., polyoxyethylene glyceryl monostearate), polyethylene glycol esters of fatty acids (eg, polyethyleneglycol distearate), polyoxyethylene alkyl ethers (eg, polyoxyethylene lauryl ether), polyoxyethylene-polyoxypropylene alkyl ethers (eg, polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene nonylphenyl ether), polyoxyethylene hydrogenated castor oils (eg, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylene wax derivatives (eg, polyoxyethylene sorbitol wax), polyoxyethylene lanolin derivatives (eg, polyoxyethylene lanolin) and polyoxyethylene fatty acid amides (eg, polyoxyethylene stearic acid amide); C12-C18 alkyl sulfates (eg, 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 lauryl sulfosuccinate ester salt); as well as natural surfactants such as lecithin, glycerophospholipid, sphingophospholipids (e.g., sphingomyelin) and sucrose esters of C12-C18 fatty acids.

In one embodiment, the surfactant in the composition is a nonionic surfactant. In certain embodiments, the surfactant in the composition is a polyoxyethylene sorbitan fatty acid ester, such as polysorbate 20, 40, 60, 80, or a combination of one or more thereof. In one embodiment, the surfactant in the composition is polysorbate 80 (Tween 80). In another embodiment, the surfactant in the composition is polysorbate 60. In another embodiment, the surfactant in the composition is polysorbate 40. In another embodiment, the surfactant in the composition is polysorbate 20 (Tween 20). The concentration of the surfactant in the solution can 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 comprise the 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, 0.5 or more)%. In another embodiment, the aqueous solution contains no more than 0.2 (e.g., no 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)% of a pharmaceutically acceptable surfactant. In a particular embodiment, the surfactant is 0.05% polysorbate 80.

In another embodiment, the solution contains a preservative. Illustrative preservatives include, but are not limited to, 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 set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, at a concentration of 100 ± 20 (e.g., 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 (e.g., 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 (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; 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 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 comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, at a concentration of 100 ± 20 (e.g., 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 (e.g., 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 (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; 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 set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, at a concentration of 100 ± 20 (e.g., 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 (e.g., 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; and also 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 (e.g., 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 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 comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, at a concentration of 100 ± 20 (e.g., 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 (e.g., 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; and 0.05 ± 0.03 (e.g., 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 (e.g., 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 (e.g., sterile solution), wherein the solution contains (a) an antibody to C5 (e.g., 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 (e.g., sterile solution), wherein the solution contains (a) an antibody to C5 (e.g., ravulizumab) at a concentration of about 100 mg/mL, (b) about 50 mM phosphate buffer; (c) about 5% sucrose, and (d) about 25 mM arginine. In another embodiment, the stable aqueous solution comprises 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 comprises (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 comprises (a) an 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 comprises (a) an anti-C5 antibody at a concentration of about 100 mg/ml, (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 comprises (a) an 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 comprises (a) an 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 comprises (a) an anti-C5 antibody at a concentration of 100 mg/ml, (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 comprises (a) an anti-C5 antibody at a concentration of 100 mg/ml, (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) an anti-C5 antibody at a concentration of about 100 mg/ml, (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) an anti-C5 antibody 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 consists of (a) an anti-C5 antibody at a concentration of about 100 mg/mL, (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) an anti-C5 antibody at a concentration of 100 mg/mL, (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 comprises (a) an anti-C5 antibody at a concentration of about 100 mg/mL, wherein the anti-C5 antibody 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 comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, (b) about 50 mM phosphate buffer, (c) about 5% sucrose, and (d) about 25 mM arginine.

In another embodiment, the stable aqueous solution comprises (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 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 set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in 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 about 100 mg/mL, wherein the anti-C5 antibody 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 comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, (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 comprises (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 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 set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in 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 about 100 mg/mL, wherein the anti-C5 antibody 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 comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, (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) 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 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 set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in 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 particular 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 administration by a parenteral route (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 comprises an anti-C5 antibody at a concentration of 100 mg/mL and is formulated for subcutaneous administration. In another particular embodiment, the solution is formulated for intravenous administration. For example, in one embodiment, the stable aqueous solution comprises 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, the anti-C5 antibody (e.g., ravulizumab) remains at least 95% (eg, at least 96, 97, 98, or 99%) monomeric during storage at 2°C-8°C for at least six months when determined by SEC-HPLC (e.g., gel permeation HPLC). In another embodiment, the anti-C5 antibody is retained 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 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, 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 two years, as determined by SEC-HPLC.

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

In another embodiment of any of the solutions described herein, the 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 the anti-C5 antibody 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 reference anti-C5 antibody corresponding to the anti-C5 antibody before storage. In another embodiment, the 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 the anti-C5 antibody before storage. In another embodiment, the 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 the anti-C5 antibody 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 two years, compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody 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 three years, compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody before storage.

In another embodiment of any of the solutions described herein, the 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 the anti-C5 antibody before storage. In another embodiment of any of the solutions described herein, the 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 anti-C5 antibody corresponding to the anti-C5 antibody before storage. In another embodiment of any of the solutions described herein, the 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 the anti-C5 antibody before storage. In another embodiment of any of the solutions described herein, the 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 anti-C5 antibody corresponding to the anti-C5 antibody before storage. In another embodiment of any of the solutions described herein, the 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 the anti-C5 antibody before storage. In another embodiment of any of the solutions described herein, the 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 the anti-C5 antibody before storage.

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

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

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

iii) concentrating 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 for producing a stable concentrated antibody solution is provided, comprising 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, wherein the method comprises:

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

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

iii) concentrating 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 complement-associated condition comprising administering to the patient a stable aqueous solution (e.g. , subcutaneously or intravenously) described herein in an amount effective to treat the complement-associated condition. Illustrative conditions associated with the complement system include, but are not limited to, rheumatoid arthritis, antiphospholipid antibody syndrome, lupus nephritis, ischemia/reperfusion injury, atypical hemolytic uremic syndrome (aHUS), typical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria (PNH), dense deposit disease, neuromyelitis optica, multifocal motor neuropathy, multiple sclerosis, macular degeneration, HELLP syndrome, spontaneous abortion, thrombotic thrombocytopenic purpura, pauci-immune vasculitis, epidermolysis bullosa, recurrent miscarriage, traumatic brain injury, myocarditis, cerebrovascular disorder, peripheral vascular disorder, renovascular disorder, mesenteric/enteric vascular disorder, vasculitis, purpura nephritis Henoch-Schonlein vasculitis, lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis, immune complex vasculitis, Takayasu disease, dilated cardiomyopathy, diabetic angiopathy, Kawasaki disease, venous gas embolism, post-stent restenosis, rotational atherectomy, percutaneous transluminal coronary angioplasty, myasthenia gravis, cold agglutinin disease, dermatomyositis, paroxysmal cold hemoglobinuria, antiphospholipid syndrome, Graves' disease, atherosclerosis, Alzheimer's disease, septic systemic inflammatory response, septic shock, spinal cord injury, glomerulonephritis, transplant rejection, Hashimoto's thyroiditis, type 1 diabetes mellitus, psoriasis, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpasture's syndrome, Degos disease, and catastrophic antiphospholipid syndrome. In a specific embodiment, the complement-associated condition is atypical hemolytic uremic syndrome (aHUS). In another embodiment, the complement-associated condition is paroxysmal nocturnal hemoglobinuria (PNH).

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

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

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

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

Figure 3 shows the dynamic light scattering results for titration of ravulizumab (ALXN1210) at 50 mg/mL with phosphate buffered saline.

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

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

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) (T=3 weeks through T=2 months 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 the stability data for ravulizumab (ALXN1210) (T=1 month through T=2 months at 23-27°C).

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

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

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

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

Figure 14 shows size exclusion chromatography (SEC) stability data 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 histidine (T=0) samples.

Figure 16 shows dynamic light scattering stability data for ravulizumab AS samples 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 ravulizumab (ALXN1210) samples in phosphate buffer (T=2 months, 2-8°C).

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

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

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

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

Figure 23 shows the stability data for ravulizumab (ALXN1210) prototypes (T=3 months through T=6 months at 2-8°C).

Figure 24 shows the stability data for ravulizumab (ALXN1210) prototypes (T=9 months through T=12 months at 2-8°C).

Figure 25 shows the stability data for ravulizumab (ALXN1210) prototypes (T=1 month through T=2 months at 23-27°C).

Figure 26 shows the stability data for ravulizumab (ALXN1210) prototypes (T=3 months through T=6 months at 23-27°C).

Figure 27 shows the stability data for ravulizumab (ALXN1210) prototypes (T=9 months through T=12 months at 23-27°C).

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

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

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

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

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

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

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

Figure 35 shows the size exclusion chromatography (SEC) stability data of the assays as % monomers for ravulizumab (ALXN1210) (from T=0 to T=12 months inclusive at 37°C).

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

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

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

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

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

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

Figure 42 shows the stability data of the test samples obtained by size exclusion chromatography (SEC) as % monomers for ravulizumab (ALXN1210) in 4-5 freeze-thaw cycles at T=1 month, -20°C.

Figure 43 shows the stability data of the test samples obtained by size exclusion chromatography (SEC) as % monomers for ravulizumab (ALXN1210) in 1-3 freeze-thaw cycles at T=3 months, -80°C.

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

Figure 45 shows the stability data of the test samples obtained by size exclusion chromatography (SEC) as % monomers for ravulizumab (ALXN1210) in freeze-thaw cycles 1-5 at T=1 month, -20°C.

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

Figure 47 shows the overall design of a phase 1 study designed to evaluate the safety, tolerability, PK, PD, and immunogenicity of a single 400 mg dose of ravulizumab (ALXN1210) administered subcutaneously compared to a single 400 mg dose of ravulizumab (ALXN1210) administered 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 ALXN1210 serum concentrations plotted against nominal time using a linear scale.

Figure 50 is a graph showing individual ALXN1210 serum concentrations plotted against nominal time using a linear-logarithmic scale.

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

Figure 53 is a graph showing the mean (±SD) percent change in chicken red blood cell (cRBC) hemolysis from baseline over time for subjects administered 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 (e.g. , ravulizumab). The solutions can be used in a variety of therapeutic applications, such as methods for treating or preventing complement-related disorders. Exemplary solutions, formulations, therapeutic kits, and methods for preparing and using any of the above are disclosed below and illustrated in demonstrative examples and are in no way intended to be limiting.

I. Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and compositions similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and compositions are described herein.

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

The term "approximately", particularly in relation to a given quantity or number, is intended to cover variations of plus or minus ten percent (±10%) (e.g. , ±5%).

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

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

An "isotonic" formulation is one that has essentially the same osmotic pressure as human blood. Isotonic formulations will typically be characterized by an osmotic pressure of approximately 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 a freezing point osmometer. A "tonicity adjusting agent" is a compound that ensures the isotonicity of a formulation.

In the context of this document, the "osmolality" of a solution is the number of osmoles of 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 a solution property that depends solely on particle concentration. These properties include vapor pressure depression, freezing point depression, boiling point elevation, and osmotic pressure, and are collectively referred to as colligative properties.

A "sterile" composition is aseptic or contains no or virtually no living microorganisms or their spores.

A "stable" formulation, as used herein, is one in which the antibody substantially retains its physical stability and/or chemical stability and/or biological activity upon storage. Various assays 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 periods of time. For example, an increase in aggregate formation after storage is an indicator of instability of an aqueous anti-C5 antibody formulation. In addition to aggregate formation, retention of original clarity, color, and odor throughout the shelf life are indicators used to monitor the stability of the aqueous solutions of anti-C5 antibodies described herein.

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

The term "aggregation" refers to the assembly of natively 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 about 1%, and no more than about 0.5% aggregates by weight of protein when measured using 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 time is such that the antibody is considered to still retain its biological activity, as defined below. Chemical stability can be assessed by identifying and quantifying chemically altered forms of the antibody. Chemical changes can include size modification (e.g. , truncation), deamidation, racemization, hydrolysis, oxidation, beta-elimination, and disulfide exchange, which can be assessed using known techniques, such as 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 retained if the biological activity of the antibody in the pharmaceutical formulation is within approximately 30%, approximately 20%, or approximately 10% (within the assay error) of the biological activity exhibited at the time of preparation of the pharmaceutical dosage form (e.g., as determined in an antigen binding assay). As used herein, "biological activity" of a monoclonal antibody refers to the ability of the antibody to bind to an antigen. This may further include the binding of the antibody to the antigen and resulting 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 can be stored before degradation occurs. For example, the shelf life may be defined as the time it takes for 0.1%, 0.5%, 1%, 5%, or 10% of the product to degrade.

As used herein, the term "antibody" describes polypeptides comprising at least one antigen-binding region derived from an antibody (e.g. , a VH/VL region, or Fv, or CDR). Antibodies include known forms of antibodies. For example, an antibody can be a human antibody, a humanized antibody, a bispecific antibody, or a chimeric antibody. An antibody can also be a Fab, Fab'2, ScFv, SMIP, Affibody®, nanobody, or domain antibody. An antibody can also be 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 using protein engineering technology (e.g. , by mutation, deletion, substitution, conjugation with a non-antibody moiety). For example, an antibody may contain one or more amino acid variants (compared to a naturally occurring antibody) that alter a property (e.g. , a functional property) of the antibody. For example, numerous such variations are known in the art that affect, for example , the half-life, effector function, and/or immune responses to the 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,""selectivebinding,""selectivelybinds," and "specifically binds" refer to binding of an antibody to an epitope on a predetermined antigen but not to other antigens. Typically, the antibody (i) binds with 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, as determined, for example , by surface plasmon resonance (SPR) technology using a BIACORE® 2000 surface plasmon resonance instrument using a predetermined antigen, such as C5, as an analyte and the antibody as a ligand, or a Scatchard assay of the antibody binding to antigen-positive cells, and (ii) binds to the predetermined antigen with an affinity that is at least two-fold higher than 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 less than about 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M, or even lower.

The term "surface plasmon resonance" as used herein refers to an optical phenomenon that enables real-time analysis of biospecific interactions by detecting changes in protein concentrations in a biosensor matrix, such as those used with the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). 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. Recognit. 8:125-131; and Jonsson, B., et al. (1991) Anal. Biochem. 198:268-277.

The term "K _off " in the context of this document is intended to refer to the rate constant for dissociation 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 (e.g. , a human patient with a condition associated with the complement system).

The terms "treat," "treating," and "treatment" as used herein refer to the therapeutic measures described herein. Methods of "treating" involve administering to a subject the combination disclosed herein to cure, delay, reduce the severity, or ameliorate one or more symptoms of a disease or disorder or a recurrent disease or 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 a treatment that produces a beneficial effect, such as,a decrease in the intensity of at least one symptom of a disease or disorder. A beneficial effect may take the form of an improvement over baseline, such as An improvement compared to a measurement or observation made before the initiation of therapy according to 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 an agent that provides the desired biological, therapeutic, and/or prophylactic result. This result may include the alleviation, amelioration, temporary alleviation, 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 a biological system. In one example, an "effective amount" is an amount of a stable aqueous solution sufficient to alleviate at least one symptom of a disease or condition. The 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.

As used herein, the terms "maintenance" and "maintenance phase" are used interchangeably and refer to the second phase of treatment. In certain embodiments, treatment continues until clinical benefit is observed or until uncontrollable toxicity or disease progression occurs.

II. Antibodies to C5

The anti-C5 antibodies described herein bind to the C5 component of the complement system (e.g. , 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 (e.g. , 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 can be used. Antibodies that compete for binding to C5 with any of the antibodies known in the art can also be used.

An exemplary anti-C5 antibody is ravulizumab, which comprises heavy and light chains having the sequences shown in 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 U.S. Patent No. 9,079,949, the teachings of which are incorporated herein by reference. The terms ravulizumab, BNJ441, and ALXN1210 may be used interchangeably throughout this document. Ravulizumab selectively binds to the human complement protein C5, inhibiting its cleavage into C5a and C5b during complement activation. This inhibition prevents the release of the proinflammatory mediator C5a and the formation of the cytolytic pore-forming membrane attack complex (MAC) C5b-9 while preserving proximal or early components of the complement pathway (e.g. , C3 and C3b) essential for microbial opsonization and immune complex clearance.

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 set forth in SEQ ID NO: 12, and the CDR1, CDR2, and CDR3 domains of the ravulizumab VL region having the sequence set forth in SEQ ID NO: 8. In another embodiment, the antibody comprises the heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 19, 18, and 3, respectively, and the light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. In another embodiment, the antibody comprises VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 8, respectively.

Another exemplary anti-C5 antibody is BNJ421, which comprises heavy and light chains having the sequences shown in 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 U.S. Patent 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 VH region of BNJ421 having the sequence set forth in SEQ ID NO: 12, and the CDR1, CDR2, and CDR3 domains of the VL region of BNJ421 having the sequence set forth in SEQ ID NO: 8. In another embodiment, the antibody comprises the heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 19, 18, and 3, respectively, and the light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. In another embodiment, the antibody comprises VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 8, respectively.

The precise boundaries of the CDRs have been defined in various ways according to various methods. In some embodiments, the positions of the CDRs or framework regions in the variable domain of the light chain or heavy chain may 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 "Kabat CDRs" (e.g. , "Kabat LCDDR2" or "Kabat HCDR1"). In some embodiments, the positions of the CDRs of the variable region of the light or heavy chain may be determined according to Chothia et al. (1989) Nature 342 :877-883. Accordingly, these regions may be referred to as "Chothia CDRs" (e.g. , "Chothia LCDDR2" or "Chothia HCDR3"). In some embodiments, the CDR locations of the variable region of the light and heavy chains may be determined using the combined definition 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 Immun o 33(17/18) :1389-1401] illustrates the identification of CDR boundaries according to the definition of 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:

QVQLVQSGAEVKKPGASVKVSCKASG H IFSNYWIQWVRQAPGQGLEWMGEILPGSG H TEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSS (SEQ ID NO: 12).

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

DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIK (SEQ ID NO: 8).

The anti-C5 antibody described herein may, in some embodiments, comprise a human Fc constant region variant that binds to the human neonatal Fc receptor (FcRn) with greater affinity than the native human Fc constant region from which the human Fc constant region variant was derived. For example, the Fc constant region may comprise 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 human Fc constant region variant was derived. The substitutions may increase the binding affinity of the IgG antibody comprising the Fc constant region variant to FcRn at pH 6.0, while maintaining pH-dependency 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 dependence of the interaction) are known in the art and are illustrated in the demonstration examples. See, for example , PCT/US2015/019225 and U.S. Patent No. 9,079,949, the disclosures of each of which are incorporated herein by reference in their entireties.

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 Bio l Chem 281 : 23514-23524; (2) the M428L or T250Q/M428L substitutions described in Hinton et al. (2004) J Bio l Chem 279 :6213-6216 and in Hinton et al. (2006) J Immun l 176 :346-356; and (3) the N434A or T307/E380A/N434A substitutions described in Petkova et al. (2006) Int Immun o l 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 Bio o l 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 amino acid residue position 312 according to EU. 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 comprises no more than 30 (e.g. , no more than 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 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, a human Fc constant region variant comprises a methionine at position 428 and an asparagine at position 434, each indicated according to EU numbering. In some embodiments, a human Fc constant region variant comprises a 428L/434S double substitution, as described, for example , in U.S. Patent No. 8,088,376.

In some embodiments, the precise location of these mutations may be shifted relative to the position 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 discovered for ravulizumab (BNJ441) and described in U.S. Patent 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 a native human Fc constant region. In some embodiments, the substitution is selected from the group consisting of methionine in place of glycine at position 237; alanine in place of proline at position 238; lysine in place of serine at position 239; isoleucine in place of lysine at position 248; alanine, phenylalanine, isoleucine, methionine, glutamine, serine, valine, tryptophan, or tyrosine in place of threonine at position 250; phenylalanine, tryptophan, or tyrosine in place of methionine at position 252; threonine in place of serine at position 254; glutamic acid in place of arginine at position 255; aspartic acid, glutamic acid, or glutamine in place of threonine at position 256; alanine, glycine, isoleucine, leucine, methionine, asparagine, serine, 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 in place of threonine at position 307; alanine, phenylalanine, isoleucine, leucine, methionine, proline, glutamine or threonine in place of valine at position 308; alanine, aspartic acid, glutamic acid, proline or arginine in place of leucine or valine at position 309; alanine, histidine or isoleucine in place of glutamine at position 311; alanine or histidine in place of aspartic acid at position 312; lysine or arginine in place 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 in place of methionine at position 428; lysine in place 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 indicated according to EU numbering.

Suitable anti-C5 antibodies for use in the methods described herein, in some embodiments, comprise a polypeptide heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 14 and/or a polypeptide light chain comprising the amino acid sequence set forth in SEQ ID NO: 11. Alternatively, anti-C5 antibodies for use in the methods described herein, in some embodiments, comprise a polypeptide heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 20 and/or a polypeptide light chain comprising the amino acid sequence set forth in 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, 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 blotting, dot blotting, surface plasmon resonance (SPR) analysis (e.g. , the BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ), or enzyme-linked immunosorbent assay (ELISA). See, e.g. , 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 (e.g. , dissociation and association constants) are outlined in the demonstration examples.

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

In one embodiment, an anti-C5 antibody or antigen-binding fragment thereof blocks the formation or activity of active fragments C5a and/or C5b of the C5 protein (e.g. , human C5 protein). By virtue of this blocking effect, the antibodies inhibit, for example , the pro-inflammatory effects of C5a and the formation of the membrane attack complex (MAC) 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 cell-killing capacity of the complement system in the subject's body fluids. Such reductions in the cell-killing capacity of the complement system present in the body fluid(s) can be measured using methods well known in the art, such as, for example, using a conventional hemolysis assay, such as the hemolysis assay described by Kabat and Mayer (eds.), "Experimental Immunochemistry, 2^andEdition”, 135-240, Springfield, IL, CC Thomas (1961), pages 135-139, or a traditional version of such an assay, such as the method using hemolysis of chicken red blood cells described, for example, in Hillmen et al. (2004)N Engl J Med 350(6):552. Methods for determining whether a candidate compound inhibits the cleavage of human C5 into the 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 can also be used. Using these or other suitable types of assays, candidate agents capable of inhibiting the C5 component of the human complement system can be screened.

Immunological assays, 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, the formation of C5a is measured. In some embodiments, antibodies specific to 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 its antigen-binding fragment on complement activation. To determine the effect of an anti-C5 antibody or its antigen-binding fragment on classical pathway-mediated complement activation hemolysis in a test serum solution in vitro , for example, hemolysin-coated sheep red blood cells or anti-chicken red blood cell-sensitized chicken red blood cells are used as target cells. The percentage of lysis is normalized by considering 100% lysis to be equal to the lysis occurring in the absence of inhibitor. In some embodiments, the complement system is activated via the classical pathway by a human IgM antibody, such as that used in the Wieslab® Classical Pathway Complement Activation Kit (Wieslab® COMPL CP310, Euro-Diagnostica, Sweden). Briefly, the test serum is incubated with an anti-C5 antibody or its antigen-binding fragment 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 its antigen-binding fragment. In some embodiments, the test serum is C5-deficient serum reduced with a C5 polypeptide.

To determine the effect of an anti-C5 antibody or antigen-binding fragment thereof on alternative pathway-mediated hemolysis, unsensitized rabbit or guinea pig red blood cells 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 is normalized by considering 100% lysis to be equal to the lysis occurring in the absence of inhibitor. In some embodiments, the complement system is activated via the alternative pathway by lipopolysaccharide molecules, such as those used in the Wieslab® Alternative Pathway Complement Activation Kit (Wieslab® COMPL AP330, Euro-Diagnostica, Sweden). Briefly, the test serum is incubated with the 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 antibody to C5b-9 and a fluorogenic substrate and measuring the fluorescence at the appropriate wavelength. As a control, the test serum is incubated in the absence of the antibody to C5 or its antigen-binding fragment.

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 classical complement system in serum. This test is a lysis assay that utilizes antibody-sensitized red blood cells as a classical complement activator and various dilutions of test serum to determine the amount required to achieve 50% lysis (CH50). The percentage 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 assay is well known and commonly used by specialists in the field. Briefly, to activate the complement system via the classical pathway, undiluted serum samples (e.g., reconstituted human serum samples) are added to microplate wells containing antibody-sensitized red blood cells, thus producing TCC. The activated serum samples are then diluted in microplate wells coated with a capture reagent (e.g., (An antibody that binds to one or more components of TCC). TCC present in the activated samples binds to monoclonal antibodies coating the surface of the microplate wells. The wells are washed, and a detection reagent that has a detectable label and recognizes the bound TCC is added to each well. The detectable label can be, for example, fluorescent label or enzymatic label. The analysis results are expressed in units of CH50 equivalents per milliliter (CH50 equivalent units/mL).

Inhibition, for example, insofar as it relates to the activity of the terminal components of the complement system, includes at least 5% (e.g., at least 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60%) decrease in the activity of terminal components of the complement system, such as in a hemolysis assay or CH50eq assay compared to the effect of a control antibody (or its antigen-binding fragment) under similar conditions and at an equimolar concentration. Significant inhibition in the context of this document refers to inhibition of the specified activity (e.g., activity of 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, the anti-C5 antibody described herein comprises one or more amino acid substitutions compared to the CDRs 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 the hemolysis assay or the CH50eq assay.

The anti-C5 antibody described herein has a half-life in human serum 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, the anti-C5 antibody described herein has a half-life in human serum of at least 40 days. In another embodiment, the anti-C5 antibody described herein has a half-life in human serum of about 43 days. In another embodiment, the anti-C5 antibody described herein has a half-life in human serum of 39-48 days. Methods for measuring the serum half-life of an antibody 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., at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 400, 500%) exceeding the half-life of eculizumab in blood serum, for example, when measured in one of the mouse model systems described in the demonstration examples (e.g., in a model system using C5/NOD/SCID-deficient mice or hFcRn-transgenic mice).

In one embodiment, the antibody competes for binding to and/or binds to the same epitope on C5 as the antibodies described herein. The term "binds to the same epitope" with respect to two or more antibodies means that the antibodies bind to the same segment of amino acid residues as determined by the method. Methods for determining whether antibodies bind to the "same epitope on C5" as the antibodies described herein include, for example, epitope mapping methods such as X-ray crystal analysis of antigen:antibody complexes, which provide atomic resolution of the epitope, and hydrogen-deuterium exchange mass spectrometry (HDX-MS). Other methods monitor antibody binding to peptide fragments of the antigen or mutant versions of the antigen, where loss of binding due to modification of an amino acid residue within the antigen sequence is often considered indicative of an epitope component. In addition, computational combinatorics methods can also be used for epitope mapping. These methods rely on the ability of the antibody of interest to affinity-select specific short peptides from combinatorial phage-display peptide libraries. Antibodies with identical VH and VL sequences or identical CDR1, 2, and 3 sequences are expected to bind to the same epitope.

Antibodies that "compete with another antibody for target binding" refer to antibodies that inhibit (partially or completely) the binding of another antibody to the target. Whether two antibodies compete with each other for target binding, 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 the 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 incubated with the target first). Competing antibodies bind to the same epitope, an overlapping epitope, or adjacent epitopes (e.g., as evidenced by steric discrepancy).

The anti-C5 antibodies or antigen-binding fragments thereof described herein for use in the methods described herein can be produced using a variety of techniques known in the art. Monoclonal antibodies can be produced using a variety of techniques known to those skilled in the art. Briefly, spleen cells from an animal immunized with the desired antigen are immortalized, typically 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 arising from single immortalized cells are screened for the production of antibodies with 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 peritoneal cavity of a vertebrate host. Alternatively, DNA sequences encoding a monoclonal antibody or its binding fragment 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 solutions of antibodies to C5

This document provides stable aqueous solutions containing the antibody to C5 (e.g., ravulizumab). The aqueous solutions described herein may be sterile pharmaceutically pure compositions, such as for administration to a subject for the treatment or prevention of a complement-associated disorder, such as PNH or aHUS. The solutions described herein can be formulated according to standard methods. The preparation of pharmaceutical formulations is a well-developed 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) “Handbokoof Pharmaceutical Excipients American Pharmaceutical Association”, 3^rdEdition (ISBN: 091733096X). Suitable methods for preparing highly concentrated antibody solutions described in this document are illustrated in the demonstration examples.

The aqueous solutions described in this document contain 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 in this document as "highly concentrated antibody solutions." For the purposes of this document, a "high concentration" of an anti-C5 antibody (e.g., ravulizumab) in an 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 the solution at a concentration of greater than 100 (e.g., greater 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 the solution at a concentration of greater than 200 (e.g., greater 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 the solution at a concentration of greater than 300 mg/mL. In another embodiment, the antibody is present in the solution at a concentration of, for example, from 40 mg/ml to 200 mg/ml, from 50 mg/ml to 200 mg/ml, from 60 mg/ml to 200 mg/ml, from 70 mg/ml to 200 mg/ml, from 80 mg/ml to 200 mg/ml, from 90 mg/ml to 200 mg/ml, from 100 mg/ml to 200 mg/ml, from 110 mg/ml to 200 mg/ml, from 120 mg/ml to 200 mg/ml, from 130 mg/ml to 200 mg/ml, from 140 mg/ml to 200 mg/ml, from 150 mg/ml to 200 mg/ml, from 40 mg/ml to 100 mg/ml, from 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, 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/ml to 50 mg/ml, 40 mg/ml to 250 mg/ml, 50 mg/ml to 250 mg/ml, 60 mg/ml to 250 mg/ml, 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 to 250 mg/ml, 130 mg/ml to 250 mg/ml, 140 mg/ml to 250 mg/ml, 150 mg/ml to 250 mg/ml, from 160 mg/ml to 250 mg/ml, from 170 mg/ml to 250 mg/ml, from 180 mg/ml to 250 mg/ml, from 190 mg/ml to 250 mg/ml, from 200 mg/ml to 250 mg/ml, from more than 200 mg/ml (e.g., from at least 201 mg/ml) to 250 mg/ml, or from more than 200 mg/ml (e.g., from 201 mg/ml or more) to 300 mg/ml.

As described herein and illustrated in the demonstration examples, the disclosed aqueous solutions provide significant physical and chemical stability, as well as functional stability, to the anti-C5 antibody formulated herein. For example, the formulations described herein are capable of maintaining the structural integrity of the anti-C5 antibody (e.g., ravulizumab) present in solution at 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 for storage at temperatures below 0°C (e.g., at -20°C or -80°C). In another embodiment, the solution is formulated for storage for a period of up to three years (e.g., for 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 demonstration examples described herein, the solutions described herein are suitable for maintaining an anti-C5 antibody at about 100 mg/mL in a predominantly monomeric form for a period of up to two years at about 2°C-8°C. As used herein, an anti-C5 antibody formulated at a high concentration in the present aqueous solution is "predominantly monomeric" or is in a "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 greater) monomeric, such as when determined by size exclusion high performance liquid 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, the anti-C5 antibody (e.g., ravulizumab) remains at least 95% (eg, at least 96, 97, 98, or 99%) monomeric during storage at 2°C-8°C for at least six months when determined by SEC-HPLC (e.g., gel permeation HPLC). In another embodiment, the anti-C5 antibody is retained 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 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, 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 two years, as determined by SEC-HPLC.

In another embodiment, less than 5% (e.g., 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 the whole antibody that have a lower molecular weight than the whole antibody. Such forms of fragmentation include, but are not limited to, a free monomeric polypeptide heavy chain, a dimeric polypeptide heavy chain (e.g., a polypeptide heavy chain stabilized by disulfide bonds), a dimeric polypeptide heavy chain associated with a single polypeptide light chain, a monomeric polypeptide heavy chain associated with a single polypeptide light chain, or additional degradation product(s) or fragment(s) of a polypeptide light chain or heavy chain. In some embodiments, less than 2% (e.g., 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% of the 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% (e.g., 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1%) of the antibodies are fragmented 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. Methods for determining the amount of monomeric antibody, as well as the amount of oligomeric, aggregated or fragmented forms of antibody to C5, present in solution are described herein and illustrated in demonstration examples. For example, one skilled in the art can determine the percentage of intact, fragmented, unfolded intermediates, and/or aggregated antibody molecules present in a given solution using, for example, size exclusion high performance liquid chromatography (SEC-HPLC, such as gel permeation HPLC), static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding techniques, intrinsic tryptophan 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 (e.g. , ravulizumab) in the solution are aggregated when determined by SEC-HPLC (e.g. , gel permeation HPLC). In another embodiment, less than 4% of the anti-C5 antibodies in the solution are aggregated when determined by SEC-HPLC. In another embodiment, less than 3% of the anti-C5 antibodies in the solution are aggregated when determined by SEC-HPLC. In another embodiment, less than 2% of the anti-C5 antibodies in the solution are aggregated when determined by SEC-HPLC. In another embodiment, less than 1% of the anti-C5 antibodies in the solution are aggregated when determined by SEC-HPLC.

As described herein and illustrated in the demonstration examples, solutions containing the anti-C5 antibodies 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 (e.g., 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, the anti-C5 antibody (e.g., ravulizumab) present in the solution described herein may 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 in a present solution retains its activity are described herein and are known in the art, for example, a hemolysis assay using avian or porcine red blood cellsin vitroSuitable 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, the anti-C5 antibody (e.g., ravulizumab) retains at least 80% (eg, 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 the anti-C5 antibody before storage. In another embodiment, the anti-C5 antibody (e.g.,ravulizumab) retains at least 80% (eg,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 the anti-C5 antibody before storage. In another embodiment, the anti-C5 antibody (e.g.,ravulizumab) retains at least 80% (eg,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 the anti-C5 antibody before storage. In another embodiment, the anti-C5 antibody (e.g.,ravulizumab) retains at least 80% (eg,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 the anti-C5 antibody before storage. In another embodiment, the anti-C5 antibody (e.g.,ravulizumab) retains at least 80% (eg,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 the anti-C5 antibody before storage. In another embodiment, the anti-C5 antibody (e.g.,ravulizumab) retains at least 80% (eg,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 the anti-C5 antibody before storage.

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

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

In one embodiment, the aqueous solution comprises one or more buffering agents. As used herein, the term "buffering agent" refers to one or more components that, when added to an aqueous solution, are capable of protecting the solution from changes in pH upon the addition of an acid or base or upon dilution with a solvent. In one embodiment, the solution comprises at least one or more buffering agents. Non-limiting examples of typical buffers that can be included in the wash solution(s) include tris (tris(hydroxymethyl)methylamine), bis-tris, bis-tris-propane, histidine, triethanolamine, diethanolamine, formate, acetate, MES (2-(N-morpholine)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 (e.g., L-histidine), serine (e.g. L-serine) and glycine (eg. L-glycine). In another embodiment, the solution comprises two or more buffering agents. In a specific embodiment, the buffering agent is sodium phosphate. In one embodiment, the present solutions do not contain a free amino acid as a buffering agent. In another embodiment, the present solutions contain one free amino acid (e.g., histidine) as a buffering agent. In another embodiment, the present solutions may contain two or more (e.g., two, three, four, five, six, or seven or more) different amino acids as buffering agents, such as, serine and histidine.

The buffer concentration is sufficient to maintain the desired pH and may also vary, for example, to maintain isotonicity of the formulation. Typical concentrations of conventional buffering agents used in parenteral formulations can be found in: Pharmaceutical Dosage Form: Parenteral Medications, Volume 1, 2nd 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 comprises at least one buffering agent at a concentration of from 10 mM to 200 mM, inclusive. In another embodiment, the aqueous solution described herein comprises a buffering agent at a concentration of at least 10 (e.g., 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 comprises a buffering agent at a concentration of from about 10 mM to 50 mM, from 15 mM to 50 mM, from 20 mM to 50 mM, from 25 mM to 50 mM, from 30 mM to 50 mM, from 40 mM to 50 mM, from 10 mM to 100 mM, from 15 mM to 100 mM, from 20 mM to 100 mM, from 25 mM to 100 mM, from 30 mM to 100 mM, from 40 mM to 100 mM, from 10 mM to 150 mM, from 15 mM to 150 mM, from 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, 10 mM to 250 mM, 15 mM to 250 mM, 20 mM to 250 mM, 25 mM to 250 mM, from 30 mM to 250 mM, from 40 mM to 250 mM, from 50 mM to 250 mM, from 60 mM to 250 mM, from 70 mM to 250 mM, from 80 mM to 250 mM, from 90 mM to 250 mM, from 100 mM to 250 mM, from 150 mM to 250 mM, or from 200 mM to 250 mM. In another embodiment, the concentration of the buffer in the formulation is about 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 the solution at a concentration of at least or equal to 20 mM. In another embodiment, the buffering agent is present in the solution at a concentration of at least or equal to 25 mM. In another embodiment, the buffering agent is present in the solution at a concentration of at least or equal to 50 mM. In embodiments in which the present solution comprises two or more different buffering agents (e.g., at least two, three, four, five, six, seven, eight, nine or 10 or more), each of the two or more buffering means may be independently present, for example, in one of the concentrations described above.

In one embodiment, the aqueous solution has or can be adjusted to a neutral pH. As used herein, the term "neutral pH" means a pH of between 7 and 8, inclusive. Accordingly, as used herein, 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 a pH of 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 pH 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, or 7.1). That is, in some embodiments, the neutral pH may be, for example, at least a pH of 7 but less than a pH of 7.5. In some embodiments, the neutral pH may be between pH 7 and 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 between 7.0 and 7.4. In another embodiment, the pH of the solution is between 7.2 and 7.8. In another embodiment, the pH of the solution is between 7.2 and 7.6. In some embodiments, the neutral pH may be, for example, pH 7. One skilled in the art will also understand 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 approximately 7.35 to 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 from about 6.4 to about 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 particular embodiment, the pH of the solution is 7.4.

In one embodiment, the solution comprises one or more surfactants, such as an anionic, cationic, or nonionic surfactant. As used herein, the term "surfactant" refers to a surface-active molecule that contains 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, but are not limited to, fatty acid esters (e.g., sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate), sorbitan trioleate, glycerol esters of fatty acids (eg, glycerol monocaprylate, glycerol monomyristate, glycerol monostearate), polyglycerol esters of fatty acids (eg, decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate), polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate), polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan tetrastearate, polyoxyethylene sorbitan tetraoleate), polyoxyethylene glycerol esters of fatty acids (e.g., polyoxyethylene glyceryl monostearate), polyethylene glycol esters of fatty acids (eg, polyethyleneglycol distearate), polyoxyethylene alkyl ethers (eg, polyoxyethylene lauryl ether), polyoxyethylene-polyoxypropylene alkyl ethers (eg, polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene nonylphenyl ether), polyoxyethylene hydrogenated castor oils (eg, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylene wax derivatives (eg, polyoxyethylene sorbitol wax), polyoxyethylene lanolin derivatives (eg, polyoxyethylene lanolin) and polyoxyethylene fatty acid amides (eg, polyoxyethylene stearic acid amide); C12-C18 alkyl sulfates (eg, 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 lauryl sulfosuccinate ester salt); as well as natural surfactants such as lecithin, glycerophospholipid, sphingophospholipids (e.g., sphingomyelin) and sucrose esters of C12-C18 fatty acids.

In one embodiment, the surfactant in the composition is a nonionic surfactant. In certain embodiments, the surfactant in the composition is a polyoxyethylene sorbitan fatty acid ester, such as polysorbate 20, 40, 60, 80, or a combination of one or more thereof. In one embodiment, the surfactant in the composition is polysorbate 80 (Tween 80). In another embodiment, the surfactant in the composition is polysorbate 60. In another embodiment, the surfactant in the composition is polysorbate 40. In another embodiment, the surfactant in the composition 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 particle formation in the formulation. For example, the surfactant may be present in the formulation 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 comprise the 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 (e.g., no 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)% of a pharmaceutically acceptable surfactant.

In another embodiment, the compositions comprise 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% (weight/volume). In certain embodiments, the composition comprises 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% (weight/volume). In certain embodiments, the surfactant is present in the composition in an amount of 0.02% or about 0.04% (weight/volume). In one embodiment, the surfactant is present in the composition in an amount of 0.05% (w/v).

In one embodiment, the formulation comprises 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 particular embodiment, the formulation comprises polysorbate 80 at a concentration of from about 0.01% to about 0.5%, from about 0.01% to about 0.3%, from about 0.001% to about 0.2%, from about 0.02% to about 0.5%, from about 0.02% to about 0.3%, from about 0.02% to about 0.2%, from about 0.05% to about 0.5%, from about 0.05% to about 0.3%, from 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 about 0.2%. In a further embodiment, the composition comprises 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 comprises about 0.05% polysorbate 80. In one embodiment, the composition comprises about 0.04% polysorbate 80. In one embodiment, the composition comprises about 0.03% polysorbate 80. In one embodiment, the composition comprises about 0.02% polysorbate 80. In one embodiment, the composition comprises about 0.01% polysorbate 80.

In one embodiment, the aqueous solution comprises one or more salts, such as sodium chloride, potassium chloride, or magnesium chloride. In some embodiments, the aqueous solution described herein comprises a salt at a concentration of at least 10 (e.g., 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 some embodiments, the aqueous solution described herein may comprise a salt at a concentration of less than or about 200 (e.g., less than or about 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, or 10) mM. In some embodiments, the aqueous solution described herein may comprise 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, 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, 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, 100 mM to 250 mM, 150 mM to 250 mM, or 200 mM to 250 mM. In embodiments in which the present solution comprises 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, in 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, but are not limited to, 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 comprises two or more different carbohydrate excipients (e.g., at least two, three, four, five, six, seven, eight, nine or 10 or more), each filler may independently be present in any of the above-described concentrations.

In another embodiment, the stable aqueous solution contains one or more stabilizing agents. Illustrative stabilizers include, but are not limited to, polyols, sugars (e.g., sucrose or trehalose), amino acids (eg, arginine), amines and salting-out salts. In one embodiment, the solution comprises at least one stabilizing agent at a concentration of 2-10%, inclusive. In one embodiment, the solution comprises 5% sucrose. In another embodiment, the solution comprises 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 the solution at a concentration of at least or equal to 20 mM. In another embodiment, the stabilizing agent is present in the solution at a concentration of at least or equal to 25 mM. In another embodiment, the stabilizing agent is present in the solution at a concentration of at least or equal to 50 mM. In another embodiment, the solution comprises 25 mM arginine.

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

As used herein, the term "preservative" refers to an agent that reduces bacterial growth and may optionally be added to the formulations described herein. The addition of a preservative may, for example, facilitate the preparation of a multiple-dose formulation. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyl dimethyl ammonium 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 comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, at a concentration of 100 ± 20 (e.g., 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 (e.g., 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 (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; 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 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 comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, at a concentration of 100 ± 20 (e.g., 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 (e.g., 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 (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; 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 set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, at a concentration of 100 ± 20 (e.g., 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 (e.g., 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; and also 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 (e.g., 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 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 comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, at a concentration of 100 ± 20 (e.g., 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 (e.g., 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; and 0.05 ± 0.03 (e.g., 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 (e.g., 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 (e.g., sterile solution), wherein the solution contains (a) an antibody to C5 (e.g., ravulizumab) at a concentration of about 100 mg/mL, (b) about 50 mM phosphate buffer; (c) about 5% sucrose, and (d) about 25 mM arginine. In another embodiment, the stable aqueous solution comprises (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 comprises (a) an anti-C5 antibody at a concentration of about 100 mg/mL, (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 comprises (a) an anti-C5 antibody at a concentration of 100 mg/mL, (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) an anti-C5 antibody at a concentration of about 100 mg/ml, (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) an anti-C5 antibody 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 consists of (a) an anti-C5 antibody at a concentration of about 100 mg/mL, (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) an anti-C5 antibody at a concentration of 100 mg/mL, (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 comprises (a) an anti-C5 antibody at a concentration of about 100 mg/mL, wherein the anti-C5 antibody 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 comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, (b) about 50 mM phosphate buffer, (c) about 5% sucrose, and (d) about 25 mM arginine.

In another embodiment, the stable aqueous solution comprises (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 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 set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in 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 about 100 mg/mL, wherein the anti-C5 antibody 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 comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, (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 comprises (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 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 set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in 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 about 100 mg/mL, wherein the anti-C5 antibody 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 comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, (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) 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 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 set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in 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 in this document are methods for producing a highly concentrated solution of an antibody to C5. In one embodiment, methods are provided for producing a stable concentrated solution of an antibody comprising an antibody to C5 at a concentration of 100 mg/ml, 50 mM phosphate buffer, 5% sucrose, and 25 mM arginine, wherein the method comprises:

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

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

iii) concentrating 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 for producing a stable concentrated antibody solution is provided, comprising 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, wherein the method comprises:

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

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

iii) concentrating 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 in this document can be administered to a patient by a number of routes, which depend in part on the route of administration. The route of administration may be parenteral, such as intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, intraocular injection, intra-articular injection, or intramuscular injection (IM). The terms "parenteral administration," "administered parenterally," and other grammatically equivalent phrases, as used herein, refer to routes of administration other than enteral and local administration, typically by injection, and include, without limitation, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, 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 accomplished using a device. The device may be a syringe, prefilled syringe, disposable or reusable autoinjector, pen, patch injector, body injector, ambulatory syringe infusion pump with a subcutaneous infusion set, or other device.

In one embodiment, the solution described herein is delivered to the subject via topical administration. As used herein, the term "topical administration" or "local delivery" refers to delivery that does not rely on the transport of the composition or active agent (e.g., antibodies to C5) to their intended target tissue or site through the vascular system. After local administration near the target tissue or site, the solution or one or more of its components can diffuse into the intended target tissue or site.

For example, the solution may be delivered by injection or by implanting a device containing the solution. The implant may be made of a porous, nonporous, or gel-like material, including membranes such as silastic membranes or fibers. The implant may be configured to release the solution into the subject's body in a sustained or intermittent manner. See, for example, U.S. Patent Application Publication No. 20080241223; U.S. Patent Nos. 5,501,856; 4,863,457; and 3,710,795; EP488401; and EP430539, the disclosures of each of which are incorporated herein by reference in their entireties. The solution described herein may be delivered to a subject via an implantable device based on, for example, diffusion, erodibility, or convection systems, such as osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmotic systems, vapor pressure pumps, electrolytic pumps, blowing agent pumps, piezoelectric pumps, erodibility systems, or electromechanical systems.

In one embodiment, the solution described herein can be topically administered to a joint (e.g., a hinge joint). For example, in embodiments where the disorder is arthritis, a therapeutically suitable solution can be administered directly to the joint (e.g., to the joint space) or near the joint. Examples of joint joints into which the composition described herein can be topically administered include, for example, the hip, knee, elbow, wrist, sternoclavicular, temporomandibular, carpal, tarsal, ankle, and any other joint susceptible to arthritic conditions. The composition described herein can also be administered to a bursa, such as, for example, the acromioclavicular, biceps-radialis, ulna-radialis, deltoid, infrapatellar, sciatic, and any other bursa known in the medical art.

In another embodiment, the solution described herein can be administered topically to the eye. As used herein, the term "eye" refers to all anatomical tissues and structures associated with the eye. In one embodiment, the solution described herein is administered into the posterior chamber of the eye. In another embodiment, the solution described herein is administered into the vitreous humor. In another embodiment, the solution described herein is administered through the sclera.

In some embodiments, for example, In embodiments for treating or preventing a disorder such as COPD or asthma, the solution described herein can be administered to a subject via the lung. Pulmonary delivery of the drug can be by inhalation, and administration by inhalation herein can 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 liquefied aerosol or mist. The inhaler can be, for example, a jet inhaler (e.g., an air-jet or liquid-jet inhaler) or an ultrasonic inhaler. Additional devices and methods for intrapulmonary administration are described, for example, in U.S. Patent Application Publication Nos. 20050271660 and 20090110679, the disclosures of each of which are incorporated herein by reference in their entireties.

In other embodiments, the solutions described herein are present in a single-dose dosage form that 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, such as the dispensing device described in U.S. Patent No. 6,302,855, may also be used. The injection system may comprise a delivery pen as described in U.S. Patent No. 5,308,341. Pens, which are most widely used for self-delivery of insulin by patients with diabetes mellitus, are well known in the art. Such devices may comprise at least one injection needle (e.g., 31 gauge needles (approximately 5-8 mm in length) are typically pre-filled with one or more single therapeutic doses of solution and are suitable for rapidly delivering the solution to the subject while causing the least possible pain.

VI. Treatment methods

The disclosed 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 complement-associated disorder, including but not limited to rheumatoid arthritis (RA); antiphospholipid antibody syndrome; lupus nephritis; ischemia/reperfusion injury; atypical hemolytic uremic syndrome (aHUS), typical or infectious hemolytic uremic syndrome (tHUS); dense deposit disease (DDD); paroxysmal nocturnal hemoglobinuria (PNH); neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); macular degeneration (e.g., age-related macular degeneration (AMD)); hemolysis, elevated liver enzymes, and thrombocytopenia syndrome (HELLP); thrombotic thrombocytopenic purpura (TTP); spontaneous abortion; pauci-immune vasculitis; epidermolysis bullosa; recurrent miscarriage; and traumatic brain injury (see, for example, Holers (2008),Immunological Reviews 223:300-316, and Holers and Thurman (2004) Molecular immunityology 41:147-152).

In another embodiment, the complement system-associated disorder is a complement system-associated vascular disorder, such as, but not limited to, a diabetes mellitus-associated vascular disorder (e.g., in the eye), central retinal vein occlusion, cardiovascular disorder, myocarditis, cerebrovascular disorder, peripheral disorder (eg, musculoskeletal) vessels, renovascular disorder, mesenteric/enteric vascular disorder, revascularization of grafts and/or replants, vasculitis, nephritis in Schönlein-Henoch purpura, vasculitis associated with systemic lupus erythematosus, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu's disease, dilated cardiomyopathy, diabetic angiopathy, Kawasaki disease (arteritis), venous gas embolism (VGE) and restenosis after stenting, rotational atherectomy and percutaneous transluminal coronary angioplasty (PTCA) (see, e.g., (US 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, burn (eg, severe burn), septic systemic inflammatory response, septic shock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis, type 1 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), such as TMA associated with a complement-associated disorder, such as any of the complement-associated disorders described herein.

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

In another embodiment, the solution described herein is administered to a subject to treat, prevent, or reduce the intensity of at least one symptom of an inflammatory response associated with the complement system (e.g., aspect of a complement-associated disorder that is a complement-associated inflammatory response) in a subject. For example, the composition can be used to treat, prevent, and/or ameliorate one or more symptoms associated with a complement-associated inflammatory response, such as tissue rejection/graft-versus-host disease (GVHD), reperfusion injury (e.g., following cardiopulmonary bypass or tissue transplantation), and tissue damage following other forms of traumatic injury, such as a burn (e.g., a severe burn), blunt trauma, spinal cord injury, or frostbite. See, e.g., 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-mediated disorder is a complement-mediated vascular disorder such as, but not limited to, a cardiovascular disorder, myocarditis, cerebrovascular disorder, a peripheral (e.g., musculoskeletal) vessels, renovascular disorder, mesenteric/enteric vascular disorder, revascularization of grafts and/or replants, vasculitis, nephritis in Henoch-Schonlein purpura, vasculitis associated with systemic lupus erythematosus, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, organ or tissue transplantation, 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, e.g., (US Patent Application Publication No. 20070172483).

VII. Types of combination 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 (e.g., which are suitable for the treatment of disorders associated with the complement system). For example, combination therapy may involve administering one or more additional agents to a human patient (e.g., anticoagulants, antihypertensive agents, or anti-inflammatory drugs (eg, steroids) that 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 antibody agents 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).

Adjunctive therapies for the treatment of a complement-related disorder in a subject will vary depending on the specific disorder being treated, but may include, without limitation, one or more antihypertensive agents (e.g., angiotensin-converting enzyme inhibitor, labetalol, hydralazine, nifedipine, calcium channel blockers, 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, IgE antibodies), corticosteroids, or nonsteroidal anti-inflammatory drugs (NSAIDs). Many different NSAIDS are available, some of which are available 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 replacement, 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 before the administration of one or more additional agents and/or therapies. In another embodiment, the solution is administered after the administration of one or more additional agents and/or therapies.

If the antibody solution described in this document is used in combination with a second active agent, the agents (e.g., (Anti-C5 antibody and a second agent) can be formulated separately or together. For example, the solution and the 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., patient with a complement-associated disorder). Kits may also optionally contain instructions, such as contain administration schedules that allow the practitioner (e.g., (a physician, nurse or patient) to administer the composition contained therein for the administration of the solution to the patient.

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

Kits optionally contain multiple containers of single-dose solution, each containing an effective amount of solution for a single administration. Devices or equipment necessary for administering the solution may also be included in the kits. For example, a kit may contain one or more prefilled syringes containing the solution.

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

The contents of all literature references, Genbank records, 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 conducted early in the formulation development process to generate candidate compound screening data and to evaluate the reduction in opalescence at higher ALXN1210 concentrations. The initial ALXN1210 formulation (10 mM phosphate, 150 mM sodium chloride, pH 7.0, 0.02% Tween 80, at 10 mg/mL) was colorless and slightly opalescent. The opalescence also increased as the ALXN1210 concentration increased. Based on the candidate compound screening results, a stability study was conducted to generate stability data for the lead compounds. Following the initial stability study, stability studies were conducted on prototypes to develop the optimal bulk drug substance and drug product formulation. Preliminary and stability studies are discussed in detail below.

1. Methods

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, the human complement protein C5. A PolySorp microtiter plate was coated with human C5 protein and blocked with bovine serum albumin (BSA). A calibration curve was generated using ALXN1210 reference material. The reference material and test samples were prepared at 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 to horseradish peroxidase (HRP). The plate was washed again and then developed using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate. The amount of reacted substrate was read spectrophotometrically in 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 fit was applied to the calibration curve, and the results for the reference material and test samples were interpolated from the curve. The sample results were compared with the results for the reference material, and the relative activity (%) was recorded.

Density

Density measurements were performed using a DMA 4500 density meter, using the U-tube method. A hollow U-shaped glass tube was filled with the sample and then excited to the lowest possible amplitude. Density was determined using the following equation:

ρ=A( ^τ2 )-B

ρ=density

τ=period of oscillation

A and B are constants of the measuring instrument, determined by calibrating the instrument using two substances of known density.

Differential scanning fluorimetry

Differential scanning fluorimetry measures changes in thermal stability by generating a thermal denaturation curve in the presence of a fluorescent dye, such as Sypro orange. As the protein unfolds, exposed hydrophobic surfaces bind to the dye, increasing fluorescence and producing a stability curve with a characteristic midpoint value at the temperature of exposure of the hydrophobic surfaces, T _h .

Dynamic light scattering

Dynamic light scattering is used to measure the size and interactions of proteins, nanoparticles, and other macromolecules in situ in microwell plates. An illumination system enables visualization of the wells in the microwell plate using a built-in 3-megapixel camera. Light scattering deviations due to Brownian motion yield a diffusion coefficient, which is related to the hydrodynamic radius of the particles present in the solution.

Gel permeation HPLC

Gel permeation (size-exclusion) HPLC was used to separate monomeric IgG from larger multimeric antibody molecules that may form through monomer aggregation. Test samples were injected 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. Higher-mass multimers were identified by observing peaks eluting before the monomer peak.

Visualized capillary electrophoresis (iCE)

This method utilized the Protein Simple iCE280 or iCE3 system, which provides a pure IEF solution in a capillary column, and protein clustering was detected using a full-column UV detector. Samples were prepared by premixing ALXN1210, carrier ampholytes, and pI markers. The sample was loaded into a capillary electrophoresis cartridge, and electrolyzers at each end of the capillary were filled with acid and base. Voltage was applied, and the analytes clustered at their pIs. A CCD camera visualized the UV absorption of the entire capillary column every 30 seconds, enabling real-time monitoring of the clustering process. The resulting separation pattern was recorded and analyzed. The pIs of proteins present in the sample were interpolated based on the positions of the pI markers added to the sample.

Lab on a Chip (L o C)

This method was used to test the homogeneity and purity of the product. Unreduced samples were denatured with lithium dodecyl sulfate (LDS). Reduced samples were denatured with lithium dodecyl sulfate (LDS), and disulfide bonds were broken with dithiothreitol (DTT). Polypeptide chains were mixed with a fluorescent dye that binds to LDS and separated according to molecular size using microcapillary electrophoresis. Protein was detected and quantified using laser-induced fluorescence.

Osmolality

The osmolality of the samples was determined using an osmometer measuring freezing point depression. The osmometer was calibrated before use using commercially available certified osmolality standards at 50 mOsm/kg and 850 mOsm/kg, which cover the sample range. A standard solution of 290 mOsm/kg was used to confirm successful calibration before testing the samples. Samples were tested in triplicate, and the average value determined for each sample was recorded.

pH

pH measurements were performed using a protein-resistant, silver-free, saturated KCl combination electrode and an appropriate measuring instrument and temperature control device. Before use, the measuring instrument was calibrated using commercially available solutions in the appropriate pH range (i.e., pH 4.0 - pH 7.0).

Determination of protein concentration using S o l o VPE

To determine protein concentrations in test samples, absorbance at 280 nm was used using a technique with different optical path lengths and a theoretically determined extinction coefficient of 1.479. In this method, absorption values were determined in triplicate for each sample.

Viscosity

Viscosity measurements were determined using an AMVn viscometer using the falling ball method. A hollow tube was filled with the 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 viscosity using the following equation:

η=K*(ρb-ρs)*tr

η=dynamic viscosity (mPa*s)

K=proportionality constant

ρb = ball density (g/ml)

ρs=sample density (g/ml)

tr=time of ball fall

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

Detecting invisible particles using microflow imaging (MGI)

The goal was to quantify all particles invisible to the naked eye in the formulation using microflow imaging (MFI). A sample was collected from storage at 2-8°C and directly tested by MFI using the BOT1 autosampler. The sample was inverted six times before loading into the BOT1 to ensure complete mixing of the particles. Samples were loaded into three consecutive wells, and one measurement was performed for each well, yielding a total of three replicates. Three mixing cycles were performed in the BOT1 to further ensure mixing homogeneity.

2. Development of the composition

Figures 1-5 and Tables 1-5 show the results of experiments conducted in the early stages of development of high-concentration 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 a charge to ensure charge repulsion between antibody molecules in solution at high concentrations. A series of experiments, described below, were conducted to ensure the optimal choice of a specific 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) reduced the degree of opalescence in a sample containing 50 mg/mL ALXN1210 in sodium phosphate buffer. This conclusion was also reached through visual inspection of the vials (data not shown).

In addition, subsequent experiments were performed using the ALXN1210 IV formulation at 10 mg/mL and by concentrating the antibody and performing buffer exchange to evaluate different starting buffer systems for use in finding the highly concentrated ALXN1210 formulation. As shown in Table 1 below, all pooled samples underwent a final buffer exchange at 1:1000 to achieve the desired pH. The pooled samples ranged in concentration from 35.3 to 54.0 mg/mL. The % recoveries after buffer exchange ranged from 70.6% to 108%. Appearance analysis results 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. The visualization capillary electrophoresis (iCE) results show that the pI is in the range of 5.98 to 6.54, the major pI is in the range of 6.19 to 6.24, and the peak area % is in the range of 63.1% to 65.9%. The size exclusion chromatography (SEC) results show that the monomer % (purity) is in the range of 98.48% to 98.98%.

Table 1. ALXN1210 with buffer exchange, 10 mg/mL to 50 mg/mL (pooled sample groups 1-3) Buffer pH buffer 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 from salt titration for histidine buffer exchanged samples at pH 7 using DLS indicate that with increasing salt concentration, the self-association of ALXN1210 also increases.

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

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

As shown in Figure 4, the results obtained for the DSF-based buffer exchange samples indicate that the hydrophobic pockets of ALXN1210 are not accessible. Citrate and acetate buffers at pH 5 and 6 exhibit low 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 appearance analysis results shown in Table 2, 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-Composition C with added L-arginine 25 mM phosphate, 25 mM L-arginine (added), 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 addition, obtained using DLS, indicate that adding 25 mM L-arginine to a sample at least 100 mg/mL provides a result most closely comparable to the standard solution at 10 mg/mL. In a sample with at least 100 mg/mL without added L-arginine, higher-order molecules are formed, indicating self-association.

The osmolality analysis results presented in Table 3 show that ALXN1210 in 25 mM histidine, pH 7.2, with 8% sucrose or 4.5% sorbitol are 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 The buffer used 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-Composition C with the addition of L-arginine 25 mM phosphate, 25 mM L-arginine, pH 7 112.0 69 SPPD-14-0042-Composition C with the addition of L-arginine 25 mM phosphate, 25 mM L-arginine, pH 7 112.0 7% sucrose 302 SPPD-14-0042-Composition C with the addition of L-arginine 25 mM phosphate, 25 mM L-arginine, pH 7 112.0 4% sorbitol 317

The viscosity analysis results presented in Table 4 show that increasing the ALXN1210 concentration also increases the viscosity of ALXN1210 solutions in histidine and phosphate buffers. The density analysis results 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 different 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 100 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 added) 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 added) 80.0 2,877 1,038

As shown in Table 5, the addition of L-arginine in its base form significantly increased the pH of the sample. L-arginine in sufficient quantity with monobasic sodium phosphate added to the sample increased the pH by 1 pH unit. L-arginine HCl added to the sample decreased the pH by 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 monobasic sodium phosphate and to the addition of L-arginine in its base form.

Table 5. Effects of adding 25 mM L-arginine to L-arginine buffers on pH and appearance for ALXN1210 Sample Description pH Appearance 0.5 M solution of L-arginine in the form of a base 11.47 1210-PD-14-0042-composition C 7.04 SPPD-14-0042-Composition C with the addition of 25 mM L-arginine 8.85 0.5 M monobasic sodium phosphate solution 4.23 0.5 M solution of dibasic sodium phosphate 8.94 0.5 M solution of L-arginine in the form of a base in sufficient quantity with 0.5 M solution of monobasic sodium phosphate 8.31 SPPD-14-0042-composition C with the addition of 25 mM L-arginine solution in sufficient quantity with a solution of monobasic sodium phosphate 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) with the addition of 25 mM L-arginine⋅HCl 7.07 Highly opalescent Bulk 1210-75-25P-7.0-3So (SPAS-14-007), 25 mM L-arginine in sufficient quantity with sodium monobasic phosphate 7.55 Moderately opalescent Bulk 1210-75-25P-7.0-3So (SPAS-14-007), 25 mM L-Arginine Base Form 9.17 Slightly opalescent

3. Storage stability

Figures 6–21 show the results of the initial stability study. These results indicate that histidine-containing formulations are the least stable, and phosphate-containing formulations are the most stable after 2 months at 2–8°C, 23–27°C, and 37°C. Also, as evidenced by size exclusion chromatography results, phosphate-containing formulations with sorbitol and sucrose were comparable after 2 months at 2–8°C and 23–27°C. However, sorbitol-containing formulations were slightly more stable at 37°C compared to sucrose-containing formulations after 2 months. Dynamic light scattering results indicate no significant change in samples in phosphate buffer with 25 mM L-arginine after 2 months at 2–8°C with the addition of either sucrose or sorbitol. Dynamic light scattering results for histidine-containing samples at T=2 months may not overlap due to high polydispersity between data collection sessions, indicating a less stable composition compared to phosphate-containing samples. Results from a 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 stability study of the test samples.

These results show that all phosphate-containing formulations 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 bulk drug substance-containing formulations at 100 mg/mL were stable and showed no significant change after 5 freeze-thaw cycles at -20°C and -80°C.

5. Interim conclusions before short-term destruction testing

Based on the results of these studies, the optimal formulation for the highly concentrated ALXN1210 material was determined. Preliminary experiments suggested adding L-arginine to reduce opalescence for ALXN1210 at 100 mg/mL. An initial stability study led to the selection of a lead formulation with a phosphate buffer and L-arginine at > 50 mg/mL. Based on the stability studies of the test samples, the initial optimal formulation 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

To evaluate the suitability of the original optimal formulation of ALXN1210 at a concentration of 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 80 (PS80) or another surfactant was necessary to prevent degradation. Two grades of PS 80 were 0.05% (w/v) POLYSORBATE 80 (HX2)™ from NOF America Corporation, which is 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 fatty acid mixture containing oleic acid and palmitic acid. Both products are often referred to as TWEEN 80® and are non-ionic surfactants derived from polyethoxylated sorbitan and oleic acid with hydrophilic groups derived from ethylene oxide polymers.

Test methods used to evaluate the potential use of PS80 Avantor J.T. Baker 4117 in ALXN1210 at 100 mg/mL, are listed below in Table 6. See the individual testing method for further details and a description of the method.

Table 6. Testing methods for determining potential applications of PS80 Avantor J.T. Baker 4117 Testing Type of analysis Purpose and rationale Appearance General characteristics A change in appearance may indicate product degradation. Protein concentration Protein concentration Changes in protein concentration assay results may indicate changes in product solubility or stability. Turbidity (UV-Visible Spectroscopy) General characteristics An increase in turbidity may indicate changes in the solubility or stability of the product. SE UPLC Purity To ensure the integrity of the sample and its compliance with purity requirements. HIAC in small 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 the physical properties of the product profile that may impact safety. CE-SDS Purity A change in the CE-SDS pattern may indicate product degradation, such as cleavage of the polypeptide chain(s) or aggregation of the product. iCE Identity An unsatisfactory result may indicate protein degradation with the formation of uncharacteristic isoforms. Dynamic Light Scattering (DLS) Stability To assess the degree of aggregation/destruction. Polysorbate 80 content General characteristics To quantify the concentration of polysorbate 80 in the composition. A decrease in the concentration of polysorbate 80 may lead to antibody aggregation. Destruction of polysorbate 80 General characteristics To quantify the degree of polysorbate 80 degradation in the formulation. Increased polysorbate 80 degradation may lead to antibody aggregation.

Vials containing 100 mg/mL ALXN1210 with either PS80 Avantor J.T. Baker 4117 or PS80 HX2 NOF were visually inspected. No visible particles or obvious color changes were observed for all samples, which were subjected to degradation by storage at 45°C and additional shaking 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 cm3 vials containing PS80 Avantor J.T. Baker 4117 or PS80 HX2 NOF Sample ID Transparency Color Intermediate particle content Final particle content 100 mg/ml ALXN1210+Avantor, T=0 Transparent Slightly yellowish color Virtually particle free Virtually particle free 100 mg/ml ALXN1210+NOF, T=0 Transparent Slightly yellowish color Virtually particle free Virtually particle free 100 mg/ml ALXN1210+Avantor, T=7 days at 45°C Transparent Slightly yellowish color Virtually particle free Virtually particle free 100 mg/ml ALXN1210+NOF, T=7 days at 45°C Transparent Slightly yellowish color Virtually particle free Virtually particle free 100 mg/ml ALXN1210, Avantor, T=7 days at 45°C+5 days with shaking at 2-8°C Transparent Slightly yellowish color Virtually particle free Virtually particle free 100 mg/ml ALXN1210, NOF, T=7 days at 45°C+5 days with shaking at 2-8°C Transparent Slightly yellowish color Virtually particle free Virtually particle free 100 mg/ml ALXN1210+Avantor, T=4 days at 45°C Transparent Slightly yellowish color Virtually particle free Virtually particle free 100 mg/ml ALXN1210+NOF, T=14 days at 45°C Transparent Slightly yellowish color Virtually particle free Virtually particle free 100 mg/ml ALXN1210, Avantor, T=14 days at 45°C+5 days with shaking at 2-8°C Transparent Slightly yellowish color Virtually particle free Virtually particle free 100 mg/ml ALXN1210, NOF, T=14 days at 45°C+5 days with shaking at 5°C Transparent Slightly yellowish color Virtually particle free Virtually particle free

A slight decrease in the ALXN1210 concentration in mg/mL was observed due to the disruption condition. All concentration measurements are presented 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 88.9 100 mg/ml ALXN1210+NOF, T=7 days at 45°C 91.3 100 mg/ml ALXN1210, Avantor, T=7 days at 45°C+5 days with shaking at 2-8°C 97.1 100 mg/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 98.4 100 mg/ml ALXN1210+NOF, T=14 days at 45°C 96.5 100 mg/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 above in Table 8, 100 mg/mL ALXN1210 formulations containing 0.05% PS80 Avantor or PS80 HX2 NOF did not show a significant change in concentration when subjected to the same disruption condition. Concentrations remained comparable for 100 mg/mL ALXN1210 formulations containing 0.05% PS80 Avantor or PS80 HX2 NOF.

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

ALXN1210 formulations at a concentration of 100 mg/mL containing 0.05% PS80 Avantor or 0.05% PS80 HX2 NOF showed no significant changes in turbidity. Turbidity values remained stable at all time points and under all degradation conditions in this study.

Table 9. Turbidity 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-04 100 mg/ml ALXN1210+NOF, T=7 days at 45°C 7.31E-03 100 mg/ml ALXN1210, Avantor, T=7 days at 45°C+5 days with shaking at 2-8°C -6.80E-04 100 mg/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-03 100 mg/ml ALXN1210+NOF, T=14 days at 45°C 1.12E-03 100 mg/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

A decrease in the percentage of monomers was observed for samples 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 degradation conditions. However, the percentage of monomers did not show a significant difference between both 100 mg/mL ALXN1210 formulations containing 0.05% PS80 Avantor or 0.05% PS80 HX2 NOF at the same time points and when exposed to the same conditions.

The monomer percentage data are shown in Table 10. Figure 4 shows the reduction in % monomer that was expected after exposure to the degradation conditions and the lack of significant difference between both 100 mg/mL ALXN1210 formulations containing either 0.05% PS80 Avantor or 0.05% PS80 HX2 NOF at the same time points and when exposed to the same condition.

Table 10. Percentage of monomers for samples 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 93.0 100 mg/ml ALXN1210+NOF, T=7 days at 45°C 93.0 100 mg/ml ALXN1210, Avantor, T=7 days at 45°C+5 days with shaking at 2-8°C 92.8 100 mg/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 88.1 100 mg/ml ALXN1210+NOF, T=14 days at 45°C 88.0 100 mg/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 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 Main peak area 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

Shake load testing was performed on The initial optimal formulation of ALXN1210 at a concentration of 100 mg/mL in a formulation buffer containing 50 mM sodium phosphate, 25 mM arginine, and 5% sucrose at pH 7.4 in the presence and absence of two grades of PS 80 included in the formulation at a concentration of 0.05%. The formation of particles invisible to the naked eye was used to assess degradation. Samples were shaken at 200 rpm at a temperature of 2-8°C. The time points for measuring whether invisible to the naked eye particles formed were days 0, 1, 3, and 5. The results shown in Table 12 indicate that adding 0.05% PS80 to the formulation significantly reduces the formation of invisible to the naked eye particles when the highly concentrated formulation was subjected to a short-term stress such as shaking at 200 rpm.

Table 12. Reduction of formation of particles invisible to the naked eye by addition of PS80 and filtration Sample ID Run number Total particle concentration (number/ml) Total number of particles (number) Number of particles sized 1-10.5 µm The number of particles sized 11.5-25.5 µm Number of particles > 25.5 µm in size Analyzed volume Unfiltered, no PS80 1 869694 454725 419989 33722 1014 0.5229 ml After filtration, without PS80 1 221485 115805 96661 18111 1033 0.5229 ml Unfiltered - PS80 NOF 1 605402 316239 312238 3414 587 0.5224 ml After filtration, PS80 NOF 1 199 104 101 2 1 0.5321 ml After filtration, 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, 5% sucrose, and 0.05% PS80 at pH 7.4.

Example 2: Phase 1 study evaluating a single dose of ALXN1210 administered subcutaneously versus intravenously in healthy subjects

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

1. Objectives

The primary objectives of this study were to (1) evaluate the safety and tolerability of a single dose of ALXN1210 administered subcutaneously compared to ALXN1210 administered intravenously in healthy subjects, as assessed by physical examination, vital sign measurements, immunogenicity data, laboratory analysis data, and adverse event (AE) assessments, and (2) determine the absolute bioavailability of ALXN1210 administered subcutaneously.

The secondary objective was to evaluate the PD effects of ALXN1210 administered subcutaneously compared with ALXN1210 administered intravenously, as assessed by free C5 levels and the rate of chicken red blood cell (cRBC) hemolysis.

2. Research plan

The overall study design was as shown in Figure 47. This was a Phase 1 study designed to evaluate the safety, tolerability, PK, PD, and immunogenicity of a single 400 mg dose of ALXN1210 administered subcutaneously compared with a single 400 mg dose of ALXN1210 administered intravenously or placebo administered subcutaneously in 42 healthy subjects. All subjects were screened for study eligibility. Subjects who did not meet study inclusion criteria were not rescreened for study participation unless the condition leading to ineligibility was transient, self-limited, and readily treatable and expected to resolve by the time of dosing.

Six subjects were initially randomly assigned in a 2:1 ratio to cohort 1a in a blinded fashion to receive a single dose of 400 mg ALXN1210 administered subcutaneously or a single dose of placebo administered subcutaneously. Clinical safety data collected during the first 48 hours postdose were assessed for subjects in cohort 1a before enrollment in cohorts 1b or 2. Thirty-six subjects were then randomly assigned in a 2:1 ratio to cohort 1b (N=24) or cohort 2 (N=12). In cohort 1b, 24 subjects were additionally randomly assigned in a 5:1, blinded fashion to receive either a single dose of 400 mg ALXN1210 administered subcutaneously (20 subjects) or a single dose of placebo administered subcutaneously (4 subjects), respectively. Twelve subjects in cohort 2 received a single dose of 400 mg ALXN1210 administered intravenously in an open-label fashion.

All subjects enrolled in the study were included in the analyses accordingly. Subjects in cohorts 1a and 1b were combined 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 assess safety, PK, PD, and immunogenicity after administration of study drug.

Forty-two subjects were evaluable for the primary and secondary objectives in this study: 6 (4 received ALXN1210 subcutaneously, 2 received placebo subcutaneously) subjects in Cohort 1a, 24 (20 received ALXN1210 subcutaneously, 4 received placebo subcutaneously) subjects in Cohort 1b, and 12 (ALXN1210 IV) subjects in Cohort 2.

3. Justification for dose selection

A single 400 mg dose, equivalent to 4 mL, was administered subcutaneously as four 1 mL injections into the abdomen. Administration of a single 400 mg SC dose of ALXN1210 was expected to have an acceptable safety profile. Single 400 mg doses of ALXN1210 SC and placebo SC, administered as described in this protocol, were expected to provide data on which to model multiple dose administration for planning dosing regimens necessary 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 assessment of clinical safety data obtained during the first 48 hours post-dose from 6 subjects in cohort 1a. Enrollment into cohort 1b and cohort 2 was performed as described in Table 13.

The rules for defining toxicity in the cohorts were as follows. Toxicity refers to clinically significant adverse reaction(s) associated with the drug. "Continuation of cohort participation in the study" refers to continuation of participation in the study with sequential doses/dosing regimen, adhering to the rules for continuing doses during the study and with minimal data requirements. "Pause of clinical study" means that additional IMP was not administered at the appropriate dose level/dosing regimen, and that further participation of the cohort in the study was suspended.

Table 13. Rules for determining toxicity CTCAE degree Severity/
seriousness Number of exposed subjects Action Impact on cohort retention in the study I Easy n.d. Continuation of the dosing regimen. The study continued according to the clinical trial protocol. II Moderate ≤ 2 subjects with different SOC 2 subjects with the same SOC or 3 subjects with different SOC Continuation of the dosing regimen. The study was started for cohorts 1b and 2 if it had not already been started. ≥ 3 subjects with the same SOC or ≥ 4 subjects with different SOC All dosing regimens were suspended unless the toxicity was due to local tolerance or an injection/infusion site reaction, in which case dosing regimens were suspended only for the exposed cohort. A major adjustment was required to continue the study (if it was stopped for both cohorts). A major adjustment was required to continue the study for the exposed cohort (for local tolerance reactions or injection/infusion site reactions). 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 the toxicity was due to local tolerance or an injection/infusion site reaction, in which case dosing regimens were suspended only for the exposed cohort. A major adjustment was required to continue the study (if it was stopped for both cohorts). A major adjustment was required to continue the study for the exposed cohort (for local tolerance reactions or injection/infusion site reactions). IV Life-threatening ≥1 subject Stopping the study. A significant amendment was required to continue the study. V Deadly SAE Serious ≥1 subject Stopping the study. A significant amendment was required to continue the study.

Abbreviations: CTCAE=Common Terminology 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. Assessment Schedule: Screening to Visit 1 Visit 1 Day of the study 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 minutes after EOI 30 min after EOI 2 hours after SOI 4 hours after SOI 8 hours after SOI 24 hours 48 hours 96 hours 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 MCV4 Immunization (Day -56) ⁵ X Immunization with meningococcal vaccine from serogroup B (day -56 and day -28) ⁵ X Serum bactericidal antibody (to meningococci of serogroups A, C, W135 and Y) X History and demographic data X Physical examination X X X Height, weight and BMI X Quanti-FERON®-TB Testing X Biochemical indicators X X X X Hematological parameters 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 pregnancy test ⁸ X X Breath alcohol testing X X General urine analysis and biochemical urine analysis X X X X Urine drug screening X X Measurements of vital signs X X X ⁹ X X ⁹ X ⁹ X ⁹ X ⁹ X X X ECG X X ¹⁰ X X X X Teleelectrocardiography ¹¹ X X (X) (X) (X) (X) Randomization X Administration of the investigational drug X Samples for PK X X X X X X X X X PD assessment panel (serum C5, cRBC hemolysis) X X X X X X X X X X Infusion site assessment ¹² X X X X X X X Immunogenicity (ADA) X Consideration of potential risks associated with the safety of ALXN1210 ¹³ X X X Concomitant medications ←Continuous monitoring (after signing the ICF during screening)→ Adverse events ¹⁴ ←Continuous monitoring (after signing the ICF during screening)→ Preventive treatment with antibiotics ¹⁵ ←Antibiotic prophylaxis→ ¹ Permissible deviations for study assessments are described in the study procedures manual.
² The end of infusion (EOI) occurred approximately 15 minutes after the start of infusion (SOI).
³ Subjects were withdrawn from the clinical trial unit after completion of all assessments on Day 5. Subjects were provided with a "study patient record" containing information for healthcare providers and the study participant about the symptoms of infectious meningitis.
⁴ Signed and dated informed consent forms were obtained before any study-related screening procedures were performed.
⁵ For subjects without adequate documentation of prior MCV4 immunization or serogroup B vaccination, MCV4 immunization was administered at least 56 days prior to the first dose on Day 1, and meningococcal serogroup B vaccine was administered at least 56 days prior to the Day 1 dose, with a booster administered at least 28 days prior to the Day 1 dose.
⁶ Complement activity, confirmed by a suitable assay such as the complement alternative pathway (CAP) ELISA/C5 inhibition (hemolysis) assay, was performed during screening to confirm that subjects were not complement deficient.
⁷ The day -1 sample was stored for future analysis if the post-dose sample was shown to be non-normalized in complement function.
⁸ Serum pregnancy testing was performed on all female subjects to confirm that female subjects were not pregnant.
⁹ On Day 1, vital sign measurements were assessed pre-dose (within 15 minutes before SOI) and at completion of the infusion, 30 minutes after completion of the infusion, 2 hours after the start of the infusion, 4 hours after the start of the infusion, and 8 hours after the start of the infusion.
On Day ^1, triplicate 12-lead ECGs were performed prior to dosing and approximately 15 minutes after completion of the infusion.
¹¹ Continuous recording of cardiac activity was performed from pre-dose throughout the IV infusion period (cohort 2) and until 3 hours after SC injection (cohorts 1a and 1b).
¹² Infusion or injection site assessments were performed within 15 minutes of the start of the infusion/injection and ±15 minutes from another scheduled time on Day 1. Indurations or reactions <1 cm in size were not reported as adverse events unless they persisted for more than 24 hours. Infusion or injection site pain was assessed using a visual analogue scale (0-10). Pain was not assessed pre-dose.
¹³ The investigator or designated person met with the subject at each visit to discuss potential safety risks associated with ALXN1210 and to address any safety concerns raised by the subject.
¹⁴ Data collection on adverse events (AEs) and serious adverse events (SAEs) began at the time of signing the informed consent form.
¹⁵ For prophylactic antibiotic treatment, subjects were given penicillin V orally at 500 mg twice daily (equivalent to 1 × 106 units) starting in the evening of day -1 until complement activity was normalized, as determined by the CH50 assay.

Abbreviations: ADA=anti-drug antibody; BMI=body mass index; cRBC=chicken red blood cell; CRU=clinical investigation unit; ECG=electrocardiogram; EOI=end 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: from 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) °P °P °P °P °P °P °P °P °P °P °P °P °P Physical examination X X X X X X Measurements of vital signs 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 parameters X X X X X X X X X Coagulation indicators X X X X X X X X X General urine analysis and biochemical urine analysis X X X X X X Serum pregnancy testing X X CH50 testing X X ¹ Samples for determination of pharmacokinetic characteristics X X X X X X X X X X X X X Pharmacodynamic 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 Consideration of potential safety risks associated with ALXN1210 ² ←--- Discussion of Potential Safety Risks Associated with ALXN1210 --→ Concomitant medications ←---Continuous monitoring (after signing the ICF during screening)-- → Adverse events ³ ←---Continuous monitoring (after signing the ICF during screening)-- → Preventive treatment with ⁴ antibiotics ⁴ ←---Antibiotic prophylaxis--→ ¹ Additional samples were collected after day 57.
² The investigator or designated person met with the subject at each visit to discuss potential safety risks associated with ALXN1210 and to address any safety concerns raised by the subject.
³ Data collection on adverse events began from the moment the informed consent form was signed.
⁴ For prophylactic antibiotic treatment, subjects were given penicillin V orally at 500 mg twice daily (equivalent to 1 x 106 units) until complement activity was normalized, as determined by the CH50 assay.

Abbreviations: ADA=anti-drug antibody; cRBC=chicken red blood cell; CRU=clinical research unit; ECG=electrocardiogram; ICF=informed consent form; OP=outpatient.

5. Selection and termination of participation of subjects

To be eligible for inclusion in the study, subjects must meet all of the following criteria.

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

2. Body mass index (BMI) ranging from 18 to 29.9 kg/ ^m2 inclusive, and weight ranging from 50 to 100 kg inclusive.

3. QT interval corrected using the Fridericia formula (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 comply with the schedule of visits during the study.

5. Documented vaccination with MCV4 at least 56 days and no more than 3 years prior to administration of the study drug. Documented evidence must include a positive antibody titer to confirm an immune response prior to administration of the study drug.

6. Vaccination with a meningococcal vaccine from serogroup B at least 56 days before the dose on Day 1, with a booster injection at least 28 days before the dose on Day 1, with an interval of at least 28 days between the first and second injections.

7. Female subjects of childbearing potential, if heterosexually active, should use highly effective or acceptable contraception, as defined below, beginning at screening and continuing for at least 6 months after study drug administration. This study required antibiotic prophylaxis, which may negatively impact the effectiveness of hormonal contraceptives. Therefore, it is recommended that subjects using hormonal contraceptives also use a barrier contraceptive method (e.g., a condom or diaphragm with spermicide) during the entire antibiotic prophylaxis period. Male subjects who are heterosexually active and have a female spouse or partner of childbearing potential, or a spouse or partner who is pregnant or breastfeeding, must agree to use barrier contraception (male condom) during the treatment period and for at least 6 months after study drug administration. Barrier contraception was required even if a medically documented successful surgical vasectomy was provided. Spouses or female partners of male subjects of childbearing potential must use highly effective contraception as defined above or acceptable contraception as defined below, beginning with screening and continuing for at least 6 months after study drug administration. Male subjects must not donate sperm 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 ineligible to participate in the study.

1. Subjects who had 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 were immunocompromised, or had one of the following primary medical conditions: anatomical or functional asplenia (including sickle cell disease); congenital deficiencies of the complement system, properdin, factor D, or primary antibodies; acquired deficiencies of the complement system (e.g., 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 were regularly exposed to N. meningitidis; military personnel during recruit training (military personnel may be at increased risk of meningococcal disease when housed in close quarters); child care workers; subjects residing in college or university dormitories; and subjects who planned to travel during the study or had traveled to areas endemic for meningococcal meningitis (e.g., India, sub-Saharan Africa, Hajj pilgrimage to Saudi Arabia) within 6 months prior to dosing.

3. History of any infection caused by Neisseria .

4. History of recurrent infection of unknown etiology or infection requiring treatment with systemic antibiotics within 90 days prior to the dose.

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 study entry. Subjects with positive HBsAg results were not included in the study. For subjects with negative HBsAg results, the following testing algorithm was required. If the hepatitis B core antigen (HBcAb) antibody test result was negative, the subject was eligible for study entry. If the HBcAb test result was positive, hepatitis B surface antigen (HBsAb) antibody testing was performed. If both HBcAb and HBsAb test results were positive, the subject was eligible for study entry. If the HBcAb test result was positive and the HBsAb test result was negative, the subject was not included in the study.

7. Acute or chronic infection caused by the hepatitis C virus (as evidenced by the antibody titer).

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

9. A positive or unclear QuantiFERON®-TB test result indicating possible tuberculosis (TB) infection.

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

11. Female subjects who are breastfeeding or heterosexually active and do not consent to contraceptive use and are not postmenopausal. Postmenopausal was defined as the absence of menses for ≥12 consecutive months without other cause and documented serum follicle-stimulating hormone levels ≥40 mIU/mL and estradiol concentrations ≤110 pmol/L for the 6 months prior to study drug administration.

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

13. Serum creatinine level above the upper limit of normal (ULN) in the reference range in laboratory tests at screening or on Day -1.

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

15. Any of the following hematological results: hemoglobin < 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 × ¹⁰³ /μL, absolute neutrophil count < 2.0 × ¹⁰³ /μL, and platelet count < 150 or > 400 × ¹⁰³ /μL at Screening or on Day -1. Complete blood count (CBC) results considered clinically important and unacceptable by the investigator on Day -1.

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

17. History of malignant neoplasms other than non-melanoma skin cancer or cervical carcinoma in situ that have been treated without evidence of recurrence.

18. Participation in a clinical trial within 30 days prior to 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 trial or participation in a mAb clinical trial within 12 months prior to screening during which the subject was exposed to an active study medicinal product. Subjects who participated in only one mAb trial were considered eligible for inclusion in this trial if they completed their participation in the previous trial 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 fillers for ALXN1210 (eg, polysorbate 80) in the medical history.

23. History of documented allergy to penicillin or cephalosporin.

24. Significant allergic reaction (eg, anaphylaxis or angioedema) to any product (food, pharmaceutical, etc.) in the anamnesis.

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

26. History of illicit drug abuse, history of severe alcohol abuse within 1 year prior to the screening visit, or history of regular alcohol consumption within 6 months prior to the screening visit (more than 14 drinking units per week [1 unit = 150 ml of wine, 360 ml of beer, or 45 ml of 40% alcohol]).

27. Positive urine toxicology screen for drugs of abuse at screening or on Day -1.

28. Alcohol consumption within 48 hours prior to study drug administration or a positive breath alcohol test on Day -1.

29. Donation of blood plasma within 7 days prior to dosing. Donation or loss (excluding volume collected during screening) 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 immunosuppressant therapeutic agent within 90 days prior to administration of study drug.

31. Use of prescription medications (except oral contraceptives) within 14 days prior to administration of the investigational medicinal product, unless prior approval by the clinical trial sponsor is obtained.

32. Regular use of over-the-counter medications, including herbal remedies and dietary supplements, for 14 days prior to study drug administration. Use of multivitamins, acetaminophen ≤ 2 g per day, and topical skin medications without significant systemic absorption are permitted.

33. Any clinically diagnosed autoimmune or rheumatological disease (e.g. systemic lupus erythematosus, rheumatoid arthritis).

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

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

36. Subjects with any history, conditions, or risks that, in the opinion of the investigator, might prevent the subject from fully participating in the study or complying with the protocol or might expose the subject to any additional risks or distort the assessment of the subject or the outcome of the study.

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 the ALXN1210 dose on Day 1 (if subjects have not been vaccinated with MCV4 within the last 3 years or if subjects have been vaccinated previously but there was no documentation to confirm prior vaccination).

2. Two injections of meningococcal serogroup B vaccine. The first injection should be given at least 56 days before the Day 1 dose, with a booster injection given at least 28 days before the Day 1 dose, with an interval of at least 28 days between the first and second injections.

3. For prophylactic antibiotic treatment, penicillin V was administered orally at 500 mg twice daily (equivalent to 1 × 10 ⁶ units) until complement activity was normalized (as determined by CH50 analysis).

The first dose of antibiotic was administered orally on day 1, the evening before day 1 of study medication administration. 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. A suitable system (such as text messaging) was used to monitor subjects' daily adherence to the antibiotic prophylaxis regimen.

The following observations support the rationale for administering antibiotic prophylaxis in this single-dose study.

1. Penicillin was the drug of choice for eradicating N. meningitidis from hosts.

2. Complement-deficient patients who received monthly injections of benzathine penicillin G as prophylaxis against recurrent meningococcal disease over a 2- to 4-year period experienced significantly fewer episodes of Neisseria infection than deficient individuals not receiving prophylaxis (Figueroa JE, et al ., Clin. Micr o bi o l. 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. Micr o bi o l . 2004 Sep;53(Pt 9):821-32).

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

5. Uncertainty about the efficacy of vaccines 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 medications 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 before study drug administration) and concomitant medications (any drug or substance taken by the subject after study drug administration until the end of the last study visit) were recorded in the electronic case report form (eCRF). Previously prescribed treatments (any therapeutic intervention [e.g., surgery/biopsy, physical therapy] performed within 14 days prior to the time the subject signed the informed consent prior to administration of the study drug) and concomitant procedures (any therapeutic intervention [e.g., surgery/biopsy, physical therapy] performed after study drug administration until the completion of the last study visit) were recorded in the subject's eCRF.

A concomitant medication was defined as any drug or substance administered from the time of a subject's screening for the study until the completion of the final study visit. Subjects were instructed not to initiate any new medications, including over-the-counter medications and herbal products, unless approved by the investigator, throughout the study period. Occasional use of over-the-counter antipyretic or analgesic medications (e.g., acetaminophen).

A concomitant procedure was any therapeutic intervention (e.g., surgery/biopsy, physical therapy) or diagnostic evaluation outside the scope of the study (e.g., Blood gas measurements and microflora analysis were performed from the time the subject signed the informed consent until the final visit during the study. Concomitant procedures were not permitted unless medically indicated.

8. Randomization and masking

Subjects eligible for participation in the study who met the inclusion and exclusion criteria were assigned unique numbers for study inclusion and randomization.

This study was a partially blinded, randomized controlled trial.

Cohort 1a. Dose administration (a single 400 mg dose of ALXN1210 or placebo SC) occurred in a double-blind fashion. Subjects in cohort 1a were randomly assigned in a 2:1 ratio (4 ALXN1210 SC, 2 placebo SC; N=6).

Cohort 1b. Dose administration (a single 400 mg dose of ALXN1210 or placebo SC) occurred in a double-blind fashion. Subjects in cohort 1b were randomly assigned in a 5:1 ratio (20 ALXN1210 SC, 4 placebo SC; N=24).

Cohort 2 Dosing (a single dose of 400 mg ALXN1210 IV) was administered in an unmasked fashion (N=12).

During dosing in Cohort 2, both subjects and on-site medical/nursing staff were aware of the drug/dose to be administered.

During cohort 1a and 1b dosing, both subjects and on-site medical/nursing staff at the study site were blinded to study medication administration. Pharmacy staff who prepared SC injections and staff administering study medication were not blinded, while all other study site personnel involved in safety assessments remained blinded to study medication administration. Clinical trial sponsor personnel were unblinded when necessary (e.g., to ensure SC injections were prepared appropriately, to determine whether SAEs could be reported), and were withheld from providing any study medication administration information to study site personnel.

9. Description of the investigational medicinal product

The study drug is described in Table 16.

Table 16. Study drug Investigational drug Product name ALXN1210 IV ALXN1210 SC Placebo SC Dosage form Sterile solution for infusion Sterile solution for injection Sterile solution for injection Single dose 150 mg/vial 100 mg/vial n.d. 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 injection solution according to the European or British Pharmacopoeia Manufacturer Alexion Pharmaceuticals, Inc. Alexion Pharmaceuticals, Inc. Saline solution marketed in the UK ¹ Each vial of ALXN1210 IV drug product was slightly overfilled to allow for the withdrawal of 15 mL (150 mg ALXN1210) for administration.
² Each vial of ALXN1210 SC drug product was slightly overfilled to allow 1 mL (100 mg ALXN1210) to be withdrawn for administration.

Abbreviations: BP=British Pharmacopoeia; IV=intravenous; n.d.=not available; 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, sterile, preservative-free aqueous solution of ALXN1210 at a concentration of 100 mg/mL, supplied in single-use 2 mL vials. Each vial of ALXN1210 SC had a slight overfill to allow for the withdrawal of 1 mL (100 mg ALXN1210) for administration.

Each SC placebo dose contained 0.9% sodium chloride for injection according to the European or British Pharmacopoeia at the same volume as specified 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 compounded, sterile, preservative-free aqueous solution of ALXN1210 at a concentration of 10 mg/mL, supplied in single-use 20 mL vials. ALXN1210 IV was diluted in 0.9% sodium chloride for injection according to the European or British Pharmacopoeia and administered by IV infusion at a maximum rate of 333 mL/h, excluding interruptions for safety or technical reasons.

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

ALXN1210 SC and placebo SC were administered blinded in a SC 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 formulated for infusion 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 was 4 hours at room temperature between 15°C and 25°C (59°F and 77°F). The expiration date and time of the dosing solution were calculated from the time the first vial was opened. The dose was administered before the expiration date and time. The drawn contents of each 1 mL syringe of ALXN1210 SC or placebo SC (4 syringes per subject) were administered within 1 hour of drawing the vial contents into the syringe.

11. Introduction

All doses of ALXN1210 SC or placebo SC were administered via four 100 mg SC injections of 1 mL each (Table 17) into the abdomen. All four 1 mL injections were administered within a 15-minute period, and at least 15 minutes were allowed to elapse between the completion of one subject's injection and the start of the next subject's injection.

Table 17. Reference dosing schedule for ALXN1210 SC and placebo SC Cohort Study drug and dose Number of prepared 1 ml syringes Total volume injected 1a 1 dose 400 mg ALXN1210 SC or placebo SC 4 4 ml 1b 1 dose 400 mg ALXN1210 SC or placebo SC 4 4 ml

All doses of ALXN1210 IV were administered by IV infusion using IV administration sets with built-in filters at a maximum rate of 333 mL/h, excluding interruptions for safety or technical reasons. At least 15 minutes were allowed between the completion of one subject's injection and the start of the next.

Table 18. Reference dosage regimen for ALXN1210 IV Cohort Study drug and dose Volume of ALXN1210 per dose (ml) Volume of diluent per dose (ml) Infusion volume (ml) Maximum infusion rate (ml/h) Minimum duration of infusion, minutes (hours) 2 1 dose 400 mg ALXN1210 IV 40 40 80 333 15 (0.25) ¹ The duration of infusion was approximate.

12. Monitoring for potential adverse events during administration of the study drug

Some subjects 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 administration of study medication for any symptoms of anaphylaxis and other hypersensitivity reactions, including circulatory and/or respiratory changes and arrest, urticaria, arthralgia, myalgia, or other signs of related reactions. Appropriate treatments were immediately available. Infusion-related adverse events could occur, and depending on their type and severity, discontinuation of the infusion may be necessary. Subjects were informed about the early symptoms and signs of hypersensitivity reactions, including urticaria, swelling of the face, eyelids, lips, or tongue, or breathing difficulties. An acute infusion reaction algorithm was used to manage infusion reactions. Regular assessments were conducted throughout the study to monitor for infusion and infusion-site reactions. To ensure prompt management of these reactions, at least 15 minutes were allowed 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 six subjects per day. Any reactions were treated and considered in the rules for dose continuation/escalation and toxicity assessment. In the event of anaphylactic reactions, the current UK Resuscitation Council Treatment Guideline for Anaphylactic Reactions was followed.

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

Following the drug administration study, serum samples for determination of ALXN1210 serum concentrations and for analyses of total and free C5 concentrations, extent of cRBC hemolysis, and other potential indicators of C5 activation were collected at the following time points, with the actual dates and times of serum sampling recorded and used in PK and PD calculations.

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

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

14. Immunogenicity assessments

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

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

An immunogenicity assay was used to evaluate the production of anti-drug antibodies (ADA) against ALXN1210. Detailed instructions for the collection, processing, storage, and transportation of serum samples for immunogenicity analysis are provided in the laboratory manual.

15. Safety assessment

Safety assessments included TB testing, physical examination findings, vital sign measurements, immunogenicity testing (ADA), laboratory assessments, ECG, infusion site and injection site assessments (e.g., bleeding, bruising, redness, swelling, induration, and pain) and monitoring for 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 evaluations include hematology, biochemistry, and coagulation panels; CBC with differential; urinalysis; and serum pregnancy testing for female subjects.

To assess the safety of ALXN1210, clinical and laboratory evaluations were conducted. The timing of these evaluations is described in the evaluation schedule. Abnormal results were monitored until they resolved or stabilized.

Demographic parameters, including age, gender, race, and ethnicity, were assessed as described in the assessment schedule. A complete medical history was obtained.

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

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

A 12-lead ECG was obtained in triplicate after the subject had been at rest for at least 5 minutes. The timing of the ECG is described in the assessment schedule. In addition, continuous cardiac recording was performed at each dose administration from pre-dose to the end of the IV infusion in cohort 2 and from pre-dose to 3 hours after the end of the 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 hematology, clinical chemistry, coagulation analysis, and viral serology, as well as urine samples for urinalysis, urine biochemistry, 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; differential white blood cell (DBC) count (neutrophils, lymphocytes, monocytes, eosinophils, basophils); hemoglobin level; hematocrit; and RBC indices (mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration). The timing of hematological assessments is described in the assessment schedule.

Blood samples were analyzed for the following clinical and biochemical parameters: blood urea nitrogen; 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 the indirect bilirubin level was calculated based on the values for total and direct bilirubin, the results for indirect bilirubin were not available if the direct bilirubin level was below the limit of quantification.

Serum follicle-stimulating hormone and estradiol concentrations were measured at screening for postmenopausal female subjects to confirm their postmenopausal status.

The timeframe for performing 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 complete urinalysis includes measurements of specific gravity, pH, glucose, protein, blood, and ketone levels. Microscopic examination of urine samples was performed only if abnormalities were detected. Urine samples were also sent to a pathology lab for protein and creatinine measurements to calculate the urine protein:creatinine ratio. The timing of urinalysis and urine biochemistry assessments is described in the assessment schedule.

Blood samples collected during screening were analyzed for antibody titers to HIV-1, HIV-2, HBsAg, and HCV. All subjects were required to be tested for hepatitis B surface antigen before study inclusion. Subjects with a positive HBsAg test were not included in the study. For subjects with a negative HBsAg test, the following testing algorithm was required.

1. If the HBcAb test result was negative, the subject was eligible for inclusion in the study.

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

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

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

A urine drug screening 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 performed. If a positive result was observed before dosing, dosing was discontinued. The timing of urine drug testing and breath alcohol testing is described in the assessment schedule.

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

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

A suitable complement assay, such as the CAP ELISA/C5 inhibition (hemolysis) assay, was performed during screening to confirm that subjects were not complement deficient. Subjects found to be complement deficient were excluded from the study.

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

Serum bactericidal antibody (SBA) titers to meningococci from serogroups A, C, W135, and Y were measured during screening. These titer measurements were used to exclude subjects without an immune response from receiving doses.

Assessments were performed at the subcutaneous injection site or IV infusion site. Pain at the SC injection site or IV infusion site was assessed using a visual analog scale (0-10). Pain was not assessed before dosing. Indurations or reactions measuring ≤1 cm were not reported as adverse events unless they persisted for more than 24 hours.

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

16. Monitoring of adverse events

The investigator was responsible for identifying, assessing, documenting, and reporting all adverse events (AEs). All AEs were recorded from the time the informed consent form was signed until the study's completion. There was no time limit for AEs considered causal.

All observed or suspected AEs, regardless of causality, were reported and recorded in the data collection system. Adverse events reported by the subject and/or their parent or legal guardian, 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.

AEs were defined as any unfavorable and unexpected sign (e.g., (including abnormal laboratory findings), a symptom or condition that is time-related to the use of a medicinal product or procedure, whether or not considered related to the use of the medicinal product or procedure, that occurs during the course of a clinical investigation.

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

Abnormal test results were considered AEs. When identifying abnormal laboratory test results, investigators were encouraged to report the diagnosis or sign or symptom rather than the individual abnormal test result. An abnormal test result was documented as an AE if it met any of the following conditions: was associated with a sign or symptom; required additional diagnostic testing (repeat testing was not considered additional testing); required medical or surgical intervention; resulted in a dose change of study drug outside the protocol-specified doses or resulted in discontinuation from the study; required significant additional treatment; or did not meet any of the above conditions.

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

AE does not necessarily include the following.

Medical or surgical procedures ( eg surgery, endoscopy, tooth extraction, blood transfusion); the condition that leads to such a procedure was an AE ( for example, 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 the time of screening assessment or identified prior to screening that are not worsening

Situations where an adverse medical event has not occurred (e.g. hospitalization for non-emergency surgery if planned before the start of the study; admission to hospital for social and/or convenience reasons)

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

An SAE was described as any adverse medical event that, at any dose:

1. Leads to death

2. Threatens life ^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

Emergency department visit within 24 hours

Non-emergency or pre-planned admission to hospital/surgery/day surgery

The admission to hospital indicated in the protocol

Admission to hospital for a pre-existing condition not associated with either a new AE or worsening of a pre-existing AE

4. Resulting in permanent or significant disability/incapacity

5. Represents a congenital anomaly/birth defect

6. It is an important medical ^phenomenon

^a The term "life-threatening" in the meaning of "serious" refers to an event in which the subject had a risk of death during the event; it does not refer to an event that could hypothetically have caused death if it had been more severe.

^b Hospitalization requires the patient's admission to the hospital or prolongation of their current hospitalization. AEs associated with hospitalization or prolongation of hospitalization were considered SAEs.

^c. Important medical event. Medical and scientific judgment should be used when deciding whether urgent reporting is necessary in other situations, such as important medical events that are not likely to be immediately life-threatening or result in death or hospitalization, but may place the subject at risk or may require intervention to prevent one of the other outcomes listed in the definition above. These should also generally be considered serious. Examples of such events included intensive treatment in the emergency department or at home for allergic bronchospasm; immediate hypersensitivity or seizures that do not result in hospitalization; or the development of drug dependence or abuse.

It's important to differentiate between severity and seriousness. Severity describes the intensity of an AE, while seriousness refers to an AE that meets the criteria for an 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 little tolerance)

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

Grade 3: Severe (limitation of abilities, with inability to perform usual activities)

Level 4: Life-threatening

Level 5: Lethal

Changes in AE severity were documented to allow for an assessment of AE duration at each evaluable intensity level. Adverse events characterized as intermittent require documentation of the onset and duration of each episode if the severity of an intermittent episode changed.

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

Not associated (unrelated): This association suggests no relationship between the study drug and the reported event.

Unlikely related. This association suggests that the clinical picture was highly consistent with a cause other than the study drug, but such an attribution cannot be made with absolute certainty, and a relationship between the study drug and the AE cannot be ruled out with complete certainty.

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

Likely related. This relationship suggests a reasonable temporal sequence between the event and study drug administration, as well as a probable relationship between the event and the study drug. This is based on the known pharmacological action of the study drug, known or previously reported adverse reactions to the study drug or class of drugs, or the investigator's judgment based on clinical experience.

Definitely related. Temporally related to the study drug. Other conditions (comorbidity, concomitant drug reaction, or progression/emergence of a disease state) do not appear to explain the event; the event is consistent with a known pharmaceutical profile; improvement upon completion of treatment; recurrence upon rechallenge with drug administration.

If a subject experienced a fatal SAE, the following procedures were performed. An SAE that resulted in death was documented as a fatal outcome, with the end date being the date of death. If the subject had additional AEs/SAEs that were ongoing at the time of death, these events were documented as ongoing without an end date. Only one event was considered 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 prior to database locking.

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

The PD population consists of all subjects for whom sufficient data on total and free C5 concentrations and cRBC hemolysis rates were available. The PD population was used to obtain summary data for the PD analysis.

The immunogenicity analysis population consisted of all subjects from whom samples were collected for pre-dose and post-dose ADA analysis.

The overall estimated sample size of 36 subjects—24 ALXN1210 SC subjects in cohort 1 and 12 ALXN1210 IV subjects in cohort 2—provided >80% power to conclude that the lower bound of the 90% confidence interval for the ALXN1210 SC to IV bioavailability ratio 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. Randomization to cohort 1a was 2:1 and to cohort 1b was 5:1 to receive ALXN1210 SC or placebo SC. This brought the total planned number of subjects to N=42.

In general, descriptive statistics for continuous variables included the number of non-missing values, arithmetic mean, standard deviation, median, minimum value, and maximum value. Descriptive statistics for PK parameters included the 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 subject allocation summary, which summarized the frequency and percentage of screened and treated subjects who completed or discontinued the study, as well as the reason for discontinuation by cohort. Demographic data and baseline characteristics were summarized for all subjects within each cohort and overall.

Safety analyses were performed on the safety analysis population, and results were reported for each group. Safety analyses included analysis of all AEs, ECG, clinical laboratory data, physical examinations, and vital sign measurements and were presented using descriptive statistics. No inferential statistical analyses were planned for the safety parameters of this study. The incidence of treatment-emergent AEs and SAEs were summarized by system organ class and preferred terms for each cohort and overall by relationship to study drug. Treatment-emergent AEs were also summarized by cohort and overall by severity. Serious AEs and AEs leading to study discontinuation were listed. Subjects with multiple AEs within a category ( (e.g., overall, in the system-organ class, under the preferred term) was counted once in that category. For severity tables, the most severe event in a subject within the category was considered.

Changes in vital sign measurements and laboratory assessments (e.g., biochemistry panel, CBC with differential, and urinalysis) from baseline were summarized for each cohort. Laboratory parameter values were categorized into CTCAE grades. Cohort-specific breakout tables were generated for these laboratory parameters. These tables summarize the number of subjects with each grade at baseline relative to the reference range and the change to the worst highest grade assessed after dosing during the study.

ECG parameters were measured at the specified time points, including heart rate, PR, RR, QRS, QT, and corrected QTcF intervals. The mean ECG values were calculated for triplicate data points at the collection time points, and changes from baseline values before treatment were assessed in each cohort.

An outlier analysis was performed, summarizing the frequency and percentage of subjects who met any of the following outlier criteria at each visit across cohorts:

QT, QTcF interval > 450 ms

QT, QTcF interval > 480 ms

QT, QTcF interval > 500 ms

QT, QTcF interval increases relative to baseline > 30 ms

QT, QTcF interval increases relative to baseline > 60 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 subjects treated with ALXN1210, with actual dates and times of sample collection, 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 serum concentration-time curve 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 V _d /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 measuring changes in total and free C5 concentrations in serum, the degree of cRBC hemolysis, and other indicators of C5 activation over time. Analyses were performed on samples collected as described in the assessment schedule.

Immunogenicity measured by ADA was summarized in tabular form by cohort and by subject list.

Example 3. Results from a Phase 1 study evaluating a single dose of ALXN1210 administered subcutaneously versus intravenously in healthy subjects

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

1. Distribution of subjects

Of the 161 subjects screened, 42 (26.09%) 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 discontinued 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). Protocol deviation categories included: time window deviation, subject compliance, failure to complete assessment, exclusion criteria, and dose administration.

In two subjects in the ALXN1210 IV group, protocol deviations were assessed as significant. For one subject, ADA, PK, PD, and laboratory assessments were not performed on day 29 because the subject did not complete a follow-up visit. For another subject, PK and PD samples were not collected on day 71 because the subject did not complete a follow-up visit. Although these deviations were assessed as significant due to the nature of the clinical trial design (the primary endpoint related to PK), they were not considered to impact the interpretation of the results. No other protocol deviations were considered to impact the interpretation of the results or the safety of the subjects. Serum pregnancy testing results were negative for all subjects during the study.

3. Evaluation of pharmacokinetic characteristics, pharmacodynamic characteristics and immunogenicity

All 42 randomized subjects received study medication and were included in the safety analysis set (Table 19). All of these subjects were also included in the PD analysis set and the immunogenicity analysis set based on definitions. For 36 subjects in the safety analysis set who received ALXN1210 SC or ALXN1210 IV, sufficient serum concentration data were available to allow calculation of PK parameters and were included in the PK analysis set (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 analysis of pharmacokinetic characteristics 0 24 (100.0) 12 (100.0) Pharmacodynamic analysis group 6 (100.0) 24 (100.0) 12 (100.0) Immunogenicity Analysis Panel 6 (100.0) 24 (100.0) 12 (100.0)

Note: Percentage (%) is equal to n/N × 100.

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

4. Demographic and other baseline characteristics

Among the treatment groups, most subjects were male (66.7%), Caucasian (69.0%), with a mean age (±SD) of 35.0 (±7.65) years. The mean (±SD) BMI for the overall population was 24.035 (±3.1582). Overall, demographic data were well balanced among treatment groups (Table 20).

Table 20. Demographic data - descriptive statistics parameters by treatment type (safety analysis group) Demographic parameter Placebo SC (N=6) ALXN1210 SC (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 (%) Hispanics or Latin Americans 0 1 (4.2) 1 (8.3) 2 (4.8) Not Hispanic or Latin American 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) Caucasians 2 (33.3) 17 (70.8) 10 (83.3) 29 (69.0) Other 2 (33.3) 0 0 2 (4.8)

Note: Percentage (%) is equal to 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.

Use of previously prescribed medications was reported by 5 (20.8%) subjects in the ALXN1210 SC group. There were no reports of use of previously prescribed medications in the placebo SC and ALXN1210 IV groups. Use of concomitant medications 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, fixed-dose combination drug (7 subjects) for contraception. None of the concomitant medications reported had an impact on the study results.

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

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

PK analyses were performed in the PK analysis set, which consisted of all subjects in the safety analysis set who received 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-time profiles for healthy subjects following SC and IV administration of ALXN1210 (linear and linear-logarithmic scales). Plots of individual ALXN1210 serum concentrations versus nominal time are presented using a linear scale (Figure 49) and a linear-logarithmic scale (Figure 50), respectively.

The pharmacokinetic parameters of ALXN1210 after SC and IV administration are summarized in Table 21. A total of 24 subjects received ALXN1210 SC; the median (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 ALXN1210 SC and IV administration, respectively. ALXN1210 elimination was similar for the IV and SC routes (Figure 49).

Table 21. Summary of pharmacokinetic parameters of ALXN1210 (pharmacokinetic analysis group) C _max t _max AUC _t AUC _∞ λ _z t _½ CL or CL/F V _d or V _d /F Treatment Statistical parameter [mcg/ml] [h.] [h.*mcg/ml] [h.*mcg/ml] [/h.] [h.] [l/h] [l] ALXN1210 SC (n=24) Geometric mean 35.1 169.8 ^a 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 The median value is presented for t _max .

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

Abbreviations: AUC _t = area under the serum concentration-time curve from time 0 to the last quantifiable concentration; AUC _∞ = area under the serum concentration-time curve from time 0 extrapolated to infinity; CL or CL/F = total serum clearance of drug; 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.d. = 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. PK parameters for ALXN1210 SC (C _max , AUC _t , and AUC _∞ ) were compared to the reference (ALXN1210 IV) using a mixed model statistical analysis after log-transformation of the data. The GMR of C _max for ALXN1210 (SC/IV) was 26.1% (95% CI: 21.3, 32.0). The absolute bioavailability of ALXN1210 SC, based on the GMR of estimated AUC _∞ (SC/IV), was 60.4% (95% CI: 49.7, 73.3).

Table 22. Statistical analysis of the absolute bioavailability of ALXN1210 after subcutaneous administration (pharmacokinetic analysis group) Geometric mean Pharmacokinetic parameter ALXN1210 SC (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*μg/ml] 47653.4 78902.9 60.4 51.4-71.0 49.7-73.3

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

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

PD analyses were performed in the PD analysis set, which consisted of all subjects from the safety analysis set for whom sufficient free and total C5 concentrations and cRBC hemolysis data were available.

Figure 51 depicts the mean (±SD) percentage change in serum free C5 concentration from baseline over time for subjects administered placebo SC, ALXN1210 SC, and ALXN1210 IV. The mean free C5 concentration remained relatively constant after SC administration of placebo. Administration of a single 400 mg dose of ALXN1210 IV resulted in immediate and virtually complete inhibition of free C5 (≥99%) through day 8 after IV administration. Administration of a single 400 mg dose 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. Following administration of ALXN1210 SC, the maximum mean inhibition of free C5 (77%) was observed 1 week post-dose.

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

Figure 53 depicts the mean (±SD) percentage change in the extent of chicken red blood cell (cRBC) hemolysis from baseline over time for subjects administered placebo SC, ALXN1210 SC, and ALXN1210 IV. The mean extent of cRBC hemolysis remained relatively constant after SC administration of placebo. Administration of a single 400 mg dose of ALXN1210 IV resulted in immediate inhibition of the mean extent of cRBC hemolysis, with a maximum mean reduction of 88%. Administration of a single 400 mg dose 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, 29%, was observed approximately 1 week after dosing with ALXN1210 SC. The duration and extent of cRBC inhibition were treatment dependent.

Immunogenicity analysis was performed in the immunogenicity analysis cohort, which consisted of all subjects in the safety cohort from whom samples were collected for ADA analysis before and after dosing. Antibody testing for the drug was performed before and after dosing 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 for this subject were lower than the pre-dose titer. The positive drug-drug antibody response in this subject was not considered clinically significant or related to ALXN1210. Therefore, this subject was not included in the immunogenicity summary data presented 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 was ADA-positive on days 57, 90, 120, 150, and 200. All positive ADA values were positive for cross-reactivity to eculizumab. The second subject was ADA-positive on days 29, 57, 90, 120, 150, and 200. All positive ADA values were positive for cross-reactivity to eculizumab except on day 90, when they were negative. The third subject had a positive ADA result on days 90, 120, 150, and 200. All positive ADA values were positive for cross-reactivity to eculizumab.

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

The earliest ADA-positive responses after dosing were observed on day 29 and day 15 for SC and IV dosing, respectively. ADA titers for ADA-positive samples were low, ranging from <1.0 to 27. In the majority of ADA-positive samples, ADA was cross-reactive with eculizumab after SC dosing. After IV dosing, ADA was not cross-reactive with eculizumab. All ADA-positive subjects remained ADA-positive until the end of the follow-up period. A formal assessment of the impact of ADA on PK and PD could not be performed due to the small number of ADA-positive subjects. Examination of the limited individual PK and PD assay results in these subjects suggests that there is no clear impact of immunogenicity on ALXN1210 PK or PD.

6. Conclusions regarding pharmacokinetic characteristics, pharmacodynamic characteristics and immunogenicity

The median (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 SC and IV administration of ALXN1210, respectively.

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

The extent and duration of the PD response, assessed by serum free and total C5 concentrations and cRBC hemolysis, were treatment-dependent. A single 400 mg IV dose of ALXN1210 resulted in immediate and virtually complete inhibition of free C5 (≥99%) through day 8 after study drug administration. A single 400 mg SC dose of ALXN1210, administered 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 with IV administration. The maximum mean inhibition of free C5 was 77%, occurring approximately 1 week after SC administration. Administration of 400 mg ALXN1210 resulted in maximum mean increases in total C5 concentrations of 82% and 107% relative to baseline after SC and IV dosing, respectively. Administration of a single 400 mg IV dose of ALXN1210 resulted in immediate inhibition of the mean degree of cRBC hemolysis, with a maximum mean reduction of 87%. Administration of a single 400 mg SC dose of ALXN1210 also resulted in inhibition of cRBC hemolysis, but not to the same extent or with the same rapid onset compared to IV dosing. The maximum mean inhibition of cRBC hemolysis, 29%, was observed approximately day 8 after SC dosing.

Treatment-emergent ADAs were reported in 3/23 (13%) subjects and 1/12 (8.3%) subjects in the ALXN1210 SC and ALXN1210 IV groups, respectively, with low ADA titer values ranging from <1.0 to 27. The earliest post-dose ADA responses were observed at day 29 and day 15 for the SC and IV doses, respectively. Following SC dosing, ADAs were cross-reactive with eculizumab in the majority of ADA-positive samples. Following IV dosing, ADAs were not cross-reactive with eculizumab. All ADA-positive subjects remained ADA-positive until the end of the follow-up period. No apparent impact of immunogenicity on ALXN1210 PK or PD 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 for this subject were lower than the pre-dose titer. This subject's positive antibody response to the drug was not related to ALXN1210.

7. Degree of impact

All subjects who received a single dose of study medication were included in the safety analysis group (N=42): placebo SC (n=6); ALXN1210 SC (n=24); and ALXN1210 IV (n=12). A total infusion volume of 80 mL of study medication was administered to each subject assigned to receive ALXN1210 IV. In one subject, the infusion was interrupted for one minute because the pump was not programmed for insufficient time to complete the infusion. A total volume of 4 mL of study medication was administered to each subject who received either ALXN1210 SC or placebo SC.

8. Adverse events

Across all three treatment groups, 35/42 (83.3%) subjects experienced 75 TEAEs (all grade 1). The proportions of subjects experiencing at least 1 TEAE were 91.7%, 83.3%, and 79.2% in the ALXN1210 IV group, placebo SC group, and ALXN1210 SC group, respectively. No deaths or SAEs were reported during the study. No subjects discontinued study medication or withdrew from the study due to TEAEs (Table 23). All TEAEs resolved during the study. Most TEAEs did not require any medication, and no subjects required nonpharmacologic interventions at any time point.

Table 23. Treatment-emergent adverse events (TEAEs) - summary description (safety analysis population) Placebo SC (N=6) ALXN1210 SC (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) 1st degree 15 5 (83.3) 38 19 (79.2) 22 11 (91.7) 75 35 (83.3) 2nd degree 0 0 0 0 0 0 0 0 3rd degree 0 0 0 0 0 0 0 0 4th degree 0 0 0 0 0 0 0 0 5th 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 study drug administration ^a 0 0 0 0 0 0 0 0 Death cases 0 0 0 0 0 0 0 0

Note: Percentage (%) is equal to n/N × 100.

Grade 1 = mild; Grade 2 = moderate; Grade 3 = severe; Grade 4 = life-threatening or disabling; Grade 5 = TEAE-related death.

Associated TEAE = possibly associated, probably associated, or definitely associated TEAE; unassociated TEAE = unassociated or unlikely associated TEAE.

For ALXN1210 IV, a TEAE was considered to have occurred during study drug administration if the TEAE occurred during the 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 an AE; SC=subcutaneous; SAE=serious adverse event; TEAE=treatment-emergent adverse event.

A total of 75 TEAEs were reported in 35 subjects. Among treatment groups, the most frequently reported TEAEs 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 type in Table 24.

Table 24. Treatment-emergent adverse events - frequency table by system organ class and preferred terms (safety analysis group) Major system-organ class
Preferred term Placebo SC (N=6) ALXN1210 SC (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) 18 14 (58.3) 9 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) Candidal 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 upper respiratory tract infection 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) 5 3 (12.5) 4 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) 4 3 (12.5) 3 3 (25.0) 9 7 (16.7) Diarrhea 1 1 (16.7) 2 1 (4.2) 1 1 (8.3) 4 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) Back pain 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) Injuries, poisoning and complications of procedures 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 administration site conditions 0 0 1 1 (4.2) 0 0 1 1 (2.4) Bruising at the site of vascular punctures 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 (%) is equal to 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 comply 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=treatment-emergent adverse event.

The majority of TEAEs (72/75 TEAEs, 96%) were considered unrelated to treatment with ALXN1210. Across 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 tract infection 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 subjects died, experienced SAEs, or discontinued study medication or participation in the study due to TEAEs.

Overall, mean values for hematology, coagulation, blood chemistry, urinalysis, and urine biochemistry were within the reference range, and no clear trends in mean change from baseline were observed.

Most subjects entered the study with normal values (i.e., within the respective reference ranges) for hematology, urinalysis, coagulation, blood chemistry, and urine biochemistry parameters. No obvious trends toward changes were observed among 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; however, these shifts were not considered clinically significant. Most shifts were transient and resolved during the study.

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

First, a decrease in neutrophil count was reported in one subject. This subject's neutrophil count at baseline was 3.77 × 10^9/L. The estimated neutrophil count on day 43 was 0.95 × 10^9/L (reference range: 2.0-7.5 × 10^9/L). The neutrophil count was within the reference range on day 57.

Elevated potassium levels were reported for two subjects (reference range: 3.5-5.1 mmol/L). One subject with a baseline potassium level of 4.5 mmol/L had an estimated potassium level of 6.1 mmol/L on Day 150. Repeated potassium measurements were within the normal range on the same day (an unscheduled visit). Another subject with a baseline potassium level of 4.6 mmol/L had an estimated potassium level of 6.4 mmol/L on Day 90. This subject had abnormal potassium values at Screening (ranging from 5.2-6.2 mmol/L at various screening visits) and at most study visits. The increases in potassium levels were transient; values were within the reference range on Day 150 and Day 200.

No significant changes in vital sign measurements from baseline and no clinically significant vital sign abnormalities were observed consistently across individual subjects.

No subject had clinically significant physical examination findings other than those reported as AEs. No significant changes in mean values from baseline were observed in ECG or telemetry monitoring results.

Changes in QT intervals were adjusted using the Fridericia formula (QTcF). In one subject in the SC placebo group, a mean QT interval > 500 ms was observed 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 increases in the QT and QTcF intervals were not considered clinically significant in this female subject in the placebo group. These changes were also not reported as AEs. No significant changes from baseline in the mean QT and QTcF intervals were observed during the study.

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

9. Safety Conclusions

All subjects who received one dose of study medication (placebo SC, ALXN1210 SC, or ALXN1210 IV) were included in the safety analysis population (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 unrelated to ALXN1210 treatment. All TEAEs were mild (grade 1) and resolved during the study. Most TEAEs did not require any medication, and no subjects required non-pharmacologic interventions at any time point. 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 study medication or subject discontinuation from the study. Overall, 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. Subjects with positive ADA results did not exhibit clinical signs or symptoms associated with an allergic reaction or hypersensitivity.

10. Discussion and general conclusions

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

Administration of ALXN1210 at a dose of 400 mg was well tolerated by the SC route 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 values were 31.3 days and 29.9 days after ALXN1210 SC and IV administration. The extent and duration of the PD response, assessed by serum free and total C5 concentrations and the extent of cRBC hemolysis, were dependent on exposure.

Anti-drug antibodies were reported in 3/23 (13%) subjects and 1/12 (8.3%) subjects in the ALXN1210 SC and ALXN1210 IV groups, respectively, with positive ADA titers ranging from <1.0 to 27. The earliest post-dose responses were observed on day 29 and day 15 for SC and IV dosing, respectively. In the majority of ADA-positive samples, post-SC dosing, the ADAs were cross-reactive to eculizumab. Subjects who experienced an ADA-positive response did not have clinical signs or symptoms consistent with an allergic reaction or hypersensitivity (including anaphylaxis). Furthermore, no clear effect of ALXN1210 on PK or PD could be identified.

No unexpected safety issues were observed in any treatment group during the study. No deaths or SAEs occurred during the study, and no subjects experienced any TEAEs leading to discontinuation of the study medication or study participation.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1
amino acid sequence of the heavy chain CDR1 of eculizumab (as defined according to the combined definition of Kabat and Chothia)
GYIFSNYWIQ SEQ ID NO: 2
amino acid sequence of CDR2 of the heavy chain of eculizumab (as defined according to the Kabat definition)
EILPGSGSTEYTENFKD SEQ ID NO: 3
amino acid sequence of CDR3 of the heavy chain of eculizumab (as defined according to the Kabat combined definition)
YFFGSSPNWYFDV SEQ ID NO: 4
eculizumab light chain CDR1 amino acid sequence (as defined according to the Kabat definition)
GASENIYGALN SEQ ID NO: 5
amino acid sequence of the light chain CDR2 of eculizumab
(as defined according to Kabat's definition)
GATNLAD SEQ ID NO: 6
amino acid sequence of the CDR3 light chain of eculizumab (as defined according to the 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 antibody BNJ421
DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGA
TNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTK
VEIK SEQ ID NO: 9
amino acid sequence of the constant region of the heavy chain of eculizumab and the BNJ421 antibody
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKC
CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF
NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS
VMHEALHNHYTQKSLSLSLGK SEQ ID NO: 10
amino acid sequence of the entire heavy chain of eculizumab
QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWM
GEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
YFFGSSPNWYFDVWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYT
CNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV
DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 11
amino acid sequence of the entire light chain of eculizumab, ravulizumab, and the BNJ421 antibody
DIQMTQSPSSLSSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYG
ATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATFATCQNVLNTPLTFGQ
GTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN
ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKSFNRGEC SEQ ID NO: 12
amino acid sequence of the variable region of the heavy chain of ravulizumab and the BNJ421 antibody
QVQLVQSGAEVKKPGASVKVSCKASG H IFSNYWIQWVRQAPGQGLEW
MGEILPGSG H TEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYC
ARYFFGSSPNWYFDVWGQGTLVTVSS SEQ ID NO: 13
amino acid sequence of the constant region of the heavy chain of ravulizumab
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
YFFGSSPNWYFDVWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYT
CNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV
DKSRWQEGNVFSCSV L HEALH S HYTQKSLSLSLGK SEQ ID NO: 15
amino acid sequence of a variant of the constant region of the IgG2 heavy chain 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 comprising the heavy chain constant region set forth in 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
the amino acid sequence of the light chain CDR1 of eculizumab (as defined by the Kabat definition) with a glycine to histidine substitution at position 8 compared to SEQ ID NO: 4
GASENIY H ALN SEQ ID NO: 18
The amino acid sequence of CDR2 of the heavy chain of eculizumab is shown, 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 CDR1 of the heavy chain of eculizumab in which 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 ANTIBODIES

<130> AXJ-226PC

<140>

<141>

<150> 62/537741

<151> 2017-07-27

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

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

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Pro Gln Val Tyr Thr Leu Pro 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

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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

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

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Ser Leu Ser Leu Gly Lys

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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

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

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

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

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

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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

<210> 11

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence: synthetic

polypeptide"

<400> 11

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 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

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence: synthetic

polypeptide"

<400> 12

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

<211> 326

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence: synthetic

polypeptide"

<400> 13

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 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="Description of artificial sequence: 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="Description of artificial sequence: 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 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="Description of artificial sequence: 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="Description of artificial sequence: 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="Description of artificial sequence: 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="Description of artificial sequence: 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="Description of artificial sequence: 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 (23)

1. A stable aqueous solution for the treatment of a human patient with a condition associated with the complement protein C5, comprising: (a) an anti-C5 antibody at a concentration of 70 mg/ml ± 5%, wherein the anti-C5 antibody comprises a heavy chain polypeptide comprising the amino acid sequence presented in SEQ ID NO: 14 and a light chain polypeptide comprising the amino acid sequence presented in SEQ ID NO: 11; (b) 50 mM ± 5% phosphate buffer; (c) 5% ± 5% sucrose; and (d) 25 mM ± 5% arginine. 2. A stable aqueous solution according to claim 1, additionally containing a surfactant. 3. A stable aqueous solution according to claim 2, wherein the surfactant is 0.05% ± 5% polysorbate 80. 4. A stable aqueous solution according to any one of claims 1 to 3, wherein the antibody to C5 is ALXN1210 (ravulizumab). 5. A stable aqueous solution according to any one of claims 1 to 4, wherein the pH of the solution is from 7.2 to 7.6. 6. A stable aqueous solution according to claim 5, wherein the pH of the solution is 7.4. 7. A stable aqueous solution according to any one of paragraphs 1-6, where: (a) the solution is sterile; (b) the anti-C5 antibody remains at least 97% monomeric when stored at 2 to 8°C for at least six months or at least one year, as determined by SEC-HPLC; (c) less than 3%, less than 2%, or less than 1% of the anti-C5 antibody in solution aggregates as determined by SEC-HPLC; and/or (d) when stored at a temperature of 2 to 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 before storage; and/or at least 95% of its ability to inhibit hemolysis compared to the anti-C5 antibody before storage. 8. A stable aqueous solution according to any one of claims 1 to 7, wherein the solution is suitable for subcutaneous administration. 9. A stable aqueous solution according to any one of claims 1 to 8, wherein the solution is in a cartridge, pre-filled syringe or disposable pen. 10. The use of a stable aqueous solution according to any one of claims 1-9 in a method of treating a human with a condition associated with the complement protein C5. 11. The use of claim 10, wherein the complement-associated condition is selected from the group consisting of antiphospholipid antibody syndrome, lupus nephritis, ischemia-reperfusion injury, atypical hemolytic-uremic syndrome (aHUS), typical hemolytic-uremic syndrome, paroxysmal nocturnal hemoglobinuria (PNH), dense deposit disease, neuromyelitis optica, multifocal motor neuropathy, multiple sclerosis, macular degeneration, HELLP syndrome, spontaneous fetal loss, thrombotic thrombocytopenic purpura, pauci-immune vasculitis, epidermolysis bullosa, recurrent fetal loss, traumatic brain injury, myocarditis, cerebrovascular disorder, peripheral vascular disease, renovascular disorder, mesenteric/intestinal vascular disease, vasculitis, Henoch-Schonlein purpura, lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis, immune complex vasculitis, Takayasu's disease, dilated cardiomyopathy, diabetic angiopathy, Kawasaki disease, venous gas embolism, post-stent restenosis, rotational atherectomy, percutaneous transluminal coronary angioplasty, myasthenia gravis, cold agglutinin disease, dermatomyositis, paroxysmal cold hemoglobinuria, antiphospholipid syndrome, Graves' disease, atherosclerosis, Alzheimer's disease, septic systemic inflammatory response, septic shock, spinal cord injury, glomerulonephritis, transplant rejection, Hashimoto's thyroiditis, type 1 diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpasture's syndrome, Degos disease, catastrophic antiphospholipid syndrome and thrombotic microangiopathy (TMA), preferably atypical hemolytic uremic syndrome (aHUS) or paroxysmal nocturnal hemoglobinuria (PNH). 12. Use according to item 9 or 10, wherein the stable aqueous solution is administered to the patient subcutaneously. 13. A therapeutic kit for treating a human patient with a condition associated with complement protein C5, comprising: (i) a stable aqueous solution according to any one of claims 1 to 9 and (ii) a means for delivering the stable aqueous solution to the human. 14. The therapeutic kit according to claim 13, wherein the means is a device. 15. The therapeutic kit according to claim 14, wherein the device is a syringe, a pre-filled syringe, a disposable or reusable autoinjector, a syringe pen, a patch injector, a body injector, an outpatient syringe infusion pump with a subcutaneous infusion set, preferably an outpatient syringe infusion pump with a subcutaneous infusion set.