CN114344460B

CN114344460B - Stable preparation containing anti-PCSK 9 antibody and preparation method and application thereof

Info

Publication number: CN114344460B
Application number: CN202111184183.3A
Authority: CN
Inventors: 靳开远; 邢亚非; 阳勇
Original assignee: Xinlitai Suzhou Pharmaceutical Co ltd; Xinlitai Chengdu Biotechnology Co ltd
Current assignee: Xinlitai Chengdu Biotechnology Co ltd; Xinlitai Suzhou Pharmaceutical Co ltd
Priority date: 2020-10-13
Filing date: 2021-10-12
Publication date: 2025-09-19
Anticipated expiration: 2041-10-12
Also published as: CN121015866A; CN114344460A

Abstract

The present invention relates to stable formulations containing high concentrations of anti-PCSK 9 antibodies, comprising a therapeutically effective dose of an anti-PCSK 9 antibody or antigen-binding fragment thereof, a pharmaceutically acceptable buffer, an osmolality regulator, and/or a surfactant, said formulations having long-term stability. When the formulation is a liquid formulation or a liquid formulation after reconstitution of a lyophilized formulation, it has low viscosity and long-term stability. The invention also provides a preparation method of the preparation and application of the preparation in preparing medicines for treating, preventing and/or improving any PCSK9 related diseases or symptoms.

Description

Stable preparation containing anti-PCSK 9 antibody and preparation method and application thereof

The application claims a stable preparation containing an anti-PCSK 9 antibody, a preparation method and a priority of application of which the Chinese application number is CN202011092207.8 and the application name is 2020.10.13.

Technical Field

The invention relates to the field of medicines, in particular to a stable preparation containing an anti-PCSK 9 antibody, a preparation method thereof and application of the stable preparation in medicines for treating, preventing or improving diseases related to PCSK9 activity.

Background

Proprotein convertase subtilisin/kexin type 9 (Proprotein convertase subtilisin/kexin type 9, pcsk 9), also known as neuronal apoptosis regulating convertase 1 (NARC-1), is a prohormone-proprotein convertase in the subtilisin (S8) family of serine proteases that is expressed in cells with proliferation and differentiation capabilities, including hepatocytes, renal interstitial cells, ileum and colon epithelial cells, embryonic telencephalon, and the like. PCSK9 has been found to play a role in the differentiation of hepatocytes and nerve cells, and not only can specifically act on cholesterol biosynthesis or uptake, but also circulating PCSK9 can be directly combined with a Low Density Lipoprotein Receptor (LDLR) on the surface of the hepatocytes, and is phagocytized by the hepatocytes together with the LDLR, so that the degradation of the LDLR in the hepatocytes is promoted, the circulating use of the LDLR is hindered, the content of LDL cholesterol (LDL-C) in blood plasma is increased, and the increased LDL-C expression is closely related to human dyslipidemia and cardiovascular-related diseases. It follows that PCSK9 is a key regulator of cholesterol homeostasis and circulating Low Density Lipoprotein (LDL) levels. Furthermore, PCSK9 has a variety of mutants, including S127R, N157K, F216L, R S and D374Y, where S127R, F L and D374Y are associated with Autosomal Dominant Hypercholesterolemia (ADH), a hereditary metabolic disorder characterized by significantly increased LDL and cholesterol levels in the plasma, leading to early-onset cardiovascular disease. When PCSK9 function is inhibited, the number of LDLR on the hepatocyte surface increases to clear more circulating LDL, and plasma cholesterol levels correspondingly decrease. Therefore, the inhibition of PCSK9 function can effectively reduce the plasma LDL level, and therefore, PCSK9 becomes a potential target for treating diseases such as atherosclerosis, coronary heart disease, hypercholesteremia, dyslipidemia and the like.

The anti-PCSK 9 monoclonal antibody can specifically target and bind to PCSK9, and can obviously reduce the concentration of circulating LDL-C by inhibiting the interaction of PCSK9 and LDLR on the surface of liver cells and reducing the endocytosis and degradation of the LDLR. The anti-PCSK 9 monoclonal antibody has the advantages of strong specificity, good safety, convenient administration and the like, and has important clinical application prospect in the fields of treating hypercholesterolemia, hyperlipidemia, cardiovascular diseases related to the hyperlipidemia and the like.

CN201710816808.0 discloses a novel monoclonal antibody against proprotein convertase subtilisin Kexin type 9 (PCSK 9).

Therapeutic anti-PCSK 9 antibodies, like other macromolecular therapeutic agents, need not only be formulated as a pharmaceutical solution suitable for administration, but also need to maintain their stability during manufacture, storage and later use. However, therapeutic antibody solutions can be affected by physical and chemical instability factors (e.g., aggregation, denaturation, crosslinking, deamidation, isomerization, oxidation, and shearing, etc.) during manufacture or storage. Thus, the stability of a liquid formulation of an antibody in formulation depends not only on the type of adjuvant in the formulation, but also on the relative amounts and proportions of the effective therapeutic ingredient and the adjuvant and adjuvant.

Cardiovascular related disorders are chronic diseases, and subcutaneous injection of antibody drugs is the preferred mode of administration if patients can self-administer the drugs at home.

Because of the presence of extracellular matrix, the mobility of the drug is limited, the volume of the drug is generally limited to 1-2mL, the concentration of the antibody preparation is required to reach more than 100mg/mL to meet the requirement of clinical dosage, but the high concentration of the antibody protein can increase soluble and insoluble protein-protein aggregates and particle precipitates on the one hand, so that the stability of the preparation is poor, meanwhile, the instability of protein conformation can cause the obvious change of chemical properties such as charge heterogeneity, the quality stability during the downstream process (such as ultrafiltration and sterile filtration) and storage is adversely affected, on the other hand, the high concentration of the protein can cause the increase of the viscosity of the protein due to the interaction between the protein as a macromolecular substance and molecules, the selection of a protein drug delivery device and the problems such as difficulty in pushing, injection pain and burning sensation caused by subcutaneous administration of the drug through an injection device are limited, so that the medication compliance of patients is affected, and on the third aspect, the high concentration of the stabilizer (such as saccharides, polyalcohol, amino acid, complexing agent, surfactant and the like) is required to be added for the liquid preparation of the high concentration protein intended for subcutaneous administration, so as to realize the long-term stability. The resulting solution formulations typically cause injection pain due to tissue damage.

CN201380035382.5 discloses a stable formulation comprising at least one monoclonal antibody that specifically binds PCSK9, wherein PCSK9 comprises the amino acid of the invention patent SEQ ID No.1 in an amount of about 40mg/ml to about 300mg/ml, and a pharmaceutically acceptable buffer in an amount of about 0.05mM to about 40mM, and a pharmaceutically acceptable surfactant in an amount of about 0.01% w/v to about 20% w/v, and at least one pharmaceutically acceptable stabilizer in an amount of about 0.5% w/v to about 10% w/v, wherein the pH of the stable formulation is between about 4.0 to about 6.0.

CN201280037861.6 discloses a pharmaceutical formulation comprising (i) an antibody that specifically binds to human proprotein convertase subtilisin/human PCSK9, (ii) a buffer having a pH of 6.0±0.3, (iii) a nonionic detergent, and (iv) a stabilizer.

The antibodies described in CN201710816808.0 are significantly different from the antibodies of prior art CN201380035382.5 and CN201280037861.6 in terms of substance properties, and the problems of the aforementioned subcutaneous injection liquid preparation are difficult to solve by adopting the corresponding preparation scheme.

Therefore, there is a need to develop an anti-PCSK 9 antibody formulation that is high in concentration, low in viscosity, isotonic, stable over a long period of time, simple in preparation process, easy in quality control, and convenient for patients to administer subcutaneously.

Disclosure of Invention

The invention starts from solving the defects of the prior art, and provides a stable preparation containing an anti-PCSK 9 antibody, which has the characteristics of low viscosity, high concentration of the antibody, long-term stability, high patient compliance and the like, and is suitable for subcutaneous injection.

The above object of the present invention is achieved by the following technical scheme:

In one aspect, the invention provides a stable formulation comprising an anti-PCSK 9 antibody comprising:

(1) An anti-PCSK 9 antibody or antigen-binding fragment thereof at a concentration of 50-200mg/mL, and

(2) A pharmaceutically acceptable buffer at a concentration of 1-50mmol/L (mM), and

(3) A pharmaceutically acceptable surfactant at a concentration of 0-0.1% w/w, and

(4) One or more other pharmaceutically acceptable excipients at a concentration of 100-400mmol/L (mM) or 1-10% w/w.

The preparation can be liquid preparation, or can be freeze-dried or the like, can be reconstituted into liquid preparation, and is preferably liquid preparation.

The anti-PCSK 9 antibody or the antigen-binding fragment thereof can specifically bind to PCSK9 protein, can be a polyclonal antibody or a monoclonal antibody, and can be used for mass production of the monoclonal antibody with homogeneity. The monoclonal antibody is not limited to a monoclonal antibody of human, mouse, rabbit, sheep, camel, monkey, etc., but may be a recombinant antibody such as a chimeric antibody, humanized antibody, fully human monoclonal antibody, etc., preferably fully human monoclonal antibody produced by a host cell or transgenic animal.

Specifically, the anti-PCSK 9 antibodies or antigen-binding fragments thereof of the invention comprise a heavy chain complementarity determining region (HCDRs) and a light chain complementarity determining region (LCDRs), wherein the HCDR1, HCDR2, and HCDR3 amino acid sequences comprise at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identity to the amino acid sequences shown in SEQ ID NOs 1,2, and 3, and LCDR1, LCDR2, and LCDR3 comprise at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identity to the amino acid sequences shown in SEQ ID NOs 4,5, and 6.

The anti-PCSK 9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%, 97%,98%,99% or 100% identity to the amino acid sequence shown in SEQ ID No. 7 and a light chain variable region comprising at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%, 97%,98%,99% or 100% identity to the amino acid sequence shown in SEQ ID No. 8.

The anti-PCSK 9 antibody or antigen-binding fragment thereof comprises a heavy chain comprising a heavy chain variable region and a light chain constant region linked by disulfide bonds, and a light chain comprising a light chain variable region and a light chain constant region, wherein the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region and the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region, and the heavy chain variable region and the light chain variable region comprise the amino acid sequences described above. The heavy chain constant region is selected from a human IgG1, igG2, igG3 or IgG4 constant region, preferably an IgG1 or IgG4 constant region, more preferably an IgG4 constant region having an amino acid sequence as shown in SEQ ID NO. 9, and the light chain constant region is selected from a human kappa light chain constant region or lambda light chain constant region, preferably a kappa light chain constant region having an amino acid sequence as shown in SEQ ID NO. 10.

The anti-PCSK 9 antibody or antigen-binding fragment thereof comprises or consists of two heavy chains and two light chains, wherein each heavy chain comprises a heavy chain constant region, a heavy chain variable region, or a CDR sequence as described above, and each light chain comprises a light chain constant region, a light chain variable region, or a CDR sequence as described above. The antibody may also be a single chain variable fragment (scFv) antibody, or an antibody fragment (e.g., fab or F (ab') ₂ fragment). Preferably, the heavy chain of the anti-PCSK 9 antibody or antigen-binding fragment thereof has the amino acid sequence shown in SEQ ID NO. 11, and the light chain has the amino acid sequence shown in SEQ ID NO. 12.

In one embodiment, the concentration of the anti-PCSK 9 antibody or antigen-binding fragment thereof is 50-200mg/mL. In another embodiment, the concentration of the anti-PCSK 9 antibody or antigen-binding fragment thereof is 70-200mg/mL. In another embodiment, the concentration of the anti-PCSK 9 antibody or antigen-binding fragment thereof is 70mg/mL. In a specific embodiment, the concentration of the anti-PCSK 9 antibody or antigen-binding fragment thereof is 70mg/mL. In a specific embodiment, the concentration of the anti-PCSK 9 antibody or antigen-binding fragment thereof is 140mg/mL. In a specific embodiment, the concentration of the anti-PCSK 9 antibody or antigen-binding fragment thereof is 200mg/mL.

The pharmaceutically acceptable buffer is selected from histidine, acetate, succinate, or glutamate.

As a preferred embodiment of the present invention, the buffer is histidine buffer. In a specific embodiment, the buffer is a histidine buffer consisting of L-histidine and L-histidine hydrochloride, wherein the concentration (molar) ratio of L-histidine to L-histidine hydrochloride is from 0.2:1 to 3:1. In one embodiment, the buffer is an acetate buffer. In a specific embodiment, the buffer is an acetate buffer consisting of acetic acid and sodium acetate. The selection of phosphate and citrate as buffers for use in the formulations of the present invention is susceptible to painful reactions upon subcutaneous injection, and patient compliance with medication is therefore significantly affected.

The concentration of the pharmaceutically acceptable buffer is 1-50mmol/L. In one embodiment, the buffer concentration is 5-50mmol/L. In one embodiment, the buffer concentration is 10-30mmol/L. In a specific embodiment, the buffer concentration is 20mmol/L.

The pH value of the stable liquid preparation is 4.5-6.5. In one embodiment, the stable liquid formulation has a pH of 4.5 to 5.5. In a specific embodiment, the pH of the stable liquid formulation is 4.5,5.0 or 6.0. In one embodiment, the stable liquid formulation has a pH of 5.0 to 6.0. In a specific embodiment, the pH of the stable liquid formulation is 5.0,5.5 or 6.0. In one embodiment, the stable liquid formulation has a pH of 5.5 to 6.5. In a specific embodiment, the pH of the stable liquid formulation is 5.5,6.0,6.2,6.3 or 6.5.

Histidine has an imidazole group in its molecular structure, which has a pKa of about 6.0 and a pH sensitivity, and which is hydrophilic when the environmental pH is <6.0, and hydrophobic when the environmental pH is > 6.0.

Although histidine is pH sensitive and prone to oxidation, the inventors have surprisingly found that when the formulation of the invention is used as a buffer system and the pH is maintained between 5.5 and 6.5, it has good compliance and quality stability, for example, compared to acetate buffer systems with lower pH (pKa 4.76), it can significantly reduce pain in patients when administered subcutaneously, whereas for chronic disease patients requiring long-term administration, liquid formulations using L-histidine/L-histidine hydrochloride can improve patient compliance, and furthermore, when L-histidine/L-histidine hydrochloride buffer systems are selected and the pH is 5.5 to 6.5, it can reduce antibody viscosity of the liquid formulation of the invention to below 15cp, even below 10cp, and can reduce degradation rate of the antibody during storage, with better stability. However, when pH <5.5 is selected, the acceleration of acid-catalyzed hydrolysis results in an increased rate of hydrolysate formation.

The other excipients of the stable liquid preparation are selected from any one or combination of proline, sucrose, trehalose, sodium chloride, sorbitol, mannitol or arginine hydrochloride. In a specific embodiment, the additional excipient is sucrose at a concentration of 1% to 10% (w/w), preferably 5% to 10% (w/w), more preferably 6% (w/w). As a preferred embodiment of the present invention, the other excipient is proline. In a specific embodiment, the other excipient is proline in a concentration of 100-400mmol/L, preferably 200-300mmol/L, more preferably 250mmol/L.

The osmotic pressure of the stable liquid preparation is 220-370mOsmol/kg. In one embodiment, the stable liquid formulation has an osmotic pressure of 250 to 350mOsm/Kg. In one embodiment, the stable liquid formulation has an osmotic pressure of 300 to 330 mOsm/Kg at 25 ℃.

The present invention has found through a number of experiments that when proline is selected as one of the excipients of the present invention, not only can the osmotic pressure of the liquid formulation of the present invention be maintained at 250-350mOsm/Kg due to the biocompatibility of proline, but also a hydration layer can be formed on the surface of the anti-PCSK 9 antibody or antigen-binding fragment thereof to stabilize the antibody protein without damaging the structure thereof, and also interactions of the antibody protein itself or other substances can be prevented from acting thereon, so as to reduce the viscosity and denaturation of the high concentration antibody in the liquid formulation, thereby being advantageous for improving the stability and fluidity of the liquid formulation and compliance of patients when subcutaneously administered. Proline is used as one of the most suitable excipients of the preparation, can avoid hemolysis or irritation during administration, and effectively ensures the use safety and stability of the injection preparation.

The surfactant of the stable liquid formulation is a nonionic surfactant selected from polyoxyethylene sorbitan fatty acid esters (e.g. polysorbate 80, polysorbate 60, polysorbate 40, or polysorbate 20), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycols, polyoxyethylene-stearates, polyoxyethylene alkyl ethers, alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (e.g. poloxamer, pluronic), sodium Dodecyl Sulfate (SDS). As a preferred embodiment of the present invention, polysorbate 80 or polysorbate 20 is preferred, and polysorbate 80 is more preferred. The surfactant concentration is 0-1% (w/w), preferably 0.01-0.05% (w/w), more preferably 0.01% (w/w), 0.02% (w/w) and 0.04% (w/w). In the invention, the nonionic surfactant is used in the amount which can prevent protein adsorption and interaction between proteins and prevent interaction between proteins on the inner surface of the packaging material by reducing interface interaction, thereby achieving the purpose of stabilizing antibodies in the preparation and storage processes.

The invention discovers through a large number of experiments that when polysorbate 80 or polysorbate 20 is selected as a surfactant of the liquid preparation, aggregation of the anti-PCSK 9 antibody or the antigen-binding fragment thereof can be reduced in stirring and shaking processes, the antibody is prevented from being adsorbed to the surface of a container, and the requirement of subcutaneous injection administration of the liquid preparation is met. In addition, the polysorbate 80 has the characteristics of low freezing point (the solid state at other normal temperature) and convenient use because of double bonds in the structure. In addition, the ability of polysorbate 80 or polysorbate 20 to stabilize antibodies is also related to their concentration. The concentration of polysorbate 80 or polysorbate 20 in the liquid formulation of the present invention above 0.01% (w/w) can inhibit the aggregation of antibodies caused by shaking, and below that concentration polysorbate 80 or polysorbate 20 cannot reduce the protein instability caused by shaking, while above 0.05% (w/w) cannot significantly improve the stability of antibodies.

The viscosity of the stable liquid preparation at 25 ℃ is about 1-20 centipoise (cp) when the excipient type and concentration are selected. As a preferred embodiment of the present invention, the stable liquid formulation has a viscosity of less than 15cp at 25 ℃. In one embodiment, the stable liquid formulation has a viscosity of about 5 to 15cp at 25 ℃. In one embodiment, the stable liquid formulation has a viscosity of about 6 to 14cp at 25 ℃. In a specific embodiment, the stable liquid formulation has a viscosity of about 6.0cp, 8.0cp, 9.0cp, 10.0cp, 11.0cp, 12.0cp at 25 ℃. In a specific embodiment, the stable liquid formulation has a viscosity of about 9.0cp at 25 ℃.

The stable liquid formulation may be stored at about 5 ℃ to about 40 ℃ for at least 2 weeks to 36 months, for example at about 5 ℃, about 25 ℃, or about 40 ℃ for at least 2 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months, or 36 months. in one embodiment, the stable liquid formulation increases the aggregate of the anti-PCSK 9 antibody or antigen-binding fragment thereof by no more than 10%, such as no more than 5%, by SEC-HPLC method, as measured by the aggregation of the anti-PCSK 9 antibody or antigen-binding fragment thereof, after storage at 2-8 ℃ for at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more, or after storage at about 25 ℃ for at least 2 weeks, at least 4 weeks, at least 12 weeks, or more, or after storage at 40 ℃ for at least 2 weeks, at least 4 weeks or more, as measured by SEC-HPLC method, at least 4 weeks, or more 4%, 3%, 2%, 1.3%, 1.2%, 1.1%, 1.0%, 0.8%, 0.7%, 0.6%, 0.5% or 0.1%, the degradation product (e.g., fragment) of an anti-PCSK 9 antibody or antigen-binding fragment thereof increases by no more than 10%, e.g., no more than 5%, 4%, 3%, 2%, 1.3%, 1.2%, 1.1%, 1.0%, 0.8%, 0.7%, 0.6%, 0.5% or 0.1%. in one embodiment, the stable liquid formulation has a purity of the anti-PCSK 9 antibody or antigen-binding fragment thereof that decreases by no more than 10%, e.g., no more than 5%, as measured by a reduced CE-SDS method (rCE-SDS) and a non-reduced CE-SDS (nrCE-SDS) after storage at 2-8 ℃ for at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer, or after storage at about 25 ℃ for at least 2 weeks, at least 4 weeks, at least 12 weeks, or longer, or after storage at 40 ℃ for at least 2 weeks, at least 4 weeks, or longer, as measured by a reduced CE-SDS method (rCE-SDS) and a non-reduced CE-SDS (nrCE-SDS), 4%, 3%, 2%, 1.0%, 0.5%, 0.2% or 0.1%. In one embodiment, the stable liquid formulation is stored at 2-8 ℃ for at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer, or after at least 2 weeks, at least 4 weeks, at least 12 weeks, or longer, or after at least 2 weeks or longer, at 40 ℃, as measured by CEX-HPLC, the acid-base charge heterosomes of the anti-PCSK 9 antibody or antigen-binding fragment thereof are no more than 50%, wherein the basic antibody is no more than about 30%, e.g., no more than about 29% >, 25% or 22%, of acidic antibodies of no more than 20%, for example no more than 19%, 17%, 15% or 10%. In one embodiment, the purity of the anti-PCSK 9 antibody or antigen-binding fragment thereof decreases by no more than 5%, e.g., no more than 5%, 4%, 3%, 2%, 1%, or 0.5% as measured by a non-reducing Caliper after storage of the stable liquid formulation at about 25 ℃ or about 40 ℃ for at least 2 or 4 weeks.

The stable formulation is a liquid pharmaceutical formulation, preferably an injection, more preferably a subcutaneous injection or an intramuscular injection, most preferably a subcutaneous injection.

In a preferred embodiment, the stable liquid formulation of the present invention comprises:

(1) 70-200mg/mL of anti-PCSK 9 antibody or antigen-binding fragment thereof

(2) Histidine buffer consisting of L-histidine and L-histidine hydrochloride in a concentration (molar) ratio of 0.2:1 to 3:1, and

(3) A concentration of 0-0.05% w/w polysorbate 80 or polysorbate 20;

(4) The concentration is 200-300mmol/L proline.

The concentration of the buffer is 1-50mmol/L (mM), and the pH value of the liquid preparation is 5.5-6.5.

Wherein the anti-PCSK 9 antibody or antigen-binding fragment thereof comprises HCDR1 of the amino acid sequence shown in SEQ ID NO.1, HCDR2 of the amino acid sequence shown in SEQ ID NO.2, HCDR3 of the amino acid sequence shown in SEQ ID NO.3, LCDR1 of the amino acid sequence shown in SEQ ID NO. 4, LCDR2 of the amino acid sequence shown in SEQ ID NO. 5 and LCDR3 of the amino acid sequence shown in SEQ ID NO. 6.

More specifically, the anti-PCSK 9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region of the amino acid sequence shown as SEQ ID NO. 7 and a light chain variable region of the amino acid sequence shown as SEQ ID NO. 8.

In a more preferred embodiment, the stable liquid formulation of the present invention comprises:

(1) anti-PCSK 9 antibody or antigen-binding fragment thereof at a concentration of 70mg/mL or 140mg/mL

(2) Histidine buffer consisting of L-histidine and L-histidine hydrochloride at a concentration of 20mmol/L, and

(3) At a concentration of 0.01-0.04% w/w polysorbate 80 or polysorbate 20

(4) The concentration was 250mmol/L proline.

The pH value of the liquid preparation is 5.5-6.5. In another aspect, the present invention provides a method of preparing a stable liquid formulation comprising an anti-PCSK 9 antibody, comprising the steps of:

a) Providing an anti-PCSK 9 antibody or antigen-binding fragment thereof;

b) Adding a buffer and an osmotic pressure regulator;

c) Adding a surfactant;

d) Sterilizing and filtering;

e) And (5) sub-packaging.

Wherein, the anti-PCSK 9 antibody or the antigen-binding fragment thereof in the step a) is an antibody protein stock solution prepared by cell culture and obtained by separation and purification.

The buffer and osmolality adjusting agent in step b) are added to the anti-PCSK 9 antibody or antigen-binding fragment thereof in step a) in the form of an ultrafiltrate and the antibody or antigen-binding fragment thereof is subjected to ultrafiltration concentration until the replacement of the antibody or antigen-binding fragment thereof is complete. Before said step c), adjusting the concentration of the displaced anti-PCSK 9 antibody or antigen-binding fragment thereof.

The surfactant in step c) is provided in the form of an aqueous solution.

The storage device of the sub-packaging in the step e) is a pre-filling and sealing syringe. The anti-PCSK 9 antibody or antigen-binding fragment thereof, buffer, osmolality regulator, and surfactant are as described above, in amounts and/or concentrations as described above or as may be suitably determined.

In one embodiment, the method of preparing a stable liquid formulation of the present invention comprises the steps of:

a) Separating and purifying the harvest liquid of the anti-PCSK 9 antibody or the antigen-binding fragment thereof prepared by culture to obtain an antibody protein stock solution, wherein the anti-PCSK 9 antibody or the antigen-binding fragment thereof is as described above;

b) Adding an ultrafiltrate comprising a buffer and an osmolality adjusting agent, preferably a histidine buffer comprising L-histidine and L-histidine hydrochloride, to perform ultrafiltration concentration until the antibody protein is completely displaced, wherein the buffer and the osmolality adjusting agent are of the type, concentration and pH as described above;

c) Adjusting the concentration of the displaced antibody protein to a concentration range defined by the stable liquid formulation of the present invention;

d) An aqueous solution of surfactant, as described above, is added, preferably polysorbate 80 or polysorbate 20.

E) Sterilizing and filtering the solution of the step d);

f) Subpackaging the mixture into prefilled syringes to obtain the stable liquid preparation of the invention.

In a third aspect, the invention provides the use of a stable liquid formulation comprising an anti-PCSK 9 antibody in the manufacture of a medicament for the treatment, prophylaxis or amelioration of any disease or condition associated with PCSK9 and a method of treatment.

The present invention further provides a pre-filled syringe loaded with the aforementioned stable formulation, which is a liquid formulation.

In one aspect, the invention provides a method of treating a disease or condition associated with PCSK9 in a subject, the method comprising administering to the subject a therapeutically effective amount of a liquid formulation of the invention comprising an anti-PCSK 9 antibody or an antibody or antigen-binding fragment thereof. In some embodiments, the activity of PCSK9 binding to LDLR is modulated by administering a liquid formulation comprising an anti-PCSK 9 antibody described herein or an antibody or antigen-binding fragment thereof described herein. In some embodiments, a disease or disorder treated or prevented in a subject with a statin (statin) drug may also be treated or prevented by administering a liquid formulation of the invention containing an anti-PCSK 9 antibody or an antibody or antigen-binding fragment thereof of the invention. In some embodiments, a disease or disorder treated by preventing cholesterol synthesis or increasing LDLR expression can also be treated by administering a liquid formulation comprising an anti-PCSK 9 antibody described herein or an antigen-binding fragment thereof. The invention also provides a liquid preparation containing the anti-PCSK 9 antibody or application of the anti-PCSK 9 antibody or the antigen-binding fragment thereof in preparing medicines for treating diseases or symptoms related to PCSK 9.

In one aspect, the invention provides a method of reducing cholesterol levels in a subject, the method comprising administering to the subject a therapeutically effective amount of a liquid formulation comprising an anti-PCSK 9 antibody of the invention or an antibody or antigen-binding fragment thereof of the invention. The cholesterol includes total cholesterol, LDL-C and non-High Density Lipoprotein (HDL) cholesterol. In one embodiment, the invention relates to a method of reducing LDL-C levels in a subject, comprising administering to the subject a therapeutically effective amount of a liquid formulation comprising an anti-PCSK 9 antibody of the invention or an antibody or antigen-binding fragment thereof of the invention. The LDL-C level is reduced after administration of the liquid formulation or the antibody or antigen-binding fragment thereof. In a specific embodiment, the invention relates to a method of reducing serum LDL-C levels in a subject, comprising administering to the subject a therapeutically effective amount of a liquid formulation comprising an anti-PCSK 9 antibody of the invention or an antibody or antigen-binding fragment thereof of the invention. The serum LDL-C level is reduced after administration of the liquid formulation or the antibody or antigen-binding fragment thereof. In one embodiment, the invention relates to a method of reducing the total cholesterol level in a subject, comprising administering to the subject a therapeutically effective amount of a liquid formulation comprising an anti-PCSK 9 antibody of the invention or an antibody or antigen-binding fragment thereof of the invention. The total cholesterol level is reduced after administration of the liquid formulation or the antibody or antigen-binding fragment thereof. The invention also provides a liquid preparation containing the anti-PCSK 9 antibody or application of the anti-PCSK 9 antibody or the antigen-binding fragment thereof in preparing medicines for reducing cholesterol level of a subject.

In one aspect, the invention provides a method of treating and/or preventing a cholesterol-related disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a liquid formulation comprising an anti-PCSK 9 antibody of the invention or an anti-PCSK 9 antibody or antigen-binding fragment thereof of the invention. The cholesterol-related diseases include any one or more of hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, alzheimer's disease, and dyslipidemia (e.g., high levels of total serum cholesterol, high levels of LDL, high levels of triglycerides, high levels of Very Low Density Lipoprotein (VLDL), and/or low levels of High Density Lipoprotein (HDL)). In some embodiments, the invention relates to a method of treating and/or preventing hypercholesterolemia and/or dyslipidemia in a subject, comprising administering to the subject a therapeutically effective dose of a liquid formulation comprising an anti-PCSK 9 antibody of the invention or an anti-PCSK 9 antibody or antigen-binding fragment thereof of the invention. The invention also provides a liquid preparation containing the anti-PCSK 9 antibody or a method for preparing the anti-PCSK 9 antibody or the antigen-binding fragment thereof for treating and/or preventing cholesterol-related diseases.

In one aspect, the invention provides a liquid formulation comprising an anti-PCSK 9 antibody of the invention or a method of administering an anti-PCSK 9 antibody or antigen-binding fragment thereof of the invention in combination with another therapeutic agent. In some embodiments, an effective dose of the liquid formulation containing an anti-PCSK 9 antibody or the anti-PCSK 9 antibody or antigen-binding fragment thereof of the invention is administered prior to administration of at least one other therapeutic agent. In some embodiments, an effective dose of a liquid formulation containing an anti-PCSK 9 antibody described herein or an anti-PCSK 9 antibody or antigen-binding fragment thereof described herein is administered concurrently with the administration of at least one other therapeutic agent. In some embodiments, an effective dose of the liquid formulation containing an anti-PCSK 9 antibody or the anti-PCSK 9 antibody or antigen-binding fragment thereof described herein is administered after administration of at least one other therapeutic agent. Such other therapeutic agents include, but are not limited to, at least one agent that reduces cholesterol levels (including serum LDL-C and total cholesterol) or increases LDLR expression levels, such as a statin (selected from any one of atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin (PITAVASTATIN), pravastatin (pravastatin), rosuvastatin, simvastatin (simvastatin), or any combination thereof. In some embodiments, the additional therapeutic agents may be used to treat and/or treat hypercholesterolemia and/or dyslipidemia. In some embodiments, the additional therapeutic agents may be used to treat and/or prevent atherosclerosis and/or cardiovascular disease and/or reduce recurrent cardiovascular events. In some embodiments, the additional therapeutic agent may be used to increase HDL cholesterol levels.

The effective dosage range of the above-described liquid formulations containing an anti-PCSK 9 antibody or the anti-PCSK 9 antibody or antigen-binding fragment thereof of the invention includes about 0.5-50mg/kg.

In a fourth aspect, the invention provides a nucleotide encoding an anti-PCSK 9 antibody or antigen-binding fragment thereof of the invention. In some embodiments, the invention provides vectors comprising the nucleotides, including expression vectors. In some embodiments, the invention also provides a host cell for preparing the antibody or antigen-binding fragment thereof, said host cell being a eukaryotic cell selected from the group consisting of a mammalian cell, a yeast cell, or other cell suitable for preparing an antibody or antigen-binding fragment thereof, preferably a mammalian cell, including Chinese Hamster Ovary (CHO) cells, mouse NSO myeloma cells, COS cells, mouse myeloma SP2/0 cells, small hamster kidney (BHK) cells, and 293 cells.

In another aspect, the invention also provides a method of preparing an anti-PCSK 9 antibody or antigen-binding fragment thereof of the invention, comprising inserting nucleotides encoding an antibody or antigen-binding fragment thereof of the invention into an expression vector operably linked such that transcriptional and translational control elements within the expression vector can regulate the transcriptional and translational functions of the antibody or antigen-binding fragment nucleotide, then transfecting the expression vector encoding the antibody or antigen-binding fragment thereof into a host cell for culturing, and isolating and purifying the antibody or antigen-binding fragment thereof prepared by the host cell.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) There is provided a liquid formulation containing a high concentration of an anti-PCSK 9 antibody, having low viscosity characteristics, meeting not only the requirements of clinical doses, but also the viscosity limits of the antibody required for subcutaneous delivery using a syringe at ambient temperature, particularly suitable for subcutaneous or intramuscular administration;

(2) The invention provides a liquid preparation containing high-concentration anti-PCSK 9 antibody, which has high physical and chemical stability, is not easy to generate aggregate, particle sediment and charge heterosomes when being stored for a long time (such as more than 1 year, especially 2 years or 3 years), therefore, the liquid preparation provided by the invention is not only suitable for long-term storage, but also can ensure the biological activity of the antibody, and the quality safety, effectiveness and high consistency of the liquid preparation prepared into clinical medicines;

(3) The liquid preparation containing the high-concentration anti-PCSK 9 antibody is simple in component, safe, nontoxic, less in aggregate and particle sediment, high in biological activity, simple and feasible in production method and easy to control quality;

(4) The liquid preparation containing the high-concentration anti-PCSK 9 antibody has the characteristics of high concentration of the antibody and low viscosity, and the pH value is maintained between 5.5 and 6.5 (especially between 6.0 and 6.5), so that the liquid preparation is suitable for subcutaneous injection administration, not only meets the requirement of chronic disease patients on domestic administration, but also can reduce the administration pain to a lower value in the administration process, and improves the medication compliance of the patients.

Detailed Description

The following description of the application is merely illustrative of various embodiments of the application. Therefore, the specific modifications discussed herein should not be construed as limiting the scope of the claims. Numerous equivalents, variations and modifications will readily occur to those skilled in the art without departing from the scope of the present application, and it is to be understood that such equivalent embodiments are included within the scope of the present application. All documents cited in this application, including publications, patents and patent applications, are incorporated by reference in their entirety.

Definition of the definition

The terms used in the present invention have the definitions listed below. If no definition is given herein, the terms used in the present invention have meanings commonly understood by those of ordinary skill in the art.

The term "about" as used herein, when used in reference to a particular value or range of values listed, means that the value may vary from the particular value listed by + -20% or + -10%, including + -5%, + -1%, and + -0.1%, as appropriate for performing the disclosed method.

For purposes of explaining the present specification, the following definitions will be used, and terms used in the singular form may also include the plural, and vice versa, as appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The term "proprotein convertase subtilisin/kexin type 9 (Proprotein convertase subtilisin/kexin type 9, PCSK 9)", "PCSK9" or "NARC-1", a natural human proprotein convertase belonging to the proteinase K subfamily of the secreted subtilisin family. PCSK9 is synthesized as a plasminogen, undergoes autocatalytic intramolecular processing in the endoplasmic reticulum, and is believed to function as a proprotein convertase. The term as used in the present application refers to any naturally occurring PCSK9, preferably any natural PCSK9 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Representative amino acid sequences of human PCSK9 and representative nucleic acid sequences encoding the same are disclosed by GenBank accession numbers NP-777596.2 and FJ525880.1, respectively, or the amino acid sequence of human PCSK9 is shown as SEQ ID NO:21 of the present application. The term "PCSK9" encompasses "full-length" unprocessed PCSK9 as well as any form of PCSK9 or any fragment thereof produced by intracellular processing. The term also includes variants of naturally occurring PCSK9, e.g., splice variants, derivative variants, substitution variants, deletion variants, and/or insertion variants or allelic variants, e.g., mutants D374Y, S R and F216L.

The term "antibody" includes any immunoglobulin, monoclonal, polyclonal, multispecific, or bispecific (bivalent) antibody that can bind to a particular antigen. A natural whole antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region (VH) and three constant regions (CH 1, CH2 and CH3, respectively), wherein a hinge region is arranged between CH1 and CH2, and each light chain consists of a variable region (VL) and a constant region (CL). Mammalian heavy chains can be classified as α, δ, ε, γ, and μ, and mammalian light chains can be classified as λ or κ. The antibody is in a Y shape, the neck part of the Y-shaped structure consists of CH2 and CH3 of two heavy chains, the CH2 and the CH3 are combined through disulfide bonds of a hinge region, and each arm of the Y-shaped structure comprises a variable region of one heavy chain and CH1, and the variable region of the Y-shaped structure is connected with a variable region of one light chain and CL through disulfide bonds. The variable regions of the light and heavy chains determine the binding of antibodies to antigens. The variable region of each chain contains three hypervariable regions (hypervariable region, HVR), termed complementarity determining regions (Complementarity-DETERMINING REGION, CDRs), wherein the CDR regions of the light chain (L) comprise LCDR1, LCDR2, LCDR3 and the CDR regions of the heavy chain (H) comprise HCDR1, HCDR2, HCDR3. CDR boundaries of the antibodies or antigen binding fragments thereof disclosed in the present invention may be defined or identified by Kabat, chothia, IMGT, abM or Contact numbering system/method. (Al-Lazikani, B., chothia, C., lesk, A.M., J.Mol.Biol.,273 (4), 927 (1997); chothia, C.et Al, J Mol biol. Dec 5;186 (3): 651-63 (1985), chothia, C.andLesk, A.M., J.Mol.Biol.,196,901 (1987), chothia, C.et Al, nature. Dec 21-28;342 (6252): 877-83 (1989), kabat E.A. Et Al, national Institutes of Health, bethesda, md. (1991), marie-Paule Lefranc et Al, nucleic ACIDS RESEARCH,1998,VOL.26,NO.1 297-303). The three CDRs are separated by laterally continuous portions called Framework Regions (FR), which are more highly conserved than the CDRs and form a scaffold-supported hypervariable loop. Antibodies can be classified into several classes according to the amino acid sequence of the heavy chain constant region, e.g., antibodies can be classified into five major classes or isomers IgA, igD, igE, igG and IgM, respectively, according to whether they contain alpha, delta, epsilon, gamma, and mu heavy chains. Several major classes of antibodies can also be classified into subclasses, such as IgG1 (gamma 1 heavy chain), igG2 (gamma 2 heavy chain), igG3 (gamma 3 heavy chain), or IgG4 (gamma 4 heavy chain), among others.

Wherein each heavy chain complementarity determining region and/or each light chain complementarity determining region is defined according to a Kabat definition, a Chothia definition, a combination of a Kabat definition and a Chothia definition, an IMGT definition, an AbM definition, or a contact definition of CDRs, preferably each CDR is defined according to a CDR definition of Kabat or a CDR definition of Chothia, or a combination of both.

The term "antigen-binding fragment" refers to an antibody fragment formed from an antibody portion containing one or more CDRs or any other antibody fragment that binds an antigen but does not have the complete antibody structure. The antigen binding fragment may bind the same antigen as the whole antibody. In certain embodiments, an antigen binding fragment may contain one or more CDRs from a particular human antibody, grafted to a framework region from one or more different human antibodies. Antigen binding fragments include, but are not limited to, fab ', F (ab ') ₂, fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv) ₂, bispecific dsFv (dsFv-dsFv '), disulfide stabilized bifunctional antibodies (dsdiabody), single chain antibody molecules (scFv), scFv dimers (diabodies), diabodies (BsFv), camelized single domain antibodies (camelized single domain antibody), nanobodies, domain antibodies, and diabodies. For example, a "Fab" fragment of an antibody refers to an antibody fragment that consists of one light chain (including the light chain variable region and the light chain constant region) disulfide-bonded to the variable region of one heavy chain and CH 1. "Fab'" fragment refers to a Fab fragment which comprises part of the hinge region. "F (ab') 2" refers to the dimer of Fab. The "Fc" fragment of an antibody is an antibody fragment in which CH2 and CH3 of the heavy chain are linked by disulfide bonds. The Fc fragment of an Antibody is responsible for a number of different effector functions, such as determining the half-life of the Antibody in vivo serum, mediating immune responses, e.g., antibody-dependent cell-mediated cytotoxicity (anti-body-DEPENDENT CELL-mediated cytotoxicity, ADCC), activating complement-dependent cytotoxicity (Complement Dependent Cytotoxicity, CDC) or Antibody-dependent cell-mediated phagocytosis (Antibody Dependent Cellular Phagocytosis, ADCP), but not involved in antigen binding. The "Fv" portion of an antibody refers to the smallest antibody fragment that contains the complete antigen binding site. Fv fragments consist of a variable region of the light chain and a variable region of the heavy chain. "Single chain Fv antibody (scFv)" refers to an engineered antibody in which the light chain variable region is linked directly to the heavy chain variable region or via a single peptide chain (Huston JS et al, proc NATL ACAD SCI USA, 85:5879 (1988)). "(dsFv) 2" comprises three peptide chains, meaning that two VH groups are linked by a polypeptide linker and are bound to two VL groups by disulfide bonds. A "bispecific ds bifunctional antibody" comprises VL1-VH2 (linked by a polypeptide linker) and VH1-VL2 (also linked by a polypeptide linker), which are joined by disulfide bonds between VH1 and VL 1. A "bispecific dsFv" or "dsFv-dsFv" comprises three polypeptide chains, a VH1-VH2 fragment, wherein the heavy chains of both are joined by a polypeptide linker (e.g., a long elastic linker) and are bound to the VL1 and VL2 fragments, respectively, by disulfide bonds, each pair of heavy chain light chains paired by disulfide bonds having a different antigen specificity. "scFv dimer" is a diabody or diabody (BsFv) comprising two VH-VL (linked by a polypeptide linker) fragments that dimerize, wherein the VH of one fragment cooperates with the VL of the other fragment to form two binding sites that can be targeted to bind the same antigen (or antigen-binding epitope) or different antigens (or antigen-binding epitopes). In other embodiments, the "scFv dimer" is a bispecific bifunctional antibody comprising VL1-VH2 (linked by a polypeptide linker) and VH1-VL2 (linked by a polypeptide linker) that are linked to each other, wherein VH1 and VL1 cooperate, VH2 and VL2 cooperate, and each cooperating pair has a different antigen specificity. "Single chain antibody Fv-Fc (scFv-Fc)" refers to an engineered antibody consisting of scFv and an antibody Fc fragment. "camelized single domain antibodies (Camelized single domain antibody)", "Heavy chain antibodies" or "hcabs" refer to antibodies that contain two VH domains but no light chain (Riechmann l. And Muyldermans S.,J Immunol Methods.Dec 10;231(1-2):25-38(1999);Muyldermans S.,J Biotechnol.Jun;74(4):277-302(2001);WO94/04678;WO94/25591;U.S.Patent No.6,005,079). Heavy chain antibodies were originally found in camelidae (including camels, dromedaries and llamas). The camelized antibody (camelized antibodies) has the full function of antigen binding despite deletion of the light chain (Hamers-Casterman C. Et al, nature. Jun 3;363 (6428): 446-8 (1993); nguyen VK. et al ,"Heavy-chain antibodies in Camelidae：a case of evolutionary innovation,"Immunogenetics.Apr; 54(1):39-47(2002);Nguyen VK. et al, immunology. May;109 (1): 93-101 (2003)). The variable region (VHH domain) of a heavy chain antibody is the antigen binding unit generated by the smallest acquired immunity known to date (Koch-Nolte F. Et al, FASEB J. Nov;21 (13): 3490-8.Epub 2007Jun 15 (2007)). a "nanobody" is an antibody fragment consisting of a VHH domain from a heavy chain antibody and two constant regions CH2 and CH 3. "domain antibody" refers to an antibody fragment containing only one heavy chain variable region or one light chain variable region. In some cases, two or more VH domains are covalently bound by a polypeptide linker and form a bivalent domain antibody. The two VH domains of a diabody can target the same or different antigens. A "diabody" includes a small antibody fragment with two antigen-binding sites, which fragment contains a VH domain and a VL domain (e.g.VH-VL or VL-VH) linked together in the same polypeptide chain (Holliger P. Et al, procNatl Acad Sci USA. Jul 15;90 (14): 6444-8 (1993); EP404097; WO 93/11161). The linker between the two domains is so short that the two domains on the same strand cannot mate with each other, forcing the two domains to mate with the complementary domains of the other strand, forming two antibody binding sites. The two antibody binding sites may be targeted to bind the same or different antigens (or antigen binding epitopes).

The term "fully human antibody or antigen binding fragment" refers to an antibody or antigen binding fragment whose amino acid sequence corresponds to that of an antibody produced by a human or human immune cell or derived from a non-human source, such as a transgenic non-human animal utilizing a human antibody repertoire, or other sequence encoding a human antibody. In some embodiments, the fully human antibody does not comprise amino acid residues derived from a non-human antibody (particularly antigen binding residues). In some embodiments, the fully human antibodies are prepared using recombinant methods. For example, transgenic animals such as mice can be made to carry transgenes or transchromosomes of human immunoglobulin genes and are therefore capable of producing fully human antibodies upon immunization with a suitable antigen such as human PCSK 9. Fully human antibodies can be isolated from the transgenic animal or alternatively, can be prepared by hybridoma technology by fusing spleen cells of the transgenic animal with an immortal cell line to produce hybridoma cells that secrete the fully human antibody. Exemplary transgenic animals include, but are not limited to, omni rats whose endogenous rat immunoglobulin genes are knocked out to express inactivation while being genetically engineered to contain a functional recombinant human immunoglobulin locus, omni mice whose endogenous mouse immunoglobulin genes are knocked out to express inactivation while being genetically engineered to contain a recombinant human immunoglobulin locus with J-locus deletions and C-kappa mutations, omniFilc being transgenic rats whose endogenous rat immunoglobulin genes are knocked out to express inactivation while being genetically engineered to contain a recombinant human immunoglobulin locus (Osborn M.et al,Journal of Immunology,2013,190:1481-90;Ma B.et al,Journal of Immunological Methods400-401(2013)78-86;Geurts A.et al,Science,2009,325:433; with a single, shared, recombinant VkJk light chain and functional heavy chain, U.S. Pat. No. 8,907,157, european patent EP2152880B1, european patent EP2336329B 1. Other suitable transgenic animals may also be used, for example, huMab mice (Lonberg, N.et al Nature368 (6474): 856-859 (1994)), xeno mice (Mendez et al Nat Genet.,1997, 15:146-156), transChromo mice (Ishida et al cloning STEM CELLS,2002, 4:91-102), velocImmune mice (Murphy et al Proc NATL ACAD SCI USA,2014, 111:5153-5158), kymouse transgenic mice (Lee et al Nat Biotechnol,2014, 32:356-363), or transgenic rabbits (Flisikowska et al PLoS One,2011, 6:21045).

The term "humanized antibody or antigen-binding fragment" refers to an antibody or antigen-binding fragment that includes CDRs derived from a non-human animal, FR regions derived from a human, and constant regions derived from a human. Since humanized antibodies or antigen binding fragments have reduced immunogenicity, they are useful as therapeutic agents in humans. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In some embodiments, the humanized antibody or antigen binding fragment consists essentially of human sequences except that the CDR sequences are non-human. In some embodiments, the human derived FR region may comprise the same amino acid sequence as the human antibody from which it is derived, or it may comprise some amino acid change, e.g., no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid change. In some embodiments, the amino acid change may be present in only the heavy chain FR region, only the light chain FR region, or both chains.

The term "chimeric" refers to antibodies or antigen-binding fragments that have a portion of the heavy and/or light chain derived from one species, and the remainder of the heavy and/or light chain derived from a different species. In one illustrative example, a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse.

The term "specific binding" refers to a reaction between an antibody and an antigen. In some embodiments, the antibodies or antigen binding fragments thereof of the application specifically bind to rat, human and/or monkey PCSK9 and their binding affinity (K _D)≤10^-6 M (e.g.. 5X 10 ^-7 M, preferably 1.0X 10 ^-8 M, more preferably 1.0X 10 ^-9 M.) K _D in the present application refers to the ratio of dissociation rate to binding rate (K _off/k_on) which can be determined by means of surface plasmon resonance, e.g.using a Biacore instrument.

The term "isotype antibody" refers to a class of antibodies encoded by heavy chain constant region genes. For example, "IgG isotype antibody" refers to the IgG form to which the heavy chain constant region of an antibody belongs. The heavy chain constant regions are identical for all of the same type of antibody and differ between different types of antibodies. For example, an antibody in the form of an IgG1 refers to an Ig domain whose heavy chain constant region Ig domain is an IgG1 domain.

The term "percent sequence identity" refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to a reference sequence after an amino acid sequence (or nucleic acid sequence) alignment is performed and, if necessary, a gap is introduced to maximize the number of identical amino acids (or nucleic acids). Conservative substitutions of the amino acid residues may or may not be considered the same residue. The sequences may be aligned to determine the percent sequence identity of amino acid (or Nucleic acid) sequences by means disclosed in the art, such as BLASTN, BLASTP (national center for Biotechnology information website (NCBI), also see, altschul S.F. et al, J.mol.biol.,215:403-410 (1990), stephen F. Et al, nucleic Acids Res.,25:3389-3402 (1997)), clustalW2 (European institute of biological information website, see, higgins D.G. et al, methods in Enzymology,266:383-402 (1996), larkin M.A. et al, bioinformation (Oxford, england), 23 (21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software. The person skilled in the art can use the default parameters of the tool or adjust the parameters appropriately according to the needs of the alignment, for example by choosing an appropriate algorithm.

The term "LDL receptor (LDLR)" is a cell surface chimeric protein having 839 amino acids (after removal of the 21 amino acid signal peptide) that mediates the endocytosis of LDL-C and the removal of LDL-C from the blood. Representative amino acid sequences of human LDL-R and mRNA nucleic acid sequences encoded thereby are disclosed by GenBank accession numbers P01130.1 and NM-000527.4, respectively. When PCSK9 binds to the LDL receptor, the antibody is destroyed and LDL-C cannot be removed from the blood. Conversely, when PCSK9 is blocked, there will be more LDL receptors on the liver surface and more LDL cholesterol will be removed from the blood. As used herein, an "anti-PCSK 9 antibody" refers to an antibody capable of specifically binding PCSK9 (e.g., human or monkey PCSK 9) that has sufficient affinity to provide diagnostic and/or therapeutic uses.

The term "vector" refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted and expressed. Vectors may be used to transform, transduce or transfect host cells such that the genetic material elements carried thereby are expressed within the host cells, such vectors also being referred to herein as "expression vectors". Vectors include plasmids, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages such as lambda or M13 phages, animal viruses, and the like. Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma-virus-vacuolated viruses (e.g., SV 40). The vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. The vector may also include components that assist in its entry into the cell, including but not limited to viral particles, liposomes, or protein shells.

The term "host cell" refers to a cell that is an exogenous polynucleotide and/or vector. Host cells include "transformants" and "transformed cells" which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may comprise the mutation. Mutant progeny having the same function or biological activity, selected or selected in the initially transformed cells, are encompassed by the present application. Preferably, mammalian host cells, such as, for example, SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651), human embryonic kidney cell line (293 or suspension cultured 293 cell subclone, graham et al, J.Gen Virol.36:59 (1977)), baby rat kidney cells (BHK, ATCC CCL 10), chinese hamster ovary cells/-DHFR (CHO, urlaub et al, proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse testis support cells (TM 4, mather, biol. Reprod.23:243-251 (1980)), monkey kidney cells (CV 1ATCC CCL 70), african green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), burrole rat liver cells (BRL 3A, ATCC 1442), human lung cells (ATCC W138), human liver cancer cells (HeLa, ATCC 75, hex.H.43-251 (1985, mr. CCL 4, mr. 3, mr. 4, or Flex. 6, mr. 3, mr. 4, mr. 3, mr. 6, mr. 4, or the like.

The term "LDL-C" refers to low density lipoprotein cholesterol. "HDL-C" refers to high density lipoprotein cholesterol. LDL and HDL belong to 5 major groups of lipoproteins, chylomicrons, very Low Density Lipoproteins (VLDL), intermediate Density Lipoproteins (IDL), low Density Lipoproteins (LDL) and High Density Lipoproteins (HDL) (order from large particles to the most dense/smallest particles). LDL (also known as particulate-containing "bad" cholesterol) is capable of transporting lipid/sterol molecules, such as cholesterol (i.e., LDL-C), to the arterial wall, attracting macrophages, and thus inducing atherosclerosis. In contrast, HDL (also known as "good" cholesterol containing particles) is capable of removing lipid molecules, such as cholesterol (i.e., HDL-C), from macrophages on the animal wall. Thus, high levels of LDL-C are major risks of cardiovascular diseases (CVD), such as peripheral arterial disease, coronary Arterial Disease (CAD), such as angina pectoris, myocardial infarction (commonly known as heart disease), hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, atherosclerosis, stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, heart valve disease, myocarditis, aortic aneurysm, peripheral arterial disease, obesity, hepatobiliary disease, nephrotic syndrome, hypothyroidism, and venous thrombosis.

"Treating" or "treatment" of a disease or condition refers to alleviating a disease or condition, reducing the rate at which a disease or condition arises or progresses, reducing the risk of developing a disease or condition, or delaying the development of a condition associated with a disease or condition, reducing or terminating a condition associated with a disease or condition, producing complete or partial reversal of a disease or condition, curing a disease or condition, or a combination thereof.

"Preventing" includes inhibition of the occurrence or progression of a disease or disorder or a symptom of a particular disease or disorder. In some embodiments, the subject with a family history is a candidate for a prophylactic regimen. In general, the term "prevention" refers to administration of a drug prior to the occurrence of a sign or symptom, particularly in a subject at risk.

By "PCSK 9-associated disease or condition" is meant a disease or condition caused or characterized by a change in PCSK9, such as an alteration in expression level, activity, and/or the presence of a variant or mutation of PCSK 9. Examples of diseases or conditions associated with PCSK9 include, but are not limited to, hyperlipoproteinemia, hyperlipidemia, dyslipidemia (e.g., elevated total cholesterol, elevated LDL, elevated triglycerides, elevated VLDL and/or reduced HDL), hypercholesterolemia, heart disease, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, lameness, type II diabetes, hypertension, cardiovascular disease or condition, inflammation or autoimmune disease. Methods for identifying/diagnosing the above-mentioned diseases or symptoms are known in the art.

The term "subject" includes any human or non-human animal. "non-human animals" include all vertebrates, such as mammals (including, but not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., monkeys), rabbits, and rodents (e.g., mice and rats)), and non-mammals (e.g., poultry, amphibians, and reptiles). In some embodiments, the subject is a human.

The term "therapeutically effective amount" or "effective dose" refers to a dose or concentration effective to achieve prevention or amelioration of symptoms associated with a disease or disorder and/or lessening the severity of a disease or disorder at a desired dose for a desired period of time. The therapeutically effective amount of the formulations, antibodies, or antigen binding fragments thereof, or compositions of the invention may vary depending on a variety of factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount may also be considered to be any toxic or detrimental effect of the formulation, antibody or antigen-binding fragment thereof or composition that is less than a therapeutically beneficial effect.

The term "formulation" refers to a composition comprising at least one active ingredient and at least one inactive ingredient suitable for administration to animals, preferably mammals (including humans). "liquid formulation" refers to a formulation in liquid form. The liquid formulations of the invention comprise (1) an anti-PCSK 9 antibody or antigen-binding fragment thereof, (2) a buffer, (3) an osmolality regulator, and/or (4) a surfactant. The composition of the formulation of the present invention may be as shown in the liquid formulation embodiments described hereinbefore. The liquid preparation of the present invention is preferably an injection, more preferably a subcutaneous injection or intramuscular injection, and most preferably a subcutaneous injection.

The term "buffer" refers to a pH buffer. Preferably, the buffer is capable of maintaining the pH of the liquid formulation of the present invention at about 4.5-6.5, preferably about 5.5-6.5. The buffer is present in the liquid formulation at a concentration of about 1-50mmol/L, preferably at a concentration of 5-50mmol/L, more preferably at a concentration of 10-30mmol/L. The buffer is selected from histidine, acetate, succinate, or glutamate, and a preferred buffer is histidine buffer.

"Histidine buffer" refers to a buffer comprising histidine. Histidine buffers include histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate. In one embodiment, the histidine buffer consists of L-histidine and L-histidine hydrochloride in a concentration ratio of 0.2:1 to 3:1.

The term "excipient" refers to a non-therapeutic agent that can be added to a formulation to provide desired characteristics (e.g., consistency, increased stability) and/or to regulate osmotic pressure, which non-therapeutic agent is added to the formulation to stabilize the physicochemical and biological characteristics of the active ingredient. Examples of common excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.

The term "osmolyte" is a molecule that contributes to the osmolarity of a solution. By "osmolality adjusting agent" in accordance with the present invention is meant an agent that is capable of adjusting, altering or optimizing the osmolality of the liquid formulation of the anti-PCSK 9 antibody of the present invention, preferably, the osmolality of the liquid formulation of the present invention is adjusted, not only to maintain the isotonicity of the liquid formulation, but also to maximize the stability of the anti-PCSK 9 antibody or antigen-binding fragment thereof of the present invention while minimizing the discomfort to the patient upon administration. Examples of osmolality adjusting agents suitable for varying the osmolality include, but are not limited to, amino acids (proline, arginine, cysteine, histidine, etc.), salts (sodium chloride, potassium chloride, calcium chloride, etc.) and/or sugars (sucrose, trehalose, glucose, mannitol, sorbitol and the like), with the preferred osmolality adjusting agent being proline or sucrose, more preferably proline.

The term "surfactant" refers to a substance that, when added in small amounts, causes a significant change in the interfacial state of the solution system. For example, proteins (e.g., antibodies) can be protected from air/solution interface induced stress, solution/surface induced stress, thereby reducing aggregation of the protein or the formation of particulates in the formulation. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 80, polysorbate 60, polysorbate 40, or polysorbate 20), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycols, polyoxyethylene-stearates, polyoxyethylene alkyl ethers such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamer, pluronic), sodium Dodecyl Sulfate (SDS). Preferred surfactants of the invention are nonionic surfactants such as polysorbate 80 or polysorbate 20. The concentration of surfactant in the liquid formulation is 0.001-1% (w/w), preferably 0.01-0.05% (w/w), more preferably 0.01% (w/w), 0.02% (w/w) and 0.04% (w/w).

The term "viscosity" may be "kinematic viscosity" or "absolute viscosity". "kinematic viscosity" is a measure of the resistive flow of a fluid under the influence of gravity. When two fluids of the same volume are placed in two identical capillary viscometers, respectively, and flow by gravity, the viscous fluid takes longer to flow through the capillary than the less viscous fluid. "absolute viscosity", sometimes referred to as dynamic viscosity or simple viscosity, is the product of the kinematic viscosity and the fluid density (absolute viscosity = kinematic viscosity x density). The dimension of the kinematic viscosity is L ²/T (where L is length and T is time). Typically, kinematic viscosity is expressed in centistokes (cSt). The International units of kinematic viscosity are mm ²/s (i.e., lcSt). Absolute viscosity is expressed in centipoise (cP) units. The units in international units of absolute viscosity are millipascal-seconds (mPa-s), where 1 cp= lmPa-s.

The term "low level viscosity" refers to an absolute viscosity of less than about 15 cp. "medium horizontal viscosity" refers to an absolute viscosity of between about 15cp and about 35 cp. The absolute viscosity of the liquid formulations of the antibodies of the present application is about 6.0cp, about 8.0cp, about 9.0cp, about 10.0cp, about 11.0cp, or about 12.0cp, as measured using standard viscosity measurement techniques, indicating that the liquid formulations of the present application have a "low level viscosity". In some embodiments, the inventors of the present application have surprisingly found that proline may not only act as an osmotic regulator, but may also reduce the viscosity of the antibody liquid formulations of the present application when formulated with anti-PCSK 9 antibodies and buffers.

The term "isotonic" means that the liquid formulation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing point depression osmometer.

The term "vehicle" refers to a substance used to mix, disperse, solubilize a test or control without affecting the test results. Solvents useful in the present invention include, but are not limited to, water for injection, salts (e.g., physiological saline), saccharides (e.g., dextrose injection), organic solvents for injection (including, but not limited to, oil for injection, ethanol, propylene glycol, and the like), or combinations thereof.

By "stable" antibody formulation is meant a formulation in which the antibody substantially retains its physicochemical stability and/or biological activity during manufacturing and/or during storage. Antibody formulations can be considered stable even if the contained antibody fails to retain 100% of its physicochemical properties or biological functions after storage for a certain period of time. For example, the formulation can maintain more than 90% of the antibody structure or function after storage for a period of time, and can be considered a "stable" formulation. The criteria for stability are, for example, that the liquid formulation is colorless or clear to slightly milky, that the concentration of the antibody, the pH and the osmotic pressure of the formulation vary by no more than.+ -.10%, that the biological activity of the antibody is 60 to 140%, preferably 80 to 120%, that the antibody monomer of the formulation degrades by no more than 10%, preferably 5%, that the aggregates formed in the formulation do not exceed 10%, preferably 5%, or that the hydrolysates formed in the formulation do not exceed 10%, preferably 5% of the reference antibody.

No significant increase in aggregation, precipitation and/or denaturation of the antibodies in the formulation is indicated if the color and/or clarity of the formulation is visually observed, or detected by Differential Scanning Calorimetry (DSC), size exclusion chromatography (SEC-HPLC) and Dynamic Light Scattering (DLS), indicating that the antibodies in the formulation "retain their physical stability. An antibody "retains its chemical stability" if no significant chemical change occurs in the antibody in the formulation, its chemical structure remains intact. Most of the disruption of chemical stability can be attributed to the formation of covalent modifications of the protein (e.g., covalent aggregates, degradants, or charge isomers) and non-covalent modifications of the protein (e.g., non-covalent aggregates). The changes in the chemical structure of the antibody protein (e.g., variants of different molecular weights or charges) can be determined using methods known to those skilled in the art. Such methods include, but are not limited to, detection of hydrolysates of antibodies using SEC-HPLC and sodium dodecyl sulfate capillary gel electrophoresis (CE-SDS), detection of degraded fragments of antibodies and other protein molecules having a molecular weight less than that of antibodies using non-reducing calipers, or detection of charge isomers of antibodies using cation exchange chromatography (CE-HPLC). An antibody "retains its biological activity" if the biological activity of the antibody in the formulation during storage is still within the range of biological activity exhibited by the formulation at the time of its preparation. Methods for detecting the biological activity of an antibody include, but are not limited to, antigen binding ELISA assays or cell activity assays of an antibody.

By "pharmaceutically acceptable" is meant that the carrier, vehicle, diluent, adjuvant and/or salt is, in general, chemically and/or physically compatible with the other ingredients in the formulation and physiologically compatible with the subject.

Anti-PCSK 9 antibodies

Exemplary anti-PCSK 9 antibodies included in the liquid formulations of the present application are set forth in detail in published patent application CN201710816808.0, the disclosure of which is incorporated herein by reference in its entirety. The anti-PCSK 9 antibodies of the application are preferably fully human antibodies, preferably produced by recombinant methods. In one embodiment, a transgenic rat containing a human immunoglobulin variable region gene is immunized with a human PCSK9 protein(OMT) to obtain fully human antibodies. The non-limiting, exemplary antibody used in the examples of the present invention, designated "antibody A", is a fully human antibody that specifically binds to human PCSK9, and the amino acid sequences and nucleotide sequences of the CDR regions, heavy chain variable regions and light chain variable regions are shown in Table 1, wherein the CDR regions in Table 1-1 are defined using the kabat method and the amino acid sequences of the CDR regions defined by the different definition methods are shown in Table 1-2:

TABLE 1-1 amino acid sequences of CDR regions, heavy chain variable regions and light chain variable regions of anti-PCSK 9 antibody (antibody A) and sequence numbers thereof

TABLE 1-2 amino acid sequences of CDR regions defined by different definition methods

In one embodiment, the anti-PCSK 9 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown as SEQ ID NO. 1, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 2, the amino acid sequence of HCDR3 is shown as SEQ ID NO. 3, the amino acid sequence of LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 6. In one embodiment, an anti-PCSK 9 antibody or antigen-binding fragment of the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequence of VH is shown in SEQ ID NO. 7 and the amino acid sequence of VL is shown in SEQ ID NO. 8. In some embodiments, the anti-PCSK 9 antibody or antigen-binding fragment thereof comprises one or more CDR sequences that have at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%, or 99% identity to a CDR sequence set forth in table 1, while retaining a binding affinity similar to or even greater than that of a parent antibody (e.g., antibody a) having substantially the same sequence but whose corresponding CDR sequence has 100% sequence identity to a sequence set forth in table 1. In some embodiments, the anti-PCSK 9 antibody or antigen-binding fragment thereof has at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%, or 99% identity to a VH or VL sequence set forth in table 1, while retaining a similar or even higher binding affinity to a parent antibody to human PCSK9 than the parent antibody.

Preparation and purification of anti-PCSK 9 antibodies

The invention also provides polynucleotides encoding anti-PCSK 9 antibodies or antigen-binding fragments thereof. In some embodiments, the polynucleotide comprises one or more nucleotide sequences as set forth in table 1 that encode a CDR region sequence, a heavy chain variable region sequence, and/or a light chain variable region sequence as set forth in table 1.

In some embodiments, the polynucleotide sequence encoding the heavy chain variable region has at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identity to SEQ ID NO 19 shown in Table 1. In some embodiments, the polynucleotide sequence encoding the light chain variable region has at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identity to SEQ ID NO. 19 shown in Table 1. In some embodiments, the percentage of identity is derived from the degeneracy of the genetic code, while the encoded protein sequence remains unchanged.

In some embodiments, vectors comprising polynucleotides encoding the anti-PCSK 9 antibodies or antigen-binding fragments thereof (e.g., sequences shown in table 1) may be introduced into host cells for cloning (amplification of DNA) or gene expression using recombinant techniques well known in the art. In some embodiments, the antibodies or antigen binding fragments thereof can be made by methods of homologous recombination well known in the art. The DNA encoding the antibody or antigen binding fragment thereof may be isolated and sequenced by conventional methods, for example, oligonucleotide probes may be used which specifically bind to the genes encoding the heavy and light chains of the antibody or antigen binding fragment thereof. A variety of vectors are available for selection as described above. Vector elements typically include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer sequence, a promoter (e.g., SV40, CMV or EF-1. Alpha.), a transcriptional sequence, and a termination sequence. In some embodiments, the vector system includes mammalian, bacterial, yeast systems, and the like, and suitable vectors may include plasmids or viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses). Such plasmids include, but are not limited to, pALTER、pBAD、pcDNA、pCal、pL、pET、pGEMEX、pGEX、 pCI、pCMV、pEGFP、pEGFT、pSV2、pFUSE、pVITRO、pVIVO、pMAL、pMONO、pSELECT、pUNO、 pDUO、Psg5L、pBABE、pWPXL、pBI、p15TV-L、pPro18、pTD、pRS420、pLexA、pACT2 and the like, commercially available or commercially available vectors.

Vectors comprising polynucleotides encoding the anti-PCSK 9 antibodies or antigen-binding fragments thereof may be introduced into host cells for cloning or gene expression. Host cells suitable for cloning or expressing the DNA in the vectors of the present invention may be prokaryotic cells, yeast or eukaryotic cells as described above, preferably eukaryotic cells, more preferably mammalian host cells. In some preferred embodiments, the host cell is a CHO cell or 293 cell.

Host cells are transformed with the vectors described above for expression or cloning of the anti-PCSK 9 antibody or antigen-binding fragment thereof and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformed cells, or amplifying genes encoding the sequences of interest.

The host cells used in the present invention for producing the antibodies or antigen-binding fragments thereof may be cultured in a variety of media. Commercially available media such as Ham's F (Sigma), minimal essential media such as MEM (Sigma), RPMI-1640 (Sigma), or Dulbecco's Modified Eagle's Medium (DMEM, sigma) may be used to culture the host cells. In addition, any of the media described in Ham et al, meth.Enz.58:44 (1979), barnes et al, anal biochem.102:255 (1980), U.S. Pat. No. 3, 4,767,704, U.S. Pat. No. 4,657,866, U.S. Pat. No. 5, 4,927,762, U.S. Pat. No. 4,560,655 or U.S. Pat. No. 5,122,469, WO 90/03430, WO 87/00195, or U.S. Pat. No. 30,985 may be used as the medium for the host cells. These media may be supplemented with necessary hormones and/or other growth factors (e.g., insulin, transferrin or epidermal growth factor), salts (e.g., sodium chloride, calcium chloride, magnesium chloride and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenylate and thymine), antibiotics (e.g., gentamicin), trace elements (defined as inorganic compounds at final concentrations typically in the micromolar range), and glucose or an equivalent energy source. The medium may also contain any other necessary additives at appropriate concentrations as known in the art. The conditions of the medium, such as temperature, pH and the like, are those previously used to select host cells for expression and are well known to those of ordinary skill.

When recombinant techniques are used, the antibody or antigen-binding fragment thereof may be produced intracellularly, in the parietal membrane space, or directly secreted into the culture medium. If the antibody is generated intracellularly, the host cells or particle debris of the lysed fragments are first removed, for example, by centrifugation or sonication. The literature Carter et al, bio/Technology 10:163-167 (1992) describes a method for isolating antibodies secreted into the E.coli wall membrane space. Briefly, cell paste (CELL PASTE) was thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethanesulfonyl fluoride (PMSF) for about 30 minutes or more. Cell debris was removed by centrifugation. If the antibody or antigen binding fragment thereof is secreted into the cell culture medium, the supernatant of the expression system is typically first concentrated using a commercially available protein filter. Protease inhibitors such as PMSF may be added during any of the preceding steps to inhibit protein degradation, as well as antibiotics to prevent the growth of occasional contaminants.

Antibodies or antigen-binding fragments thereof produced from the cells may be purified using purification methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique. The kind of the antibody and the presence of any immunoglobulin Fc domain in the antibody determine whether protein a is suitable as its affinity ligand. Protein A can be used to purify antibodies based on the heavy chain of human gamma 1, gamma 2 or gamma 4 (LINDMARK ET al, J.Immunol. Meth.62:1-13 (1983)). Protein G is suitable for all murine isomers and human gamma 3 (Guss et al, EMBO J.5:15671575 (1986)). Agarose is the most commonly used affinity ligand attachment matrix, but other matrices may be used. Mechanically stable matrices such as controlled pore glass or poly (styrene) benzene can achieve faster flow rates and shorter processing times than agarose. If the antibody contains a CH3 domain, it can be purified using Bakerbond ABX.TM resin (J.T.Baker, phillipsburg, N.J.). Other protein purification techniques may also be determined based on the antibody obtained as desired, such as fractionation in ion exchange columns, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin sepharose chromatography based on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

After any preliminary purification steps, the mixture containing the antibody of interest and impurities may be treated by low pH hydrophobic interaction chromatography, preferably with a wash buffer having a pH of about 2.5-4.5, preferably at low salt concentrations (e.g., from about 0 to 0.25M salt concentration). Analytical methods for evaluating stability of liquid formulations containing anti-PCSK 9 antibodies.

The antibody liquid formulations of the invention contain high concentrations of anti-PCSK 9 antibody, a pharmaceutically acceptable buffer and excipients. The physicochemical properties and biological activity of the antibody should be maintained when the antibody preparation is formulated. Methods for evaluating the stability of physicochemical properties of antibody formulations include, but are not limited to DSC, DLS, UV spectrophotometry, SEC-HPLC, CE-SDS, CEX-HPLC, and/or non-reducing calipers. Wherein DSC (Pharm. Res.,15:200,1998; pharm. Res.,9:109, 1982) detects the protein denaturation temperature and glass transition temperature to analyze the stability of the protein in the natural (folded) state, DLS (American Lab., nov. 1991) detects the average diffusion coefficient of the solution, analyzes the content of soluble and insoluble aggregates, UV spectrophotometry detects the absorbance of the preparation at 278nm to analyze the concentration of antibody protein, SEC-HPLC can separate higher molecular weight aggregates or lower molecular weight degradants (such as fragments) from the natural antibody, analyze the percentage of the natural antibody (or antibody monomers), the percentage of soluble hydrolysates (or degradants with lower molecular weight, such as fragments) and the percentage of aggregates (i.e., determined based on the area of the natural antibody peak, the area of hydrolysates or aggregates compared to the total area of all antibody peaks), CE-SDS is used to detect hydrolysates in the preparation, wherein the reducing CE-SDS (rCE) can separate higher molecular weight aggregates or lower molecular weight degradants (such as fragments) from the natural antibody, the reducing protein, the reducing CE-SDS-protein can separate the reducing protein from the main antibody, the main chain (or the main chain of the reducing protein from the main chain, and the main chain (SDS-protein) can elute from the main antibody, the main chain (or the main chain) and the main chain can elute from the main chain (SDS-chain) by the main chain. The charge isomers of the natural antibodies are determined from the relative percentage change in the ratio of the areas of the main, acidic and basic peaks to the total area of all antibody peaks, the stability of the antibodies is inversely proportional to the percentage of the acidic form of the antibodies, the non-reducing calipers detect degraded fragments of antibodies and other protein molecules having a molecular weight less than that of antibodies in the formulation, the potency or biological activity of the anti-PCSK 9 antibodies can be assessed by their ability to bind to the antigen PCSK9, and the specific binding ability of the antibodies to the antigen can be quantitatively detected using methods known to those skilled in the art, such as immunoassays such as ELISA (enzyme linked immunosorbent assay).

Detailed Description

The present invention will be described in further detail with reference to examples, but embodiments of the invention are not limited thereto.

Meaning of chinese and english abbreviations:

DSC (DIFFERENTIAL SCANNING Calorimetry) differential scanning calorimetry

DLS (Dynamic light scattering) dynamic light Scattering

HPLC (High Performance Liquid Chromatography) high performance liquid chromatography

CEX (Cation-exchange) Cation exchange

SEC (Size-Exclusion Chromatography) Size exclusion chromatography

CEX-HPLC cation exchange chromatography

SEC-HPLC size exclusion chromatography

CE (Capillary Electrophoresis) capillary electrophoresis

SDS (Sodium Dodecyl Sulfate) sodium dodecyl sulfate

CE-SDS-sodium dodecyl sulfate capillary gel electrophoresis

The anti-PCSK 9 antibody used in the embodiment of the present invention is obtained by screening the method for antibody 18.156.8 in examples 1 to 3 of the disclosure patent CN201710816808.0, and a CHO cell line capable of stably expressing the anti-PCSK 9 antibody of the present invention is constructed, and after cell culture, the supernatant is taken and purified by steps such as affinity chromatography, ion exchange, ultrafiltration, etc., to obtain the stock solution of the anti-PCSK 9 antibody (also referred to as antibody a in the present invention).

Example 1 buffer System and pH screening experiment

According to the invention, anti-PCSK 9 antibody preparations under different buffer systems are prepared according to the formula of Table 2, and stability of 70mg/mL anti-PCSK 9 antibody (such as antibody A) under three buffer systems of acetic acid/sodium acetate, histidine/histidine hydrochloride and succinic acid/sodium succinate with different proportions and different pH values thereof are examined by carrying out a high-temperature acceleration stability experiment at 40+/-2 ℃ so as to select the optimal buffer system suitable for the anti-PCSK 9 antibody according to the invention by detecting appearance, pH values, DSC, protein content, SEC-HPLC, CEX-HPLC, non-reducing Caliper and the like of the anti-PCSK 9 antibody sample under different buffer systems.

Adding the stock solution of the anti-PCSK 9 antibody (antibody A) into an ultrafiltration centrifuge tube (molecular weight cut-off is 30K _D), centrifugally concentrating, adding buffer solutions with different pH values shown in table 2, diluting, continuously concentrating, repeating the process for more than 3 times until the anti-PCSK 9 antibody is completely replaced. The concentration of the replaced antibody is adjusted to 70mg/ml, the antibody is sterilized and filtered, and then is respectively filled in penicillin bottles, and a plug is added for capping, so that the F1-F7 sample is finally prepared.

TABLE 2 formulation of anti-PCSK 9 antibodies in different buffer systems

Each sample was placed at 40±2 ℃, taken out at week 0, week 2 and week 4 respectively, and analyzed and detected, the specific detection items are shown in table 3, and whether the charge properties of the protein were changed due to aggregation, degradation and modification was examined, and the percentage of the natural form (protein monomer) and the aggregated form of the anti-PCSK 9 antibody was detected by SEC-HPLC; the percentage of the acidic and basic forms of the antibody (main protein peak) was detected by CEX-HPLC, and the percentage of the anti-PCSK 9 antibody and its degraded fragments and other protein molecules having a molecular weight less than that of the antibody was detected by non-reducing calipers.

SEC-HPLC method by gel chromatography (5 μm, 7.8X100 mm), and subjecting the sample to dilution and then to isocratic elution with 20. Mu.l of sample.

CEX-HPLC method, in which a weak cation column (10 μm, 4X 250 mm) is used, 100. Mu.l of sample is introduced after dilution, and pH gradient elution is performed with 4mM 2-Methylpiperazine,4mM Imidazole,4mM Tris as mobile phase.

Non-reducing calipers. Samples were analyzed using a LabChip GXII Touch HT instrument from PerkinElmer, inc. HT Protein Express LabChip. The samples were diluted and incubated with sample denaturing solution (containing 100mM NEM and 10% SDS) for 10 minutes, and finally the samples were transferred to 96-well plates for loading analysis.

TABLE 3 anti-PCSK 9 antibody sample detection terms under different buffer systems

(X represents the appearance, pH, protein content, SEC-HPLC, CEX-HPLC, non-reducing Caliper; Y represents DSC).

Table 4 results of stability experiments at 40±2 ℃ for anti-PCSK 9 antibody samples under different buffer systems

As shown in Table 4, when the buffer system is histidine/histidine hydrochloride and the pH value is 5.5-6.5 (sample No. F4-F6), after the buffer system is placed for 4 weeks under the conditions of 40+ -2 ℃ and 75% + -5% RH, the main peak content of the sample is over 94% and the main peak content is reduced by about 1.5% as detected by SEC-HPLC, and the purity of the anti-PCSK 9 antibody is reduced by 1.5% or less as detected by non-reducing Caliper. Compared with other prescriptions, when the buffer system is histidine/histidine hydrochloride buffer and the pH value is 6.0 (sample No. F5), after the sample is placed under 40+/-2 ℃ and 75+/-5% RH conditions for 4 weeks, the SEC-HPLC main peak (or protein monomer) content of the sample is highest (about 94.8%), the main peak content is reduced minimally (about 1.5%), the non-reducing Caliper purity is reduced minimally (about 0.9%), and in addition, the stability of F5 is also found to be better from detection results of DSC and CEX-HPLC. When succinic acid/sodium succinate was chosen as the buffer system, the sample had a slight floc precipitation and was stored at 40±2 ℃ with the SEC-HPLC and CEX-HPLC main peak levels dropping the most (around 4.5% and 5.0%, respectively). When acetic acid/sodium acetate is selected as a buffer system, the sample has inferior purity, DSC stability and SEC-HPLC protein monomer content as compared with histidine/histidine hydrochloride in the buffer system. Based on these results, histidine/histidine hydrochloride buffer at pH 5.5-6.5 was selected as the buffer system for the liquid formulation of anti-PCSK 9 antibodies of the present invention.

Example 2 excipient screening experiments

Based on the experimental results of the screening of the buffer system in example 1, the invention screens 9 different auxiliary material prescriptions and the influence thereof on the stability, viscosity, osmotic pressure and other aspects of the high-concentration anti-PCSK 9 antibody through a high-temperature acceleration experiment at 25+/-2 ℃ and at 40+/-2 ℃, and the specific auxiliary material prescriptions and the information items of corresponding detection items are shown in table 5. The preparation method of the different auxiliary materials formula comprises the steps of respectively adding the different auxiliary materials shown in the table 5 or the combination thereof into a solution containing 140mg/mL of anti-PCSK 9 antibody (antibody A) and 20mM of histidine/histidine hydrochloride buffer (pH 6.0) or acetic acid/sodium acetate buffer (pH 5.0), then adding the surfactant shown in the table 5 into the solution, subpackaging the prepared preparation solution into penicillin bottles, adding a stopper rolling cover, respectively placing the penicillin bottles at 25 ℃ and 40 ℃, and taking out and analyzing and detecting the stability of the antibody, the viscosity of the preparation solution, the osmotic pressure and the like at the 2 nd week and the 4 th week.

ELISA is adopted to determine biological activity, namely, anti-PCSK 9 antibody is diluted to 1.5 mug/ml by coating liquid, 100 mug/well is added into a 96-well ELISA plate, and the temperature is 2-8 ℃ and the coating is carried out overnight. After plate washing and sealing, the standard and the test sample are diluted to the same series of concentrations, 100 μl/well is added to the ELISA plate, the plate is sealed, and incubated at 25deg.C and 220rpm for 1h. Wash plate 100 μl/well of horseradish peroxidase labeled anti-human immunoglobulin was added to each reaction well of the ELISA plate, and the plate was sealed with a sealing plate membrane and incubated at 25℃and 220rpm for 1h. After washing the plate, TMB substrate solution was added for color development, and after color development, A ₄₅₀ was measured after termination with 2M sulfuric acid. Four-parameter curve fitting was performed and the biological activity was calculated from EC ₅₀ of the standard and test. The test results are shown in Table 5.

Table 5 screening prescriptions for different auxiliary materials and detection items thereof (X represents detection items of appearance, pH value, protein content, SEC-HPLC, CEX-HPLC, non-reducing calipers, DLS; Y represents detection items of DSC, osmotic pressure, viscosity; Z represents detection items of activity; and "[ ]" represents optional detection items)

TABLE 6 stability test results of anti-PCSK 9 antibody samples with different adjuvant formulations at 25+ -2 ℃ and 40+ -2 DEG C

As shown in Table 6, when the excipient is proline, the osmotic pressure of the antibody liquid preparation can be maintained within the range of 300-340 mOsm/Kg, the viscosity of the preparation (< 15 cp) and the average particle size of the anti-PCSK 9 antibody can be remarkably reduced to be less than 15d.nm, the antibody has strong thermal stability, namely, after the liquid preparation is placed at a high temperature of 40 ℃ for 4 weeks, the content of antibody monomers (or main peaks) detected by SEC-HPLC is more than 94%, the content of the monomers is reduced by about 1.6-3.2%, the content of the main peaks of the electric charges of the antibody detected by CEX-HPLC is more than 47%, the content of the main peaks of the electric charges is reduced by about 0.2-0.5%, and the purity of the antibody detected by non-reducing clamp is more than 97%, and the purity of the antibody detected by non-reducing clamp is reduced by about 1.3-1.7%. When the excipient is sucrose, the viscosity, osmotic pressure, rubber plug sliding performance, average particle size of the antibody, CEX-HPLC charge main peak content reduction rate after being placed at a high temperature of 40 ℃ for 4 weeks, and non-reducing Caliper antibody purity reduction rate are all higher than those of the excipient which is proline.

Further, when the excipient of the antibody liquid preparation is proline, the buffer system is histidine/histidine hydrochloride buffer solution, and the pH is 6.0 (namely the prescriptions of samples F13 and F16), the fluidity of the preparation and the compliance of patients in the administration are most suitable, and the stability of the antibody is the strongest. Specifically, the liquid formulation had an osmotic pressure of about 330mOsm/Kg, an antibody viscosity of <10cp, a SEC-HPLC antibody monomer content drop of about 1.6% and 2.2% respectively, a CEX-HPLC main peak content drop of about 0.3% and 0.5% respectively, and a non-reducing Caliper purity drop of about 1.3% and 1.5% respectively after 4 weeks of high temperature storage at 40 ℃.

Again, when the excipient of the antibody liquid formulation was proline, the buffer system was acetic acid/sodium acetate buffer, pH 5.0 (i.e. the formulation of sample F18), the viscosity of the formulation (about 11.63 cp) was slightly higher than that of F13 and F16, the plug sliding performance was inferior to that of F13 and F16, and after 4 weeks of standing at 40 ℃ high temperature, the SEC-HPLC antibody monomer content was reduced by about 3.2%, the antibody monomer content reduction rate was higher than that of F13 and F16, and the non-reducing Caliper purity reduction rate was also slightly higher than that of F13 and F16, indicating that acid catalysis might be the main cause of hydrolysate formation. In addition, acetic acid/sodium acetate buffer, a liquid formulation at pH 5.0, can produce subcutaneous pain, reducing patient compliance when administered.

In addition, when the excipient of the antibody liquid preparation only contains proline, the concentration is 200-300 mM, and the buffer system is histidine/histidine hydrochloride buffer solution, the concentration ratio of L-histidine to L-histidine hydrochloride is 0.2:1-3:1, the liquid preparation of the invention can be ensured to be isotonic solution, the pH value of the liquid preparation is maintained within 5.5-6.5, and the liquid preparation does not contain other stabilizer components, so that the auxiliary material components of the liquid preparation of the invention are simple and easy to control quality.

It follows that when the excipient of the liquid preparation of an antibody of the present invention is proline, proline can serve not only as a stabilizer but also as a viscosity-reducing agent and an osmotic pressure regulator. Particularly, when the buffer system is histidine/histidine hydrochloride buffer solution and the excipient is proline, the degradation and aggregation of the high-concentration anti-PCSK 9 antibody can be obviously reduced, the stability of the antibody can be ensured, the viscosity of the high-concentration antibody can be obviously reduced, the liquid preparation of the invention can be ensured to be an isotonic solution, and the antibody protein is uniformly distributed, so that the compliance of patients in the process of drug administration is obviously improved.

Example 3 surfactant screening experiments

Based on the buffer system and excipient screening, the invention further develops screening research of the surfactant. According to the formulation recipe shown in Table 7, 0.01% (w/w) polysorbate 80 or no polysorbate 80 was added to a formulation solution containing 20mM histidine/histidine hydrochloride (pH 6.0), an auxiliary material (250 mM proline or 6% (w/w) sucrose) and 140mg/mL of an anti-PCSK 9 antibody (antibody A), respectively, and the stability of the antibody was analyzed by cold temperature and room temperature freeze-thaw cycle experiments at-70.+ -. 10 ℃ to examine whether the antibody liquid formulation of the present invention requires the addition of a surfactant.

Table 7 liquid formulation formulations for antibodies in surfactant screening experiments

TABLE 8 results of investigation of the Effect of surfactants on antibody stability by freeze thawing cycle experiments

As shown in Table 8, samples F19-F21 show no obvious change in appearance, protein content, pH value, SEC-HPLC main peak purity, non-reducing calipers purity and CEX-HPLC charge main peak purity after 3 times and 5 times of freeze thawing cycles respectively, no obvious difference exists among the samples, the antibody activities of F19, F20 and F21 are kept between 80% -120% after 5 times of freeze thawing cycles, no obvious difference exists among the samples, and insoluble particles of sample F20 are slightly less than prescribed F21 after 5 times of freeze thawing. Therefore, the liquid preparation of the antibody of the invention does not contain surfactant, so that the stability of the antibody can be ensured. Because the liquid preparation contains high-concentration antibody, the surfactant is added into the liquid preparation, so that the antibody can be protected from the stress induced by an air/solution interface and the stress induced by a solution/surface in the storage and transportation processes, the aggregation of the antibody or the formation of particles in the preparation can be reduced, and the stability of the antibody can be improved more conveniently.

In addition, the concentration of the surfactant (such as polysorbate 80) was screened and analyzed by a room temperature shaking stability test, namely, the surfactant was prepared according to the formula shown in table 9 at room temperature and a rotation speed of 300rpm, the sample solution was shaking for 1 day and 3 days after being filled by a pre-filled syringe, the storage and transportation processes were simulated, and each sample was taken out and tested and analyzed, and the detection results are shown in table 10.

Table 9 liquid formulations of anti-PCSK 9 antibodies with varying concentrations of surfactant

Table 10 results of stability experiments on liquid formulations of anti-PCSK 9 antibodies containing surfactants at different concentrations

As shown in Table 10, samples F22, F23 and F24 remained good in antibody stability after shaking at room temperature at 300rpm for 1 day and 3 days, and there was no significant difference in appearance, protein content, pH, SEC-HPLC main peak purity, non-reducing Caliper purity, CEX-HPLC charge main peak purity for each sample. As can be seen, the antibody liquid preparation of the invention contains 0.01% -0.04% (w/w) polysorbate 80, which can effectively maintain the stability of the antibody.

In addition, polysorbate 20 can also reduce aggregation of the antibody of the invention during shaking, prevent the antibody from being adsorbed on the surface of a container, and meet the requirement of subcutaneous injection administration of the liquid preparation of the invention.

EXAMPLE 4 stability validation study of liquid formulations containing anti-PCSK 9 antibodies

Semi-finished products were prepared according to the formulation in example 2 sample F13 (i.e., 140mg/mL anti-PCSK 9 antibody protein, 20Mm histidine/histidine hydrochloride buffer system, ph6.0,250Mm proline, 0.01% polysorbate 80) and filled into 1mL elongate pre-filled syringes (with needles) with matched plugs, and subjected to 3 month storage stability, 3 month acceleration stability and 1 month high temperature stability studies at 5±3 ℃,25±2 ℃ and 40±2 ℃ respectively, with specific study contents shown in table 11, wherein the reduced CE-SDS electrophoresis method mainly detects the sum of the contents of heavy chain, non-glycosylated heavy chain and light chain of the antibody, and the non-reduced CE-SDS electrophoresis method mainly detects the major peak content. The stability test results are shown in tables 12-14.

Table 11 antibody liquid formulation stability validation study protocol

(X represents that the detection items are appearance, pH value, protein content, SEC-HPLC, CEX-HPLC, reducing CE-SDS and non-reducing CE-SDS; Y represents that the detection items are osmotic pressure and viscosity; Z represents that the detection items are insoluble particles (MFI) and the sliding performance of the rubber plug is tested; W represents that the detection items are active; and "[ ]" represents that whether detection is carried out or not is determined according to the detection result of the later time point).

TABLE 12 results of 3 month storage stability experiments for liquid formulations of anti-PCSK 9 antibodies at 5.+ -. 3 °c

TABLE 13 results of 3 month accelerated stability experiments at 25.+ -. 2 ℃ for liquid formulations of anti-PCSK 9 antibodies

TABLE 14 results of 1 month high temperature stability experiments at 40.+ -. 2 ℃ for liquid formulations of anti-PCSK 9 antibodies

As shown in Table 12, the stability of all detection items of the antibody liquid preparation of the invention is good after the antibody liquid preparation is stored for 3 months at 5+/-3 ℃ in a dark place, and the antibody liquid preparation of the invention is proved to meet the requirement of the quality stability of medicines. As can be seen from the experimental results in Table 13, the liquid formulations of the antibodies of the present invention were stored at 25.+ -. 2 ℃ for 3 months at room temperature, and the protein content, appearance, pH, insoluble particles, and biological activity of the samples were tested to show good stability, and the monomer content (SEC-HPLC), the purity of the reducing CE-SDS, and the purity of the non-reducing CE-SDS of the antibodies were slightly decreased, 0.6%, 0.5%, and 1.2%, respectively, but the degree of decrease was less than 5%, indicating that the liquid formulations of the present invention remained stable at room temperature for at least 3 months. As can be seen from the experimental results in Table 14, the antibody liquid preparation of the present invention was stored at 40.+ -. 2 ℃ for 1 month, and the protein content, appearance, pH value, insoluble particles, biological activity test results of the samples all had good stability, and the antibody monomer content (SEC-HPLC), the purity of the reducing CE-SDS and the purity of the non-reducing CE-SDS all had a tendency to decrease, which were 1.7%, 1% and 1.4%, respectively, indicating that the antibody liquid preparation of the present invention could be stably stored at high temperature for a short period of time.

EXAMPLE 5 Long-term stability Studies of liquid formulations containing anti-PCSK 9 antibodies

Based on the stability verification experiment of example 4, the present invention further investigated the long-term stability of three batches of antibody liquid formulations under the condition of 5±3 ℃ and the results of the investigation are shown in tables 15 to 17.

TABLE 15 results of 5+ -3 ℃ Long-term stability study of anti-PCSK 9 antibody liquid formulation (lot number: antibody A-001)

TABLE 16 results of 5.+ -. 3 ℃ Long-term stability study of anti-PCSK 9 antibody liquid formulation (lot number: antibody A-002)

TABLE 17 results of 5+ -3 ℃ Long-term stability study of anti-PCSK 9 antibody liquid formulation (lot number: antibody A-003)

As can be seen from tables 15-17, three batches of the antibody liquid formulations of the present invention showed no significant changes in appearance, pH, protein content, SEC-HPLC, non-reducing CE-SDS and biological activity after 3 months, 6 months, 12 months, 18 months, 24 months and 36 months of storage at 5+ -3 ℃, no significant changes in foreign matter, insoluble particles and sterility were in compliance with the pharmacopoeia specifications, and slight changes in the heterogeneity of the antibody charge in CEX-HPLC assay (sample subjected to CPB cleavage) were caused mainly by deamidation and heavy chain N-ring cleavage, which are typical modifications and conformational changes of the antibody, and which did not affect its biological activity. Therefore, the antibody liquid preparation can be stored for 36 months at the low temperature of 5+/-3 ℃, so that the long-term stability of the medicine stored at the low temperature is ensured, the difference among preparation batches is small, the antibody biological activity is high, and the quality safety of long-term administration of patients is met.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Sequence listing

<110> Xinlitai (Chengdu) Biotechnology Co., ltd

<120> Stabilized formulations containing anti-PCSK 9 antibodies, methods of making and uses thereof

<150> 2020110922078

<151> 2020-10-13

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Val Ile Trp Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala Asp Ser Val Lys

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Glu Lys Gly Leu Asp

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Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Thr Asn Lys Asn Tyr Leu

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Trp Ala Ser Thr Arg Glu Ser

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Gln Gln Tyr Tyr Ser Thr Pro Trp Thr

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

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Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

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Ala Val Ile Trp Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

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

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Ala Arg Glu Lys Gly Leu Asp Trp Gly Gln Gly Thr Leu Val Thr Val

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

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

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

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

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Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

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

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Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

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Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

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

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

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

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

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

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

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

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

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

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

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Leu Ser Leu Ser Leu Gly Lys

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Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

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Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

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Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

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Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

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

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Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

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Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

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Ala Val Ile Trp Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

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

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Ala Arg Glu Lys Gly Leu Asp Trp Gly Gln Gly Thr Leu Val Thr Val

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

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Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys

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

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

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Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

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

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Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

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

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

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

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Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

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Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

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Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

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

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

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Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

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Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

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

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

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Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

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Ser Thr Asn Lys Asn Tyr Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln

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

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

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Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

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Tyr Tyr Ser Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

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Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

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Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

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Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

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Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

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Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

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

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Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

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aagtccagcc agagtgtttt atacagctcc accaataaga actacttagt t 51

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tgggcatcta cccgggaatc c 21

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cagcaatatt atagtactcc gtggacg 27

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caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg gatggcagtt atatggtatg atggaactaa taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacggtgtat 240

ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagagaag 300

gggctggact ggggccaggg aaccctggtc accgtctcct ca 342

<210> 20

<211> 339

<212> DNA

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<400> 20

gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60

atcaactgca agtccagcca gagtgtttta tacagctcca ccaataagaa ctacttagtt 120

tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggc atctacccgg 180

gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240

atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttatagtact 300

ccgtggacgt tcggccaagg gaccaaggtg gaaatcaaa 339

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Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu

20 25 30

Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu

35 40 45

Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe

50 55 60

His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val

65 70 75 80

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

85 90 95

Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu

100 105 110

His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly

115 120 125

Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu

130 135 140

Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg

145 150 155 160

Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly

165 170 175

Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp

180 185 190

His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val

195 200 205

Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp

210 215 220

Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly

225 230 235 240

Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln

245 250 255

Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg

260 265 270

Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro

275 280 285

Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu

290 295 300

Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp

305 310 315 320

Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val

325 330 335

Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly

340 345 350

Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile

355 360 365

Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln Ser Gly

370 375 380

Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu

385 390 395 400

Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile

405 410 415

His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp

420 425 430

Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr

435 440 445

His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His

450 455 460

Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg Cys Ala Pro Asp

465 470 475 480

Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg

485 490 495

Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala His

500 505 510

Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu

515 520 525

Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala

530 535 540

Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr

545 550 555 560

Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro

565 570 575

Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg

580 585 590

Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys

595 600 605

Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu Gln Val Thr Val

610 615 620

Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly

625 630 635 640

Thr Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val

645 650 655

Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val

660 665 670

Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser

675 680 685

Gln Glu Leu Gln

690

Claims

1. A stable formulation comprising:

(1) An anti-PCSK 9 antibody or antigen-binding fragment thereof at a concentration of 70-140mg/mL, and

(2) A pharmaceutically acceptable buffer at a concentration of 20mmol/L, which is a histidine buffer consisting of L-histidine and L-histidine hydrochloride in a concentration ratio of 0.2:1 to 3:1, having a pH of 5.5 to 6.5, and

(3) A pharmaceutically acceptable surfactant selected from polysorbate 80 at a concentration of 0.01-0.04% w/w, and

(4) And other pharmaceutically acceptable excipients at a concentration of 250mmol/L, wherein the excipient is selected from the group consisting of proline, and wherein the anti-PCSK 9 antibody or antigen-binding fragment thereof comprises HCDR1 of the amino acid sequence shown in SEQ ID NO. 1, HCDR2 of the amino acid sequence shown in SEQ ID NO. 2, HCDR3 of the amino acid sequence shown in SEQ ID NO. 3, LCDR1 of the amino acid sequence shown in SEQ ID NO. 4, LCDR2 of the amino acid sequence shown in SEQ ID NO. 5 and LCDR3 of the amino acid sequence shown in SEQ ID NO. 6.

2. The stable formulation of claim 1, wherein the anti-PCSK 9 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID No. 7 and a light chain variable region comprising an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID No. 8.

3. The stable formulation of claim 2, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO. 7 and the light chain variable region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO. 8.

4. The stable formulation of any one of claims 1-3, wherein the anti-PCSK 9 antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from the group consisting of human IgG1, igG2, igG3, and IgG4 constant regions and a light chain constant region selected from the group consisting of human kappa light chain constant region or lambda light chain constant region.

5. The stable formulation of claim 4, wherein the heavy chain constant region is selected from the group consisting of an IgG4 constant region, the amino acid sequence of the IgG4 constant region is set forth in SEQ ID No. 9, and the amino acid sequence of the kappa light chain constant region is set forth in SEQ ID No. 10.

6. The stable formulation of any one of claims 1-3, wherein the anti-PCSK 9 antibody or antigen-binding fragment thereof comprises or consists of two heavy chains having the amino acid sequence set forth in SEQ ID No. 11 and two light chains having the amino acid sequence set forth in SEQ ID No. 12.

7. The stable formulation of claim 1, wherein the formulation is a liquid formulation having a viscosity of less than 15cp at 25 ℃, and wherein the liquid formulation has an osmotic pressure of 220-370 mosm/Kg at 25 ℃.

8. The stable formulation of claim 7, wherein the liquid formulation has a viscosity of less than 10cp at 25 ℃, and the osmotic pressure is 250 to 350mOsm/Kg.

9. The stable formulation of claim 1, wherein the liquid formulation is stable for at least 2 weeks at a temperature of about 5 ℃ to about 40 ℃.

10. The stable formulation of claim 9, wherein the liquid formulation is stable for at least 6 months at a temperature of about 5 ℃ to about 40 ℃.

11. The stable formulation of claim 1, wherein the formulation has a purity of the anti-PCSK 9 antibody or antigen-binding fragment thereof that decreases by no more than 10%, and/or an acid-base charge heteroplast that increases by no more than 50%, and/or an aggregate that increases by no more than 10%, and/or a degradation that increases by no more than 10% when stored at 2-8 ℃ for at least 3 months.

12. The stable formulation of claim 11, wherein the formulation has a purity of the anti-PCSK 9 antibody or antigen-binding fragment thereof that decreases by no more than 5% and/or an aggregate that increases by no more than 5% and/or a degradation product that increases by no more than 5% when stored at 2-8 ℃ for at least 12 months.

13. The stable formulation of claim 1, wherein the formulation is administered parenterally, wherein the parenteral administration is selected from subcutaneous or intramuscular injection.

14. A process for the preparation of a stable formulation according to any one of claims 1 to 13, comprising the steps of:

a) Providing an anti-PCSK 9 antibody or antigen-binding fragment thereof;

b) Adding a buffer and an osmotic pressure regulator;

c) Adding a surfactant;

d) Sterilizing and filtering;

e) And (5) sub-packaging.

15. A prefilled syringe loaded with a stable formulation according to any one of claims 1 to 13, said formulation being a liquid formulation.

16. Use of a stable formulation according to any one of claims 1-13 in the manufacture of a medicament for the treatment, prevention or amelioration of any PCSK 9-related disease or condition selected from the group consisting of hyperlipidemia, hypercholesteremia, stroke, atherosclerosis, heart disease.