WO2021139720A1

WO2021139720A1 - Purification method for adalimumab and stable composition thereof

Info

Publication number: WO2021139720A1
Application number: PCT/CN2021/070654
Authority: WO
Inventors: 杨忠华; 马一冬; 邢振荣; 蔺智勇
Original assignee: 信达生物制药(苏州)有限公司
Priority date: 2020-01-08
Filing date: 2021-01-07
Publication date: 2021-07-15
Also published as: CN114929725A

Abstract

A purification method for adalimumab, a stable adalimumab composition obtained by the purification method, and pharmaceutical use of the composition.

Description

Adalimumab purification method and stable composition thereof

Technical field

The present invention relates to the field of antibodies. Specifically, the present invention relates to a purification method of adalimumab, a stable adalimumab composition obtained by the purification method, and the medical use of the composition.

Background technique

Stability of antibody products

In the entire production process of biopharmaceuticals, from the preparation of protein drugs to purification, formulation, and packaging, there is the possibility of introducing impurities into the finished drug products. These impurities include, for example, process-related impurities and product-related impurities. The impurities formed by the instability of the target protein drug in the formulation are called product-related impurities in the art. These product-related impurities are molecular variants of the target product, but often do not have the activity, efficacy and safety equivalent to the target product, for example, various degradation products and charge variants formed during the production, purification and/or storage process Wait. Therefore, it is necessary to control the presence and content of these product-related impurities in pharmaceutical products.

Monoclonal antibodies generally exhibit several heterogeneities, including, for example, size heterogeneity, charge heterogeneity, and glycosylation pattern heterogeneity. These heterogeneities can be caused by many different factors. These factors may include, but are not limited to, impurities that may be introduced into the drug product during any stage of drug preparation, purification, and storage, as well as the interaction between impurities, and the interaction between impurities and the target protein. On the other hand, the complexity of the structure and properties of antibody drugs as macromolecules has further aggravated the difficulty of analyzing destabilizing factors in antibody preparations. Therefore, the stability of monoclonal antibodies is complicated, and it is often related to specific proteins and related to specific antibody production processes.

The generation of charge heterogeneity is due to a series of post-translational modification and degradation of antibody drugs during the production process, which leads to changes in surface charge characteristics such as space charge distribution. Studies have found that the formation of charge heterogeneity may have an impact on the in vivo and in vitro characteristics of antibody drugs, such as the binding capacity, pharmacokinetics, immunogenicity, biological activity and stability, and thus affect the effectiveness and safety of clinical applications. Sex is an important quality attribute of antibodies. Recently, the detection of charge heterogeneity has attracted more and more attention from global pharmaceutical companies and regulatory agencies. Especially in the development of biosimilar drug production processes, the control of charge heterogeneity is one of the important links. However, there are many sources of charge heterogeneity and the formation mechanism is complex. The study of charge heterogeneity has always been one of the difficult problems in the development of antibody drugs. Dakshinamurthy P, et al., Charge variant analysis of proposed biosimilar to Trastuzumab, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2016.12.006.

It has been identified that a variety of antibody modifications can be responsible for the formation of acidic or basic components of charge variants. For acidic components, the main modifications include deamidation and glycosylation of sialic acid and asparagine residues. For basic components, the main modifications include retention of part of the leader sequence, ring opening of N-terminal glutamine, C-terminal amidation, succinimide due to aspartic acid isomerization, and C-terminal lysine. See Yu Chuanfei et al. (Pharmaceutical Analysis Impurities, 2014, 34(7), p1212, charge heterogeneity analysis of monoclonal antibody products by imaging capillary isopoint focusing electrophoresis). In addition, it has been reported that antibodies oxidized by methionine at position 24 of the Fc region appear as basic components in cation exchange chromatography. See Chumsae, C.; Gaza-Bulseco, G.; Sun, J.; Liu, H.J. Chromatogr. B. 2007, 850, 285-294.

Wu Linping et al. (Analysis of the Key Quality Attributes of the Physical and Chemical Properties of Adalimumab, China Medical Industry Impurities, 2018,49(3)), studied the stability of Adalimumab injection produced by AbbVie in the United States. The characterization results of charge heterogeneity showed that the main modifications of Adalimumab injection were N deamidation and terminal K modification, of which N deamidation modification was the main source of acidic variability; and the terminal K modification included Variants of 0, 1, and 2 K residues are the main source of major alkaline variability.

Some methods for stabilizing antibody products have been proposed, which mainly include adding a certain amount and type of excipients to the antibody product. However, the addition of excipients may cause problems in product safety or efficacy. For example, SHUXIA ZHOU et al. disclosed that the addition of 0.05mg-1mg/ml metal chelate to the monoclonal antibody preparation can provide the stability of the preparation (Comparative Evaluation of Disodium Edetate and Diethylenetriaminepentaacetic Acid as Iron Chelators to Prevent Metal-Catalyzed Destabilization of a Therapeutic Monoclonal Antibody, JOURNAL OF PHARMACEUTICAL Science, VOL.99,NO.10,2010,pp4239-4250). However, not all formulations are suitable for adding metal chelating agents. On the one hand, the addition of a considerable amount of excipients (such as metal chelating agents) will increase the possibility of clinical adverse reactions. For example, it has been found that adding a small amount of EDTA to the preparation of the biological drug Leukine (recombinant GM colony stimulating factor) can cause clinical adverse reactions. On the other hand, due to the complexity of antibody stability issues, there is no universal excipient solution applicable to any antibody formulation. For example, CN 104768576A uses SEC, RP HPLC, cIEF and CE-SDS methods, by keeping it at 40°C for one week or at 25°C for two weeks to study the use of various excipients and excipients in the aqueous pharmaceutical composition of adalimumab The results show that EDTA is more likely to be a destabilizer for adalimumab preparations; and it is proposed that metal ions (such as zinc, magnesium and calcium) can be used as stabilizers for adalimumab preparations.

Therefore, in the field of monoclonal antibodies, optimizing the production process of antibodies to minimize the instability factors existing in antibody drugs is a challenging direction in this field.

Adalimumab

Adalimumab is a fully human IgG1 monoclonal antibody consisting of 1330 amino acids and having a molecular weight of approximately 148 kilodaltons. The antibody acts on tumor necrosis factor alpha (TNF-α) related diseases and is mainly used to treat diseases mediated by the immune system, including rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis and Crohn's disease. Adalimumab is usually produced by recombinant DNA technology in mammalian cell expression systems, especially Chinese hamster ovary CHO cells. Adalimumab injection sold under the trade name Humira was first launched in the United States in January 2003. Its composition is: mannitol, citric acid monohydrate, sodium citrate, sodium dihydrogen phosphate dihydrate Compounds, disodium hydrogen phosphate dihydrate, sodium chloride, polysorbate 80, sodium hydroxide, water for injection, and 40mg/0.8ml adalimumab. Since then, some biosimilar drugs have also been approved for marketing, including Exemptia, Adfrar, Amjevita and Cyltezo.

In order to ensure the safety and effectiveness of monoclonal antibody drugs, a process method that can provide adalimumab drugs with stable quality attributes is still needed.

Summary of the invention

The inventor found that the adalimumab composition purified by protein A affinity chromatography and ion exchange chromatography has the problem of decreased stability during storage, which is mainly reflected in the decreased purity of SEC-HPLC and product-related impurities ——The increase in acidic charge isomers and the decrease in the content of excipient Tween 80. In order to minimize the influence of instability factors on the adalim antibody, the inventors conducted a production process optimization study. On the basis of these in-depth studies, the technical solution of the present invention is proposed, and a cost-effective and practical method for improving the stability of the purified adalimumab composition (especially the improved stability of the charge variant) is established.

Therefore, in one aspect, the present invention provides a method for preparing an adalimumab composition with improved stability through an improved purification process, wherein the stability is preferably charge variant stability, optionally also Including monomer stability, oxidation stability, or a combination thereof. In one embodiment, the method includes the following steps:

(a) Affinity chromatography: applying a mixture containing adalimumab to an affinity chromatography resin, eluting with an elution buffer, and collecting a collection solution containing the antibody;

(b) Anion exchange chromatography: applying a collection solution containing the antibody to an anion exchange resin, and collecting the penetrating solution containing the antibody; and

(c) Cation exchange chromatography: applying a collection solution containing the antibody to a cation exchange resin, eluting with an elution buffer, and collecting the collection solution containing the antibody, wherein the elution solution contains a metal ion chelate The combination agent, for example, a metal ion chelating agent selected from but not limited to the following: EDTA, DTPA, EGTA, BAPTA, DMPS, DMSA, ALA, or a combination thereof; preferably EDTA or DTPA.

Preferably, the purified adalimumab composition obtained by the method has one or more characteristics selected from the following:

-After storage, for example, after 4 weeks of storage at 40°C, the acid charge variants increase by no more than 40%, preferably no more than 30%, 25%, 20%, 15%, 10%; and optionally, the main component of charge decreases Not more than 40%, preferably not more than 30%, 25%, 20%, 15%, 10%;

-After storage, for example, after storage at 40°C for 4 weeks, the monomer purity, as measured by SEC-HPLC, is 97% or more, preferably 98% or 99% or more;

-It has oxidation stability. After storage, for example, after storage at 25°C for 4 weeks, as measured by peptide mapping, the oxidation ratio of the 256-position methionine residue (M256) of the antibody does not exceed 40%, or more preferably not More than 30%, 20% or 10%, 5%.

In a preferred embodiment, the cation exchange chromatography step includes: eluting with a cation eluent containing a metal ion chelating agent, wherein the amount of the metal chelating agent in the eluent results in the purified adalimidine obtained by the method. The anti-composition has 0.00026-0.05 mg/ml metal chelating agent, preferably 0.0005-0.03 mg/ml, more preferably 0.001-0.02 mg/ml, for example about 0.002, 0.004, 0.008, 0.01 mg/ml. In another preferred embodiment, the amount of the metal ion chelating agent in the eluent is 5-40 mmol/L, for example, 10-30 mmol/L, for example, 20 mmol/l.

In still another preferred embodiment, the cation exchange chromatography step further comprises: washing with a cation washing solution containing a metal ion chelating agent before elution. Preferably, the metal ion chelating agent in the rinse solution is 5-40 mmol/L, for example, 10-30 mmol/L, for example, 10 mmol/l.

In another aspect, the present invention also provides a method for improving the stability of the purified adalimumab aqueous composition, wherein the stability is preferably selected from charge variant stability, monomer stability, oxidation Stability, or a combination thereof.

In still another aspect, the present invention also provides a method for purifying adalimumab from a mixture containing adalimumab, the method comprising: affinity chromatography, anion exchange chromatography and cation exchange chromatography, wherein the cation Exchange chromatography uses an eluent containing a metal ion chelating agent for elution. Preferably, the purified adalimumab composition obtained by the method has stability selected from the group consisting of oxidation stability, monomer stability, and charge variant stability.

In a preferred embodiment, the method of the present invention includes the steps: (1) recovering adalimumab from the cell culture recombinantly expressing adalimumab to obtain a mixture containing adalimumab; (2) using an affinity layer Analysis (such as protein A column) to capture antibodies; (3) virus inactivation; (4) anion exchange and cation exchange chromatography; (5) virus filtration (reduce virus titer, for example, more than 4log10); (6) ultra Filtration/diafiltration (for example, to replace the antibody protein in a formulation buffer that is conducive to its stability and concentrate to a suitable concentration); and optionally (7) preparation of the purified composition (for adding suitable excipients) Wherein the method is characterized in that in cation exchange chromatography, elution is performed with an elution buffer containing a metal ion chelating agent (for example, EDTA or DTPA) to collect a collection solution containing adalimumab antibody.

In still another aspect, the present invention provides an adalimumab composition comprising adalimumab and a metal chelating agent, preferably 0.00026-0.05 mg/ml metal chelating agent, preferably 0.0005-0.03 mg/ml, More preferably 0.001-0.02 mg/ml, for example, about 0.002, 0.004, 0.008, 0.01 mg/ml of the metal chelating agent. Preferably, the adalimumab is purified by the method of any one of claims 1-8. Preferably, the composition exhibits stability selected from the group consisting of oxidation stability, monomer stability, and charge variant stability after storage.

In yet another aspect, the present invention provides the use of the antibody composition of the present invention in the treatment of diseases in which TNFα activity is detrimental.

Description of the drawings

Figure 1 shows a typical spectrum of EDAT residues detected by high performance liquid chromatography.

Figure 2 shows a typical spectrum of the polysorbate 80 content of the sample detected by the FLD-HPLC method.

Figure 3 shows a typical map of the CEX-HPLC method for detecting antibody charge heterogeneity.

Figure 4 shows the effect of process optimization on the main components of adalimumab charge isomers.

Figure 5 shows the effect of process optimization on the content of Tween 80 in the purified composition.

Detailed description of the invention

Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because the methods and conditions can be changed. In addition, the terms used herein are only for describing specific embodiments, and are not intended to be limiting.

definition:

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. For the purpose of the present invention, the following terms are defined below.

The term "about" when used in conjunction with a numerical value means to encompass a numerical value within a range having a lower limit that is 5% smaller than the specified numerical value and an upper limit that is 5% larger than the specified numerical value.

When the term "and/or" is used to connect two or more alternatives, it should be understood to mean any one of the alternatives or any two or more of the alternatives.

As used herein, the term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In this document, when the term "comprises" or "includes" is used, unless otherwise specified, it also encompasses the situation consisting of the stated elements, integers or steps.

As used herein, the phrase "CEX-HPLC" refers to cation exchange high performance liquid chromatography.

As used herein, the phrase "SEC-HPLC" refers to size exclusion high performance liquid chromatography

As used herein, the phrase "RH" refers to relative humidity.

As used herein, the phrase "SV" refers to the sample volume.

As used herein, the term "PS80" is synonymous with "Tween 80" and "Polysorbate 80".

As used herein, the term "EDTA" refers to ethylenediaminetetraacetic acid, and the EDTA described herein also includes its salts and other variants, such as disodium ethylenediaminetetraacetic acid.

As used herein, the phrase "CV" refers to column volume.

As used herein, excipients refer to auxiliary materials used in the preparation of biological products, such as adjuvants, stabilizers, excipients, etc.

As used herein, a drug substance (DS), also called bulk, refers to a uniform drug substance used to make a final formulation.

As used herein, drug products (DP), also called final products, refer to products formed by filling drug substances into the final storage container of drugs. In this process, the drug substance can be diluted and/or formulated as appropriate or not, such as adding excipients.

As used herein, the expression "stable" antibody composition is intended to encompass that the antibody in the composition retains an acceptable degree of physical and/or chemical stability after storage under specific conditions. For example, after storage for a certain period of time, if the antibody contained in the antibody composition still maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the antibody structure or function, then The antibody composition can generally be considered "stable". In some embodiments, the antibody composition of the present invention substantially retains its physical and chemical stability after storage. A variety of analytical techniques known in the art can be used to determine the stability of antibody compositions, see, for example, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). The stability can be measured at a selected temperature and selected storage time. For example, the storage time can be selected based on the expected shelf life of the formulation. Or an accelerated or forced stability test can be used. In some embodiments, a mandatory stability test is performed on the antibody composition.

As used herein, the expression "monomer stability" of an antibody composition refers to the stability of the antibody composition measured by measuring the percentage of antibody monomers in the composition after storage at a specific temperature for a specific time, wherein The higher the percentage of antibody monomers, the higher the stability of the composition. As used herein, when stored at a specific temperature for a specific time, at least about 95%, preferably at least 96%, 97%, 98% or 99% or more of the antibody monomer is detected in the composition, then the composition It can be regarded as having improved monomer stability. In some embodiments, storage at a specific temperature for at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months After months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more, "improved monomer stability" It means that at least about 95%, 96%, 97%, 98%, 99% of antibody monomers are maintained in the composition. When evaluating the stability of the monomer, the specific temperature used for the storage of the composition may be any temperature from about -80°C to about 45°C, for example, about -80°C, about -30°C, about -20°C, or about 0°C. , About 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, about 40°C, about 42°C, or about 45°C. In a preferred embodiment, if the composition is stored at about 40°C±2°C for 1 month, at least about 95%, 96%, 97%, 98%, 99% of antibody monomers are detected, then the combination The material is considered to have improved monomer stability. The measurement of antibody monomer percentage can be carried out in various ways known in the art, such as SEC-HPLC. In a preferred embodiment, the antibody composition produced by the method of the present invention has improved monomer stability compared to the antibody composition produced by the control method. Preferably, the difference between the control method and the method of the present invention is only the lack of the EDTA use step of the method of the present invention.

As used herein, the expression "charge variant stability" of the antibody composition refers to the determination of the antibody charge variants (for example, acidic charge variants, basic charge variants, etc.) in the composition after storage at a specific temperature for a specific period of time. , The main component of the charge variant, or a combination thereof) to measure the stability of the antibody composition, wherein compared to before storage, the lower the percentage of change of the charge variant in the composition, the higher the stability of the composition . In a preferred embodiment, the main component of the charge variant is an antibody variant containing 0, 1, and 2 terminal lysines. As used herein, when stored at a specific temperature for a specific time, no more than about 30%, preferably no more than 25%, 24%, 23%, 22%, 21%, and more preferably no more than 20% are detected in the composition. % Charge variant change (for example, acid charge variant change), the composition can be considered to have improved charge variant stability (or more specifically, acid charge variant stability). When evaluating the stability of charge variants, the composition can be stored at a specific temperature for at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more. The specific temperature used for the storage of the composition may be any temperature from about -80°C to about 45°C, for example, about -80°C, about -30°C, about -20°C, about 0°C, about 4°C to 8°C, About 5°C, about 25°C, about 35°C, about 37°C, about 40°C, about 42°C, or about 45°C. In a preferred embodiment, if the composition is stored at about 40°C±2°C for 1 month, no more than about 30% is detected, preferably no more than 25%, 24%, 23%, 22%, 21%, More preferably, no more than 20% acidic variant change, the composition is considered to have improved charge variant stability or improved acidic charge variability stability. In another preferred embodiment, if the composition is stored at about 40°C ± 2°C for 1 month, no more than about 30% is detected, preferably no more than 25%, 24%, 23%, 22%, 21% , And more preferably no more than 20% of the charge variant main component change, the composition is considered to have improved charge variant stability. The measurement of the percentage of charge variants of an antibody can be carried out in a variety of ways known in the art, such as the CEX-HPLC method. In a preferred embodiment, the antibody composition produced by the method of the present invention has improved charge variant stability compared to the antibody composition produced by the control method. Preferably, the difference between the control method and the method of the present invention is only the lack of the EDTA use step of the method of the present invention.

As used herein, the expression "oxidative stability" of the antibody composition refers to the determination of the oxidation ratio of the 256-position methionine residue of the antibody in the composition after storage at a specific temperature for a specific time (ie, oxidation residue/( The stability of the antibody composition measured by oxidized residues + non-oxidized residues)), wherein the lower the oxidation ratio in the composition, the higher the stability of the composition compared to before storage. The measurement of the oxidation ratio at position M256 of the antibody can be carried out, for example, in various ways known in the art, such as peptide mapping. In one embodiment, the antibody composition produced by the method of the present invention has improved oxidative stability compared to the antibody composition produced by the control method. Preferably, the difference between the control method and the method of the present invention is only the lack of the EDTA use step of the present invention.

As used herein, the expression "polysorbate 80 stability" refers to the stability of the antibody composition measured by determining the content of the excipient polysorbate 80 in the composition after storage at a specific temperature for a specific time, wherein the relative Compared with before storage, the less the content of the excipient polysorbate 80 decreases, the more stable the polysorbate 80 is. The content of polysorbate 80 can be carried out by various methods known in the art, such as high performance liquid chromatography-fluorescence detection method (FLD-HPLC method). In one embodiment, the antibody composition produced by the method of the present invention has improved polysorbate 80 stability compared to the antibody composition produced by the control method. Preferably, the difference between the control method and the method of the present invention is only the lack of the EDTA use step of the present invention.

I. The method of the present invention

The inventors found that by optimizing the purification process of adalimumab, the storage stability problem of the purified antibody composition produced can be effectively solved, especially the problem of charge heterogeneity. Therefore, on the basis of in-depth research, the inventors proposed the purification method of the present invention. The adalimumab composition obtained by the method of the present invention has improved adalimumab purity and charge isomer stability (for example, forced stability at 40° C. for 4 weeks, the main component decreased by less than 26%), and the combination The content of polysorbate 80 is more stable (40°C forced stability decreased less than 10% for 4 weeks).

Therefore, in one aspect, the present invention provides a method for improving the stability of an adalimumab composition through a purification process, wherein the method includes the steps of: affinity chromatography; anion exchange chromatography; and cation exchange layer Analyze; wherein, in the cation exchange chromatography step, a cation eluent containing a metal ion chelating agent (for example, EDTA or DTPA) is used for elution. Preferably, the method of the present invention further includes: a virus inactivation step, an ultrafiltration and a diafiltration step, and a preparation step of the purified adalimumab composition.

The various aspects of the method of the present invention will be further described below.

Adalimumab

In this article, the term "adalimumab" is the active pharmaceutical ingredient in Humira, and is intended to cover biosimilar or bio-modified variants of the Adalimumab protein used in Humira. For the purpose of the present invention, the term also covers adalimumab protein with slightly modified amino acid sequence structure but does not significantly affect the function of the antibody. The amino acid sequence of adalimumab and its pharmacological and therapeutic properties have been disclosed in, for example, WO97/029131, which is hereby incorporated by reference. The full-length sequences of the light and heavy chains of adalimumab are shown below:

Full-length sequence of adalimumab light chain:

Full-length sequence of adalimumab heavy chain:

EVQLVESGGGLVQPGRSLRLSCAASGFTFD DYAMH WVRQAPGKGLEWVS AITWNSG HIDYADSVEG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAK VSYLSTASSLDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL M ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2, a heavy chain constant region is in italics, underlined HCDR1-3, located in the Fc region 256 Met)

In some embodiments of the present invention, adalimumab can be produced in host cells (such as mammalian cells, especially CHO cells) by genetic recombination methods. Adalimumab can be produced in cells, or preferably secreted into the culture medium. Adalimumab can be recovered from the cells or culture medium by conventional methods in the art. This recovery step may include, for example, one or more centrifugation or deep filtration steps, thereby providing a mixture containing adalimumab that can be used in the purification method of the present invention. In one embodiment, after the end of the fermentation, the fermentation broth is centrifuged to clarify impurities such as cells to obtain a supernatant containing adalimumab.

Affinity chromatography:

In some embodiments, affinity chromatography is performed on the Adalimumab mixture recovered from the producer cells. In one embodiment, affinity chromatography fillers, such as protein A affinity fillers, are used for affinity chromatography. In one embodiment, a loading capacity of 10-45 g/L is used. In one embodiment, the sample flow rate is 100-250 cm/h. In one embodiment, before loading the sample, a neutral buffer is used for equilibration; and after the sample is loaded, the neutral buffer is used for equilibration again. An example of a suitable neutral buffer is a Tris/NaCl buffer with a pH of, for example, about 7.2. In one embodiment, the neutral buffer used for equilibration contains a low concentration of salt, such as NaCl at a concentration of 50-200 mmol/L, such as 150 mmol/L. In one embodiment, before elution, rinse with a rinse solution containing high salt. Preferably, the rinse solution contains 300 mmol/L-1000 mmol/L, for example, 500 mmol/L NaCl. An example of a suitable flushing solution is a neutral Tris buffer containing 500 mM NaCl. After loading and washing, you can use an appropriate elution buffer to elute the protein A column. In one embodiment, the elution buffer has a pH of 3.3-3.7. An example of a suitable elution buffer is a citric acid/NaCl buffer, such as 20 mmol citric acid + 100 mmol/L NaCl. The eluate can be monitored using techniques known to those skilled in the art. For example, the absorbance at 280 nm can be tracked. In one embodiment, after elution, the main peak at UV 280 nm is collected.

In a preferred embodiment, the affinity chromatography step includes: using a protein A affinity filler, loading the sample after equilibration in a neutral buffer, with a loading capacity of 10 to 45 g/L, and a loading flow rate of 100 to 250 cm/h; After loading the sample, balance 3CV with neutral buffer, rinse 3CV with high-salt washing solution, and balance 3CV with neutral buffer; then start elution, the pH range of elution buffer is 3.3～3.7, and collect UV The main peak at 280nm.

The protein A affinity filler that can be used in the present invention is not limited. Several commercial sources of protein A resin are known, including, but not limited to: MabSelect from GE Healthcare and ProSep Ultra Plus from Millipore. In a preferred embodiment, the protein A affinity filler used in the method of the present invention is a strong alkali-resistant chromatography medium, such as Mabselect SuRe affinity filler.

The affinity column that can be used in the affinity chromatography step of the present invention is not limited. For example, a column bed height of about 20 cm can be used. For example, a non-limiting example of a suitable column packed with MabSelect is a column of about 1.0 cm diameter x about 21.6 cm length (~17 mL bed volume). Columns of this size can be used for small-scale purification. The present invention can also use a larger column size, for example, a 20cm x 21cm column (the column bed volume is about 6.6L), or larger.

Virus inactivation:

In one embodiment, virus inactivation is performed in the affinity collection solution obtained by affinity chromatography. Preferably, an acid inactivation method is used for the virus. The acid inactivation of the virus depends on the protein concentration, pH and inactivation time, and these parameters can be selected through routine experiments. In some embodiments, the pH of the collected protein A eluate containing adalimumab is adjusted to reach a pH of about 3-4, for example, pH 3.2 to 3.7. In one embodiment, an acidic solution, such as citric acid, acetic acid, etc., is used to adjust the pH of the collection solution. Preferably, the protein concentration in the collection solution is ≤25mg/ml. Preferably, the acid inactivation treatment is carried out at 18 to 26°C for 90 to 180 minutes. After the acid inactivation treatment, an alkaline solution, such as Tris, Tris-bis, histidine, etc., is preferably used to adjust the pH of the sample to neutral.

In a preferred embodiment, the virus inactivation step includes: using an acidic solution, such as citric acid, of the affinity chromatography collection solution, adjusting the pH to 3.2-3.7, and the protein concentration ≤ 25 mg/ml, at 18-26°C. Let it stand for 90-180 minutes, and then use an alkaline solution, such as 2M Tris, to adjust the pH of the sample to neutral, for example, the pH is about 6.5-8.5.

Deep filtration:

Depth filtration can remove host cell protein impurities from the recombinant monoclonal antibody process stream by adsorption. The depth filtration can be performed before the protein A chromatography capture, or after the affinity chromatography capture, for example, after the virus is inactivated. In a preferred embodiment, after affinity chromatography, depth filtration is performed. It can be carried out using any depth filtration device known in the art. In a preferred embodiment, a depth filtration membrane package such as X0HC or the like is used for filtration. In a preferred embodiment, the step of deep filtration includes: using X0HC membrane package to perform filtration with a flux ≤ 150 LMH and a load capacity of 65-160 L/m2.

Ion exchange chromatography:

In some embodiments, the adalimumab purification method of the present invention includes performing ion exchange chromatography after the affinity chromatography step. The inventors have found through in-depth research that in this ion exchange chromatography step of the purification process, the use of an eluent containing a metal chelating agent such as EDTA or DTPA for antibody elution can well overcome purified adalimumab The stability of the composition, especially the charge heterogeneity of the antibody composition during storage.

Therefore, in one embodiment, at least one ion exchange separation step is performed on the antibody-containing collection from the previous purification step, wherein an eluent containing a metal chelating agent is used to obtain an adalimumab-containing antibody from ion exchange chromatography. The elution collection fluid. Optionally, before the ion exchange separation step, other chromatographic separation steps may be implemented, including but not limited to ion exchange chromatography, hydrophobic interaction chromatography, and mixed mode chromatography.

In a preferred embodiment, the method of the present invention involves the sequential combined use of anion and cation exchange chromatography, and the use of an eluent containing a metal chelating agent in the cation exchange chromatography for elution of the antibody. In a more preferred embodiment, in ion exchange chromatography, a flushing solution containing a metal chelating agent can also be used to flush the chromatography column.

A variety of clinically available metal chelating agents are known in the art, and all of these metal ion chelating agents can be used in the present invention. Preferably, the metal chelating agent is selected from: EDTA (ethylenediaminetetraacetic acid), DTPA (diethyltriaminepentaacetic acid), EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid), BAPTA (1 ,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid), DMPS (dimercaptopropanesulfonic acid), DMSA (dimercaptosuccinic acid), ALA ( Alpha lipoic acid), or a combination thereof. More preferably, the metal chelating agent is EDTA or DTPA. The amount of metal chelating agent used in the eluent can be determined by those skilled in the art according to the teachings of this specification. For example, those skilled in the art can calculate the amount of metal chelating agent used in the eluent based on the ultrafiltration/diafiltration dialysis volume used for concentration and exchange of the purified antibody composition. In a preferred embodiment, the metal ion chelating agent in the eluent is 5-40 mmol/L, for example, 10-30 mmol/L, for example, 20 mmol/l. Preferably, the amount of metal chelating agent in the eluate results in a purified antibody composition, such as a drug substance or drug product having 0.00026-0.05 mg/ml metal chelating agent, preferably 0.0005-0.03 mg/ml, more It is preferably 0.001-0.02 mg/ml, for example about 0.002, 0.004, 0.008, 0.01 mg/ml. In still another preferred embodiment, in addition to using an eluent containing a metal chelating agent, a metal chelating agent can also be added to the flushing solution used for ion exchange chromatography. In a preferred embodiment, the rinsing liquid contains 5-40 mmol/L, such as 10-30 mmol/L, for example, 10 mmol/l of a metal ion chelating agent, especially EDTA or DTPA.

The ion exchange separation packing that can be used in the present invention is not particularly limited. It is known in the art that anionic or cationic substituents can be attached to the matrix to form an anionic or cationic support for chromatography. Non-limiting examples of anion exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and quaternary amine (Q) groups. Cation exchange substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphoric acid (P) and sulfonic acid (S). Chromatographic membranes, such as ion exchange membranes available from Sartorius, can be used for membrane chromatography. Examples of usable chromatography membranes include, but are not limited to, for example Sartobind Q, Sartobind S (Sartorius AG), Mustang Q (Pall), ChromaSorb, and

HD-Q (millipore). Polysaccharide-based ion exchange resins can also be used. For example, cellulose ion exchange resins such as DE23, DE32, DE52, CM-23, CM-32 and CM-52 are available from Whatman Ltd. Maidstone, Kent, UK. Values based on dextran gel, such as ion exchange resins based on SEPHADEX and SEPHAROSE are also known. For example, DEAE-, QAE-, CM- and SP-SEPHADEX and DEAE-, Q-, CM- and S-SEPHAROSE and SEPHAROSE Fast Flow can be obtained from multiple suppliers. In addition, synthetic polymethacrylate resins such as

EMD "tentacles" ion exchange media can also be used in the present invention. Preferably, in one embodiment, in the method of the present invention, anion exchange chromatography uses Q Sepharose Fast Flow, for example, Q Sepharose Fast Flow from GE Company in the United States. In one embodiment, in the process of the present invention, the cation exchange resin Fractogel polymethacrylate chromatography using a sulfonic acid group attached SO ₃ ^-, for example, from MERCK company Fractogel EMD SO ₃ ^- (M).

Anion exchange chromatography:

In a preferred embodiment, the method of the present invention includes: after affinity chromatography and before cation exchange chromatography, anion exchange packing is used to carry out antibody purification in a flow-through mode. In a preferred embodiment, Q Sepharose Fast Flow anion exchange packing is used. In one embodiment, before performing anion exchange chromatography loading, adjust the pH of the collection solution containing the target antibody from the previous purification step to 6.8-7.4, and the conductivity ≤ 7 mS/cm. Preferably, in an embodiment where the depth filtration is performed, the collected solution of the depth filtration is diluted with an anion balance buffer. In one embodiment, before loading the sample on anion exchange chromatography, it is preferable to flush the chromatography column with a high-salt buffer, and then balance the chromatography column with an anion balance solution. An example of a suitable equilibration buffer is Tris/NaCl, pH approximately 7. In one embodiment, the loading capacity is ≤30g/L. In one embodiment, the absorption peak at 280 nm is monitored by ultraviolet after the start of sample loading. In one embodiment, the penetrating fluid is collected when the absorption value rises to greater than or equal to 25 mAU/mm optical path. In one embodiment, after loading the sample, rinse with an anion balance buffer, continue to collect the absorption peak at 280 nm, and stop the collection when the absorption value drops to less than or equal to 1000 mAU/mm optical path. Preferably, the collecting liquid is adjusted to pH 6.0 using an acidic solution, for example, a 2mol/L citric acid solution.

In a preferred embodiment, the anion exchange chromatography step includes: diluting the collected solution of depth filtration with an anion balance buffer, adjusting the pH to 6.8-7.4 with an alkaline solution, and the conductivity ≤ 7mS/cm; before loading the sample, the anion exchange layer The column is first rinsed with a high-salt buffer, and then balanced with an anion balance solution; load the sample and collect the penetrating solution, for example, when the absorbance rises to greater than or equal to 25mAU/mm optical path, the penetrating solution will be collected; after the sample is loaded , Rinse with anion balance buffer, continue to collect the absorption peak at 280nm ultraviolet, preferably stop collection when the absorption value drops to less than or equal to 1000mAU/mm optical path; preferably, the collection solution is adjusted to pH with 2mol/L citric acid solution 6.0.

Cation exchange chromatography:

In a preferred embodiment, the method of the present invention includes: after anion exchange chromatography, cation exchange chromatography is used to purify adalimumab in an adsorption-elution mode. Preferably, in cation ion exchange chromatography, a cation eluent containing a metal chelating agent such as EDTA or DTPA is used. More preferably, a metal ion chelating agent is used for the rinse and eluent of cation exchange chromatography. In one embodiment, prior to elution, a linear gradient wash is performed. For example, use Rinse A solution and Rinse B solution for linear gradient flushing, where Rinse A is a cation balance solution (for example, 20mmol/L phosphate buffer, pH 6.0); Rinse B has higher ions than the cation balance solution Strength and/or pH (for example, 70 mmol/L phosphate buffer, pH 7.5). In a preferred embodiment, the flushing solution also includes the step of flushing the chromatography column with a flushing solution containing a metal chelating agent such as EDTA or DTPA or other metal ion chelating agents; preferably, it is performed before the linear gradient flushing. Preferably, before sample loading and before elution, the chromatography column is equilibrated with a cation balance solution. In one embodiment, the UV monitoring chromatography eluent starts to collect the elution peak when the absorption peak at UV 280 nm rises to greater than or equal to 50 mAU/mm optical path, and stops collecting when it drops to less than or equal to 1000 mAU/mm optical path.

In a preferred embodiment, the cation exchange chromatography step includes: equilibrate the column with a cation balance solution and load the sample; rinse with a cation rinse solution containing EDTA (or other metal ion chelating agent); balance with a cation balance solution Afterwards, use washing liquid A and B for linear gradient washing, where washing A is a cation balancing liquid, and washing B is a cation balancing liquid with increased ionic strength and/or pH; after washing, balance the column with a cation balancing liquid, and use The cation eluent containing EDTA (or other metal ion chelating agent) begins to elute; preferably, the elution peak is collected when the absorption peak at UV 280nm rises to greater than or equal to 50mAU/mm optical path, and the absorption peak drops to Stop collecting when the optical path is less than or equal to 1000mAU/mm.

In addition to virus filtering:

In the method of the present invention, in an optional embodiment, the antibody collection solution obtained by ion exchange chromatography is subjected to virus removal filtration. Preferably, a virus-removing filter is used to filter the sample, for example, at a working pressure ≤ 4 bar. In a preferred embodiment, multiple filters can be connected in series for sample filtration. For example, you can change

Prefilter and

Pro filters are connected in series.

Ultrafiltration and diafiltration :

In a preferred embodiment, the method of the present invention further includes ultrafiltration and diafiltration steps for, for example, substituting the antibody protein in a formulation buffer that facilitates its stability and concentrating it to a suitable concentration.

Therefore, in the method of the present invention, in one embodiment, it further includes subjecting the antibody collection solution obtained by ion exchange chromatography to ultrafiltration and diafiltration. In one embodiment, an ultrafiltration membrane is used for ultrafiltration and concentration. In one embodiment, the sample is diafiltered after being concentrated by ultrafiltration. The diafiltration buffer can be used for continuous liquid exchange. Preferably, the diafiltration buffer is used in an amount of 5-12 times the volume after ultrafiltration and concentration. Preferably, the antibody composition obtained by the ultrafiltration/diafiltration step has 10-150 mg/ml, preferably 10-100 mg/ml, more preferably, 10-60 mg/ml, for example, 40-50 mg/ml adalim antibody.

In one embodiment, the diafiltration buffer used in the present invention may include buffers, salts, and polyols. In a preferred embodiment, the diafiltration buffer contains: about 10-30 mM L-histidine, about 50-100 mM sodium chloride, about 1-2% (w/v) sorbitol, pH 6.±0.5. More preferably, the diafiltration buffer contains: about 25 mM L-histidine, about 100 mM sodium chloride, about 1.2% (w/v) sorbitol, pH 6.

Preparation of purified antibody composition

In some embodiments, the method of the present invention further includes the step of sterilizing and filtering the purified adalimumab harvested in the above purification step to form DS (drug substance). The drug substance may be further added with or without adjuvants, such as stabilizers or excipients (for example, polysorbate surfactants, for example, polysorbate 80). In addition, in some embodiments, the method of the present invention further includes dispensing the drug substance into a sterile final container to form a drug product DP. Drug substances and drug products can be tested to verify the purification process, analyze product-related impurities, and characterize product stability.

II. Characterization of the stability of the purified composition

The stability of the purified antibody composition produced by the process during storage is related to the quality attributes of the product. For example, the aggregation, degradation or chemical modification of antibodies can lead to antibody heterogeneity, including size heterogeneity and charge heterogeneity, and thus affect the quality of antibody drug products. Therefore, it is necessary to monitor the stability of the purified antibody composition produced by the process. Various methods are known in the art that can be used to test the stability of an antibody composition. For example, non-reduced CE-SDS and SEC-HPLC methods can be used to analyze the purity changes of the antibody composition during storage; capillary isoelectric focusing electrophoresis (cIEF), imaging capillary isoelectric focusing electrophoresis (iCIEF), and Ion exchange chromatography (IEX), etc., analyze the changes in the charge variant components of the antibody composition during storage. In addition, methods known to those skilled in the art can be used to monitor other product-related variants of the antibody composition during storage.

Stability studies generally include long-term stability studies under actual storage conditions, accelerated stability studies, and forced-condition test studies. Under accelerated and forced conditions, degradation is accelerated, and degradation products can be monitored at an earlier point in time and in larger amounts. Therefore, accelerated and forced condition tests can be used to clarify the degradation pathway of the target antibody product, help understand the nature (mechanism) of the target antibody product degradation, and provide the stability of the product under deviating from the optimal storage conditions and extreme conditions. In a preferred embodiment of the present invention, the antibody composition sample is placed at an elevated temperature, such as about 40°C±2°C, 25°C±2°C, for forced or accelerated stability studies.

Purity testing

Methods for monitoring changes in the purity of the antibody composition can include, but are not limited to, non-reduced or reduced SDS-PAGE gel electrophoresis, or CE-SDS (sodium dodecyl sulfate-polyacrylamide capillary electrophoresis), molecular Methods such as size exclusion chromatography (SEC-HPLC). These methods can be used for quantitative analysis of antibody monomers, polymers or fragments.

Size exclusion high performance liquid chromatography, namely SEC-HPLC, is an important method for the purity analysis of monoclonal antibodies. This method is mainly based on the size of the molecule or the difference in hydrodynamic radius to separate the molecules. Through SEC-HPLC, antibodies can be separated into three main forms: high molecular weight form (HMMS), main peak (mainly antibody monomer), and low molecular weight form (LMMS). Antibody purity can be calculated as the percentage of the main peak area on the chromatogram to the sum of all peak areas. Through the SEC-HPLC method, the percentage of antibody monomers in the preparation product can be measured, and the content information of soluble aggregates and shears can be given. For a further description of the SEC-HPLC method, see, for example, J. Pharm. Scien., 83: 1645-1650, (1994); Pharm. Res., 11: 485 (1994); J. Pharm. Bio. Anal. , 15:1928 (1997); J. Pharm. Bio. Anal., 14: 1133-1140 (1986).

Preferably, after the purified adalimumab composition obtained by the method of the present invention is stored, for example, after 4 weeks of storage at 40°C, the monomer purity, as measured by SEC-HPLC, is 97% or more, preferably 98% or 99% the above.

Detection of charge variants

Charge variants are one of the main quality attributes of recombinant monoclonal antibodies and are closely monitored and controlled during the production process. Methods commonly used to analyze charge variants include: cation exchange chromatography, anion exchange chromatography, isoelectric focusing electrophoresis, and capillary focusing electrophoresis, such as, but not limited to, cIEF, IEX-HPLC, hydrophobic high performance liquid chromatography (HIC-HPLC), Reversed-phase high performance liquid chromatography (RP-HPLC) and so on. See, for example, Kahle J, etc., J Pharm Biomed Anal. 2019 Sep 10; 174:460-470, Comparative charge-based separation study with various capillary electrophoresis (CE) modes and cation exchange chromatography (C EX) for the analysis of monoclonal.

Charge variants are classified as acidic or alkaline components relative to the main component. The charge variant eluted from the early stage of cation exchange chromatography or the charge variant eluted from the late stage of anion exchange chromatography is generally referred to as acidic component (acid charge variant). The charge variant eluted later from the cation exchange chromatography or the charge variant eluted early from the anion exchange chromatography is generally referred to as the basic component (basic charge variant).

In a preferred embodiment of the present invention, the charge variant of the antibody in the antibody preparation is determined by cation exchange high performance liquid chromatography (CEX-HPLC). In this assay, the peaks that eluted from the CEX-HPLC column earlier than the retention time of the main peak are marked as "acidic peaks", and those that eluted from the CEX-HPLC column later than the retention time of the main peak The peaks are labeled as "basic peaks". Preferably, using the CEX-HPLC measurement method, after the purified adalimumab composition obtained by the method of the present invention is stored, for example, after storage at 40°C for 4 weeks, the main component of charge decreases by no more than 40%, preferably no more than 30%, 25%, 20%, 15%, 10%, where the main component is defined as the sum of the main peak detected by CEX-HPLC and the basic peaks 1 and 2, wherein the main peak, basic peak 1, and basic peak 2 are respectively It is an antibody variant containing 0, 1, and 2 terminal lysines. In another preferred embodiment, using the CEX-HPLC measurement method, after the purified adalimumab composition obtained by the method of the present invention is stored, for example, after storage at 40°C for 4 weeks, the acid charge variant does not increase by more than 40%, preferably No more than 30%, 25%, 20%, 15%, 10%.

Detection of product-related impurities

Product-related impurities include, but are not limited to, oxidation products, deamidation products, or other structurally incomplete molecules. For example, peptide mapping methods can be used to detect related variants of antibodies, such as deamidation, disulfide bond mismatches, and oxidation. See, for example, Li X et al. J Chromatogr A. 2016 Aug 19; 1460: 51-60, High throughput peptide mapping method for analysis of site specific monoclonal antibody oxidation. In a preferred embodiment, the product-related impurities include adalimuma antioxidant products, especially oxidation products at position M256. In a further preferred embodiment, the amount of the adalimuma antioxidant product in the purified antibody composition of the present invention is reduced relative to the composition obtained without using the purification process of the present invention.

III. The antibody composition of the present invention

In one aspect, the present invention provides a stable adalimumab composition, especially a stable adalimumab composition produced by the antibody purification method of the present invention. In yet another aspect, the present invention also provides an article of manufacture comprising packaging material, the adalimumab composition of the present invention, and a label or package insert.

In one embodiment, the present invention provides an adalimumab composition comprising adalimumab and a metal chelating agent, preferably 0.00026-0.05 mg/ml metal chelating agent, preferably 0.0005-0.03 mg/ml , More preferably 0.001-0.02 mg/ml, for example, about 0.002, 0.004, 0.008, 0.01 mg/ml of the metal chelating agent. Preferably, the metal ion chelating agent is selected from: EDTA, DTPA, EGTA, BAPTA, DMPS, DMSA, ALA, or a combination thereof; more preferably, EDTA or DTPA.

In one embodiment, the present invention provides a stable liquid adalimumab composition comprising (i) adalimumab, especially an adalimumab composition produced by the purification method of the present invention; (ii) a buffer , (Iii) polyol, and (iv) surfactant, the pH of the antibody composition is about 5.0-7.0. Optionally, the composition of the present invention also contains a salt, such as sodium chloride.

The amount of the antibody contained in the antibody composition of the present invention may vary according to the specific purpose characteristics of the composition, the specific environment, and the specific purpose for which the composition is used. In some embodiments, the antibody composition is a liquid, which may contain about 1-150 mg/mL, preferably about 10-100 mg/mL, for example, about 15, 20, 25, 30, 35, 40, 45, 50 , 55, 60mg/mL antibody. In one embodiment, the present invention relates to antibody drug substances produced at a concentration of 10-50 mg/mL. In another embodiment, the present invention relates to a drug product formed from the drug substance in combination with pharmaceutically acceptable excipients.

In a preferred embodiment, the adalimumab composition of the present invention comprises:

(i) 10-150mg/ml, for example 20-50mg/ml, such as about 40mg/ml adalimumab;

(ii) 0.001-0.02mg/ml metal chelating agent, such as EDTA or DTPA;

(iii) 0.8-1.5mg/ml; more preferably 1.0-1.2mg/ml of polysorbate non-surfactant, such as polysorbate 80;

(iv) 10-50mM histidine buffer, such as about 25mM histidine, and

Preferably, the composition of the present invention has a pH of about 5-7, such as 6.0±0.5, such as about 6.0.

In another preferred embodiment, the adalimumab composition of the present invention further comprises:

(vi) 50-100 mM sodium chloride, such as 100 mM sodium chloride; and

(vii) About 1-2% (w/v) sorbitol, such as about 1.2% (w/v) sorbitol.

The antibody liquid composition of the present invention may or may not contain other excipients. The excipients include, for example, flavoring agents, antimicrobial agents, sweeteners, antistatic agents, antioxidants, gelatin and the like. These and other known pharmaceutical excipients and/or additives suitable for the formulation of the present invention are well known in the art, for example, listed in "The Handbook of Pharmaceutical Excipients, 4th Edition, edited by Rowe et al., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21st edition, edited by Gennaro, Lippincott Williams & Wilkins (2005)".

In a preferred embodiment, the composition of the invention has stability. Preferably, the composition has stability selected from one or more of the following: oxidation stability, monomer stability, and charge variant stability, and polysorbate 80 stability. More preferably, after the composition of the present invention is stored, for example, after 4 weeks of storage at 40°C, the main component of charge decreases by no more than 40%, preferably no more than 30%, 25%, 20%, 15%, 10%; preferably, The increase in acid charge variants is not more than 40%, preferably not more than 30%, 25%, 20%, 15%, 10%. More preferably, after the composition of the present invention is stored, for example, after 4 weeks of storage at 40°C, the monomer purity, as measured by SEC-HPLC, is 97% or more, preferably 98% or 99% or more. Preferably, the composition of the present invention does not have significantly increased oxidation products of M265 after storage, for example, 4 weeks at 25°C, for example, the oxidation rate of the residue is less than 40%, more preferably less than 20%, more preferably It is less than 10%, more preferably less than 5%. Preferably, after the composition of the present invention is stored, for example, after 4 weeks of storage at 40°C, the content of polysorbate 80 is reduced by no more than 10%, for example no more than 5%.

IV. Use of the antibody composition of the present invention

In another aspect, the present invention provides medical uses of the antibody composition of the present invention, and treatment methods using the antibody composition of the present invention.

Therefore, in one embodiment, the present invention provides a method of treating a disease in which TNFα activity is detrimental, the method comprising administering to a human subject a pharmaceutical composition comprising an antibody obtained by any method of the present invention. In one embodiment, the disease in which TNFα activity is detrimental is selected from autoimmune diseases, intestinal diseases, and skin diseases. In one embodiment, the disease is selected from rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis and chronic plaque psoriasis disease. In one embodiment, the composition of the present invention is administered to individuals in need in combination with another therapeutic agent, for example, with methotrexate, DMARDS, glucocorticoids, non-steroidal anti-inflammatory drugs (NSAID), and/or Analgesics.

In yet another embodiment, the present invention relates to the use of the antibody composition of the present invention, especially the antibody composition prepared by the method of the present invention, in the preparation of a pharmaceutical composition for the treatment of diseases in which TNFα activity is harmful.

The present invention is further illustrated in detail by the following examples, which should not be considered restrictive. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.

Example

material

Adalimumab

The adalimumab purified in the following examples is adalimumab expressed in Chinese hamster ovary CHO cells. The heavy chain and light chain sequences of the antibody are shown in SEQ ID NOs: 1 and 2.

Process raw materials :

General method description

EDTA residue test method

Reversed-phase high performance liquid chromatography (RP-HPLC method) was used to detect the residual amount of EDTA in the purified composition. Use Agilent 1260 high performance liquid chromatograph. Take 7.5g/L EDTA solution (manufacturer sigma, article number E4884-100G), and dilute with ultrapure water to 1.0g/L, 0.5g/L, 0.1g/L, 0.05g/L, 0.01g/L for standardization Curve sample preparation, and establish a standard curve. Take 100μl sample, add 30μl 10% trichloroacetic acid (manufacturer Aladdin, article number T104257-500g), mix well, centrifuge at 13000r/min for 5min, take 50μl supernatant, add 0.5μl 20g/L CuSO ₄ solution (manufacturer Aladdin, article number C112401-100g), mix well. Chromatographic analysis conditions: Symmetry C18 column (manufacturer Waters, article number WAT054275), flow rate: 0.4ml/min, injection volume: 10μl, column temperature: 25℃, detection wavelength: 254nm, mobile phase A is pH4.0, containing 10% tetrabutylammonium hydroxide 50mM ammonium formate aqueous solution, mobile phase B is methanol, ratio: A:B=90:10, running time: 20 minutes. Calculate the EDTA content according to the standard curve. Use the following EDTA content calculation formula:

EDTA content (g/L) = sample EDTA content value (μg/μl) × 1.3 (dilution multiple)

Figure 1 exemplarily shows a typical map of the EDTA residue test.

Polysorbate 80 content test method

The FLD-HPLC method (High Performance Liquid Chromatography Fluorescence Detection Method) was used to detect the polysorbate 80 content of the samples. Use Agilent 1260 high performance liquid chromatograph. Take 2.5% PS80 stock solution and dilute it to 1.6mg/ml, 0.8mg/ml, 0.4mg/ml, 0.2mg/ml, 0.1mg/ml with buffer without PS80 to prepare the standard curve, and establish the standard Curve and chromatographic analysis conditions: Knitted Reactor Coil column (manufacturer SUPELCO, catalog number 57405), mobile phase: 0.15mol/L sodium chloride, 0.05mol/L Tris, pH 8.0, 5% acetonitrile, 5.0μmol/L NPN, 15ppm Brij, flow rate: 1.5ml/min; collection time: 3 minutes; injection volume: 10μl; column temperature: 30°C; detection wavelength: excitation light 350nm, emission light 420nm. Calculate the polysorbate 80 content according to the standard curve. Figure 2 exemplarily shows a typical map.

Charge heterogeneity determination method (CEX-HPLC method)

Cation exchange chromatography (CEX-HPLC method) was used to detect the charge heterogeneity of the samples. Use ProPac ^TM WCX-10 ion exchange column (manufacturer Thermo, article number 054993) for separation, mobile phase A is 10mmol/L phosphate buffer, mobile phase B is 10mmol/L phosphate+200mmol/L sodium chloride buffer, flow rate 1.0ml/min, gradient elution, detection wavelength 280nm, column temperature 35°C. Dilute the sample to 2.0mg/ml with ultrapure water as the test solution. Take 50μl of the test solution and inject it into the liquid chromatograph. The temperature of the injector is 8℃ and the running time is 35min. According to the area normalization method, the acidic components, main components (main peak + alkali peak 1 + alkali peak 2) and basic components of the sample were calculated. Consistent with the research results in this field, the main peak, basic peak 1, and basic peak 2 are variants containing 0, 1, and 2 terminal lysines respectively (see, for example, Wu Linping et al., the key to the physical and chemical properties of adalimumab Quality attribute analysis, Chinese medical industry impurities, 2018,49(3)). Figure 3 exemplarily shows a typical map.

CEX-HPLC method elution gradient

Antibody purity determination method (SEC-HPLC method)

Size exclusion chromatography (SEC-HPLC method) was used to detect the molecular size variants of the samples. Use TSK-gel G3000SWxl (manufacturer TOSOH, article number 0008541) for separation, the mobile phase is 20mmol/L phosphate buffer +200mmol/L sodium chloride, the flow rate is 0.5ml/min, the detection wavelength is 280nm, and the column temperature is 25°C. Dilute the test product to 2.0mg/ml with ultrapure water as the test product solution; inject 50μl of the test product solution into the liquid chromatograph, the temperature of the injector is 8℃, and the running time is 30min. Calculate the aggregates, monomers and fragments of the sample according to the area normalization method.

Metal element content detection method

Use standard IPC-MS method for metal element content detection. The operation scheme of the method can be found in, for example, "Metal Ion Analysis Technology" Chemical Industry Press, edited by Yu Zili and Cheng Guanglei. In 2004, ISBN 750256148.

Stability experiment

The test samples are filtered, subpackaged, stoppered and capped in an ultra-clean table (Suzhou Sujing Antai, Model: SW-CJ-2FD). After that, put the sample in a constant temperature and humidity box (German binder, model: KBF P 720) RH 60% ± 5%, and set the temperature (for example, about 25°C or 40°C) for accelerating the stability test under dark conditions , Or the temperature used for long-term stability experiments (for example, about 5°C). At selected time points (for example, t=0, 1 week, 2 weeks, 3 weeks, 4 weeks), samples are taken for testing. Detection includes, but is not limited to, detection of charge variants by CEX-HPLC, detection of antibody purity by SEC-HPLC, and/or detection of polysorbate 80 content by FLD-HPLC.

Example 1:

This example describes an unoptimized purification process. The process includes the following steps:

a) Affinity chromatography: Mabselect SuRe (US GE, article number 17-5438-03, 17-5438-04, 17-5438-05) affinity filler is used. Different packing sizes can be used according to process requirements. For example, a pack size of 1-10 liters can be used, such as 1 liter, 5 liters and 10 liters. Use equilibration buffer 20mmol/L Tris+150mmol/L NaCl, pH 7.2, load the sample after equilibration, load 10~45g/L, sample flow rate 100~250cm/h. After loading the sample, equilibrate 3CV with 20mmol/L Tris+150mmol/L NaCl, pH 7.2, and then rinse 3CV with 20mmol/L Tris+0.5mol/L NaCl, pH7.2, 20mmol/L Tris+150mmol/L NaCl , PH7.2 balances 3CV. Then start elution, and the pH range of the elution buffer (20mmol citric acid + 100mmol/L NaCl) is 3.3 to 3.7. Collect the main peak at 280nm ultraviolet.

b) Virus inactivation: Use 2mol/L citric acid for the affinity collection solution, adjust the pH to 3.2～3.7, and the protein concentration ≤25mg/ml, let stand at 18～26℃ for 90～180min, then use 2mol/L Tris Adjust the pH of the sample to neutral.

c) Deep filtration: use X0HC (product of Millipore, product number: MX0HC10FS1) membrane package, flux ≤ 150LMH, load capacity 65 ~ 160L/m2, for filtration.

d) Anion exchange chromatography: Q Sepharose Fast Flow (US GE, article number 17-0510-04, 17-0510-05, 17-0510-60) anion exchange packing is used. Dilute the collected solution with anion balance buffer 20mmol/L Tris-HCl, pH 7.1, and adjust the pH to 6.8～7.4 with 2mol/L Tris. After mixing thoroughly, take a sample for detection. The conductivity is ≤7mS/cm. First flush the column with 20mmol/L Tris-HCl+1mol/L NaCl, pH 7.0, and then balance the column with an anion balance solution. Start to load the sample, the load of the sample is less than or equal to 30g/L, and when the absorption value at 280nm of ultraviolet rises to greater than or equal to 25mAU/mm optical path, the penetrating fluid will be collected. After loading the sample, rinse with anion balance buffer, continue to collect the absorption peak at 280nm, and stop the collection when the absorption value drops to less than or equal to 1000mAU/mm optical path. The collected solution was adjusted to pH 6.0 with 2mol/L citric acid solution.

e) Cation exchange chromatography: use cation exchange packing Fractogel EMD SO3-(M) (Merck, Germany, 1.16882.5000) packing. After equilibrating the column with cation balance solution 20mmol/L phosphate buffer, pH 6.0, start loading. After loading the sample, equilibrate with cation balance solution, and then start linear gradient washing. Washing solution A is cation balance solution, washing solution B is 70mmol/L phosphate buffer, pH 7.5, linear gradient washing 10CV. After washing, equilibrate the column with cation balance solution again, and start elution with cation eluent 20mmol/L phosphate buffer, 200mmol/L NaCl, pH 7.0. The elution peak is collected when the absorption peak at ultraviolet 280nm rises to greater than or equal to 50mAU/mm optical path, and the collection stops when it drops to less than or equal to 1000mAU/mm optical path.

f) In addition to virus filtering: adopt

Prefilter (Merck Millipore, United States, article number: MSPV05FS1) prefilter and

Pro (Merck Millipore, U.S., article number: VPMG201NB1) filters are connected in series to filter the samples. Working pressure≤4bar.

g) Ultrafiltration and diafiltration: Pellicon2 (30kD) (Merck Millipore, USA, catalog number: P2B030A05) ultrafiltration membrane package is used for ultrafiltration and concentration. After the sample is concentrated, start diafiltration, and use diafiltration buffer (3.9g/L L-histidine, 12.0g/L sorbitol, 6.2g/L sodium chloride, pH 6.0) to continuously change the solution 5-12 times and concentrate After the volume, the collected liquid of diafiltration was obtained.

h) Stock solution preparation: add polysorbate 80 (Germany Merck KGaA, article number: 8.17061.1000) and sterilize and filter.

Example 2

This example describes the optimized purification process. The process includes the following steps:

a) Affinity chromatography: Mabselect SuRe (US GE, article number 17-5438-03, 17-5438-04, 17-5438-05) affinity filler is used. Use 20mmol/L Tris+150mmol/L NaCl, load the sample after the pH is 7.2 equilibrated, load 10-45g/L, and load the sample flow rate 100-250cm/h. After loading the sample, equilibrate 3CV with 20mmol/L Tris+150mmol/L NaCl, pH 7.2, and then rinse 3CV with 20mmol/L Tris+0.5mol/L NaCl, pH7.2, 20mmol/L Tris+150mmol/L NaCl , PH7.2 balances 3CV. Then start elution, and the pH range of the elution buffer (20mmol citric acid + 100mmol/L NaCl) is 3.3 to 3.7. Collect the main peak at 280nm ultraviolet.

e) Cation exchange chromatography: use cation exchange packing Fractogel EMD SO3-(M) (Merck, Germany, 1.16882.5000) packing. After equilibrating the column with cation balance solution 20mmol/L phosphate buffer, pH 6.0, start to load the sample. After loading the sample, rinse 3CV with cation rinse solution 20mmol/L phosphate buffer, 10mmol/L disodium edetate, pH 6.0. Equilibrate with a cation balance solution, and then start linear gradient washing as described in Example 1, washing solution A is a cation balance solution, washing solution B is 70mmol/L phosphate buffer, pH 7.5, linear gradient washing 10CV. After washing, equilibrate the column with cation balance solution again, and start elution with cation eluent 20mmol/L phosphate buffer, 160mmol/L NaCl, 20mmol/L EDTA·2Na, pH 6.8. The elution peak is collected when the absorption peak at ultraviolet 280nm rises to greater than or equal to 50mAU/mm optical path, and the collection stops when it drops to less than or equal to 1000mAU/mm optical path.

f) In addition to virus filtering: adopt

Pro (Merck Millipore, United States, article number: VPMG201NB1) filters are connected in series to filter the samples, and the working pressure is less than or equal to 4 bar.

g) Ultrafiltration and diafiltration: Pellicon2 (30kD) (Merck Millipore, U.S., catalog number: P2B030A05) ultrafiltration membrane package is used for ultrafiltration and concentration. After the sample is concentrated, start diafiltration, use diafiltration buffer (3.9g/L L-histidine, 12.0g/L sorbitol, 6.2g/L sodium chloride, pH 6.0), and change continuously 5-12 times After concentration, the volume is obtained, and the collected liquid of diafiltration is obtained.

The stock solution obtained through the above optimization process contains: 40mg/ml adalimumab, 3.9g/L L-histidine, 12.0g/L sorbitol, 6.2g/L sodium chloride, 1mg/mL polysorbate-80 , PH6.0.

Example 3:

The structure of the antibody itself, the residual impurities, the production process, the extract of the packaging material and the formulation of the formulation, etc. will affect the stability of the formulation, including the influence of chemical and physical properties, such as deamidation, isomerization, hydrolysis, racemization, and terminal Modification, saccharification, oxidation, disulfide bond change, denaturation, aggregation, precipitation or particle formation, adsorption, etc. Therefore, antibody preparations are more sensitive to environmental factors such as temperature changes, oxidation, light, ion content, and cutting force.

This example describes the stability experiment and results of the stock solution sample produced by the production process of Example 1 to investigate the influence of the production process on the stability of the antibody preparation.

Filtering, sub-packing, plugging and capping are carried out in the ultra-clean bench (Suzhou Sujing Antai, model: SW-CJ-2FD), and placed in a constant temperature and humidity box (German binder, model: KBF P 720) RH 60%± 5%, set the temperature under accelerated and long-term conditions under dark conditions. Sampling and testing were performed at the time points shown in Table 1. The main component of charge variants in the sample was detected by CEX-HPLC method. The experimental results are shown in Table 1. Different batches of antibodies from the same process were used in the experiment.

Table 1. Stability test results before process optimization

It can be seen from the results of the stability experiment that the purified adalimumab composition produced by the pre-optimized process is unstable, the amount of the main component of the charge variant decreases during storage, and the antibody composition shows increased charge heterogeneity. Sex.

Example 4:

In this example, the peptide comparison method was used to identify the main factors causing the instability of charge heterogeneity in the adalimumab composition produced by the process of Example 1.

Peptide comparison method:

In order to confirm the reason for the decrease in the main components of CEX-HPLC under accelerated conditions, the original solution and the finished product of the composition (that is, the finished product formed after the original solution is filled into the final storage container of the drug), the original solution of batch 1 and the two finished products Samples stored for one month under long-term (5°C) and accelerated (25°C) conditions were digested into peptides with trypsin, and then liquid chromatography tandem mass spectrometry (LC-MS/MS) technology was used to compare the Differences in post-translational modification and disulfide bond pairing. The results show that the disulfide bond pairing in the two finished products is the same, there is no change, the deamidation content has no obvious change, but the oxidation content has a significant change, such as the M (256 position) in the composition heavy chain peptide (253-259) DTLMISR There is a big difference in the oxidation content of CEX-HPLC. See Table 2 for details, which confirms that M oxidation is the cause of the decrease in the main components of CEX-HPLC under accelerated conditions.

Table 2. Peptide map analysis results of the composition stock solution and finished product

Example 5:

In the production process, metal ions are a possible factor causing antibody oxidation. Therefore, in this example, the metal element content in the two batches of adalimumab composition produced by the process of Example 1 was tested.

The test results are shown in Table 3 below.

Example 6:

In this example, a subsequent process optimization study was conducted for the removal of metal ion oxides to investigate the effect of process optimization on the stability of the purified adalimumab composition.

For products produced under different optimized conditions, a forced stability test at 40°C was carried out.

Experimental sample: Adalimidine antigen solution produced by the following different process conditions

①The process conditions of Example 2, wherein the cation exchange chromatography eluent contains 20mM EDTA, and ultrafiltration/diafiltration dialysis 6SV (sample volume)

②The process conditions of Example 2, wherein the cation exchange chromatography eluent contains 20mM EDTA, and the ultrafiltration/diafiltration dialysis 10SV

③The process conditions of Example 1, wherein the cation exchange chromatography eluent does not contain EDTA, and the ultrafiltration/diafiltration dialysis 10SV

④50% sample ②+50% sample ③mix

Determination of EDTA content in samples

According to the theoretical equation of ultrafiltration/diafiltration continuous operation mode:

Calculate the concentration of EDTA in the stock solution;

among them,

n=dialysis volume multiple

Cc = Concentration of EDTA in the reflux solution

Co = Concentration of raw material EDTA

R = impurity interception rate

R is 0 without interception, so generally Cc>e^(-n)×Co

e=2.72, when n=10, Cc>4.5×10 ^-5 ×Co, the cation eluate of Example 2 contains 20 mM EDTA, so it can be estimated that the Cc EDTA content in the preparation is greater than 9×10 ^-4 mM, that is, greater than 0.00026 mg/ml.

The EDTA content in the process samples of Example 2 obtained by using different ultrafiltration/diafiltration dialysis volumes (SV), namely the aforementioned sample ① and sample ②, were determined in duplicate. The results are as follows in Table 4:

Table 4 EDTA content determination

The test sample was placed at 40°C for stability test. Samples were taken at the beginning of the experiment (T=0) and during storage for 1 week, 2 weeks, and 4 weeks to determine the content of polysorbate 80 in the antibody charge variant and composition. The results are shown in Figure 4 and Figure 5.

The results show that the purity of adalimumab and the charge isomers of the adalimumab composition with EDTA content greater than 0.00026g/L are more stable (the main component of the adalimumab is less than 26% for 4 weeks of forced stability at 40°C), polysorbate 80 content is more stable (40°C forced stability decreased less than 10% for 4 weeks).

Example 7:

This example describes the product produced by the optimized process (Example 2), and the result of the forced stability test at 40°C.

① The process conditions of embodiment 2, in which EDTA is added to the washing solution and eluent of cation exchange chromatography;

② The process conditions of Example 2, in which only EDTA was added to the washing solution of cation exchange chromatography.

The test sample was placed at 40°C for stability test. Sampling was taken at the beginning of the experiment (T=0) and at the time of storage for 1, 2, and 4 weeks to determine the antibody charge variant (CEX-HPLC method) and purity (SEC-HPLC method), and to determine the amount of polymer in the composition. The content of sorbate 80. The results are shown in Table 5 below.

Table 5. 40℃ forced stability test results before and after process optimization

The results show that, compared to only adding EDTA to the rinse solution, adding EDTA to the rinse solution and the eluent can significantly improve the stability of the purified adalimumab composition, especially the improved charge heterogeneity; At the same time, the antibody purity detected by SEC-HPLC also showed some increase. In addition, the addition of EDTA in the eluent also increases the stabilizer of the surfactant polysorbate 80 in the composition, and significantly reduces the degradation of polysorbate 80 during the storage of the composition.

Example 8

In order to further demonstrate the influence of the optimized process of the present invention on the antibody composition, the product obtained by the optimized process is compared with

The original research drug (AbbVie, USA, specification 40mg:0.8ml, batch number 45024LX01) was compared for stability.

Experimental sample:

① Two batches of Adalimidine antigen solution produced according to the optimized process conditions of Example 2.

②Adalimumab

The test sample was placed at 40°C for stability test. Sampling was taken at the beginning of the experiment (T=0) and at the time of storage for 1, 2, and 4 weeks to determine the antibody charge variant (CEX-HPLC method) and purity (SEC-HPLC method), and to determine the amount of polymer in the composition. The content of sorbate 80. The results are shown in Table 6 below.

Table 6. Forced stability test results of the original drug and the product of the present invention at 40°C after optimization of the process

The results show that the purified adalimumab composition obtained by the optimized process of the present invention is equivalent to or better than the commercially available adalimumab preparation (Humira) in terms of stability. In the forced stability test at 40°C and stored for 4 weeks, the product of the present invention and the commercially available preparation are basically equivalent in the changes in the main component and acidic component of the charge variant (main component: about 23.3% vs. about 20%; acidic component: About 21% vs. about 19%); and by SEC-HPLC detection, the product of the present invention is better than the commercially available preparation in purity (about 99% vs. about 92%).

Further, the finished product of the composition after the process optimization of the present invention was subjected to peptide mapping analysis to check the oxidation rate of the product. The detection method is the same as described above.

Table 7. Peptide map analysis results of the finished composition after process optimization

The above peptide map analysis results confirm that the optimization process of the present invention can significantly improve the oxidation stability of the adalimumab composition.

Claims

A method for preparing an adalimumab composition with improved stability, the stability is preferably charge variant stability, optionally further comprising monomer stability, oxidation stability, or a combination thereof; The method includes the following steps:

(a) Affinity chromatography: applying a mixture containing adalimumab to an affinity chromatography resin, eluting with an elution buffer, and collecting a collection solution containing the antibody;

(b) Anion exchange chromatography: applying a collection solution containing the antibody to an anion exchange resin, and collecting the penetrating solution containing the antibody; and

(c) Cation exchange chromatography: applying a collection solution containing the antibody to a cation exchange resin, eluting with an elution buffer, and collecting the collection solution containing the antibody, wherein the eluate contains a metal ion chelate Mixture (for example, EDTA or DTPA),

Preferably, after the purified adalimumab composition obtained by the method is stored, for example, after 4 weeks of storage at 40°C, the acid charge variant increases by no more than 40%, preferably no more than 30%, 25%, 20%, 15%, 10%; and optionally, the main component of charge is reduced by no more than 40%, preferably no more than 30%, 25%, 20%, 15%, 10%;

More preferably, after the purified adalimumab composition obtained by the method is stored, for example, after 4 weeks of storage at 40°C, the monomer purity, as measured by SEC-HPLC, is 97% or more, preferably 98% or 99%. %the above;

More preferably, the purified adalimumab composition obtained by the method has oxidative stability. After storage, for example, after storage at 25°C for 4 weeks, as measured by peptide mapping, the methionine residue at position 256 of the antibody The oxidation ratio of (M256) does not exceed 40%, or more preferably does not exceed 30%, 20% or 10%, 5%.
The method of claim 1, wherein after affinity chromatography and before performing anion exchange chromatography, virus inactivation and optionally depth filtration are performed.
The method of claim 1, wherein the affinity chromatography is performed using protein A affinity filler, with a loading capacity of 10 to 45 g/L; preferably, the sample flow rate is 100-250 cm/h; preferably, the pH range of the elution buffer It is 3.3 to 3.7; more preferably, before elution, rinse with a rinse solution containing high salt and balance with a neutral buffer.
The method of any one of the preceding claims, wherein the anion exchange chromatography step comprises: before performing anion exchange chromatography, adjusting the pH of the adalimumab-containing collection solution to be chromatographed to 6.8-7.4; and the conductivity ≤7mS/cm.
The method of any one of the preceding claims, wherein the cation exchange chromatography step comprises: eluting with a cation eluent containing a metal ion chelating agent, wherein the amount of the metal chelating agent in the eluent results in the amount obtained by the method The purified adalimumab composition has a metal chelator of 0.00026-0.05mg/ml, preferably 0.0005-0.03mg/ml, more preferably 0.001-0.02mg/ml, for example about 0.002, 0.004, 0.008, 0.01mg/ml;

Preferably, the amount of the metal ion chelating agent in the eluent is 5-40 mmol/L, for example, 10-30 mmol/L, for example, 20 mmol/l.
The method of any one of the preceding claims, wherein the cation exchange chromatography step further comprises: before elution, washing with a cation washing solution containing a metal ion chelating agent,

Preferably, the metal ion chelating agent in the rinse solution is 5-40 mmol/L, for example, 10-30 mmol/L, for example, 10 mmol/l.
The method of any one of the preceding claims, wherein the metal ion chelating agent is selected from: EDTA, DTPA, EGTA, BAPTA, DMPS, DMSA, ALA, or a combination thereof; preferably EDTA or DTPA.
The method according to any one of the preceding claims, wherein the method further comprises the steps of ultrafiltration and diafiltration of the purified adalimumab and the preparation step of the purified antibody composition; preferably, 5-12 times ultrafiltration is used Concentrate the volume of the diafiltration buffer to obtain the diafiltration collection; preferably, in the preparation step of the purified antibody composition, a stabilizer polysorbate (preferably polysorbate 80) is added to the composition.
An adalimumab composition comprising adalimumab and a metal chelator, preferably a metal chelator of 0.00026-0.05mg/ml, preferably 0.0005-0.03mg/ml, more preferably 0.001-0.02mg/ml, For example, about 0.002, 0.004, 0.008, 0.01 mg/ml metal chelating agent.
The adalimumab composition of claim 9, wherein the adalimumab is obtained by purification by the method of any one of claims 1-8.
The adalimumab composition of claims 9-10, wherein the composition is an aqueous composition comprising:

Adalimumab, for example 10-150mg/ml, for example 20-50mg/ml, such as about 40mg/ml adalimumab;

Metal chelating agent, for example 0.001-0.02mg/ml, for example 0.002, 0.004, 0.008, 0.01mg/ml EDTA or DTPA;

Polysorbate non-surfactant, such as polysorbate 80 or 20, such as 0.8-1.5 mg/ml; more preferably 1.0-1.2 mg/ml polysorbate 80;

Buffer, for example histidine buffer, for example 10-30 mM histidine, such as about 25 mM histidine;

Polyols, such as sorbitol, for example about 1-2% (w/v) sorbitol, such as about 1.2% (w/v) sorbitol;

Sodium chloride, for example about 50-100 mM sodium chloride, such as about 100 mM sodium chloride,

Preferably, the pH is about 5-7, for example about 6.0.
The adalimumab composition of any one of claims 9-11, wherein:

After the composition is stored, for example, after 4 weeks of storage at 40°C, the acid charge variant increases by no more than 40%, preferably no more than 30%, 25%, 20%, 15%, 10%; preferably, the main component of charge Decrease not more than 40%, preferably not more than 30%, 25%, 20%, 15%, 10%;

Preferably, after the composition is stored, for example, after 4 weeks of storage at 40°C, the monomer purity, as measured by SEC-HPLC, is 97% or more, preferably 98% or 99% or more;

Preferably, the composition has oxidation stability.
A method for improving the stability of the purified adalimumab aqueous composition, wherein the stability is preferably selected from charge variant stability, monomer stability, oxidative stability, or a combination thereof,

The method includes:

(a) Affinity chromatography: applying a mixture containing adalimumab to an affinity chromatography resin, eluting with an elution buffer, and collecting a collection solution containing the antibody;

(b) Anion exchange chromatography: applying a collection solution containing the antibody to an anion exchange resin, and collecting the penetrating solution containing the antibody; and

(c) Cation exchange chromatography: applying a collection solution containing the antibody to a cation exchange resin, eluting with an elution buffer, and collecting the collection solution containing the antibody, wherein the eluate contains a metal ion chelate Mixture (for example, EDTA or DTPA),

Preferably, after the purified adawood composition obtained by the method is stored, for example, after storage at 40°C for 4 weeks, the main component of charge decreases not more than 40%, preferably not more than 30%, 25%, 20%, 15%, 10%; preferably, the increase in acid charge variants does not exceed 40%, preferably does not exceed 30%, 25%, 20%, 15%, 10%;

Preferably, after the purified adalimumab composition obtained by the method is stored, for example, after 4 weeks of storage at 40°C, the monomer purity, as measured by SEC-HPLC, is 97% or more, preferably 98% or 99% the above;

Preferably, the purified adalimumab composition obtained by the method has oxidative stability.
A method for purifying adalimumab from a mixture containing adalimumab, the method comprising:

(a) Affinity chromatography: applying a mixture containing adalimumab to an affinity chromatography resin, eluting with an elution buffer, and collecting a collection solution containing the antibody;

(b) Anion exchange chromatography: applying a collection solution containing the antibody to an anion exchange resin, and collecting the penetrating solution containing the antibody; and

(c) Cation exchange chromatography: applying a collection solution containing the antibody to a cation exchange resin, eluting with an elution buffer, and collecting the collection solution containing the antibody, wherein the eluate contains a metal ion chelate Mixture (for example, EDTA or DTPA),

Among them, preferably, the purified adalimumab composition obtained by the method has stability selected from the group consisting of oxidation stability, monomer stability, and charge variant stability;

Preferably, after the purified adawood composition obtained by the method is stored, for example, after storage at 40°C for 4 weeks, the main component of charge decreases not more than 40%, preferably not more than 30%, 25%, 20%, 15%, 10%; preferably, the increase in acid charge variants does not exceed 40%, preferably does not exceed 30%, 25%, 20%, 15%, 10%;

Preferably, after the purified adalimumab composition obtained by the method is stored, for example, after 4 weeks of storage at 40°C, the monomer purity, as measured by SEC-HPLC, is 97% or more, preferably 98% or 99% the above;

Preferably, the purified adalimumab composition obtained by the method has oxidative stability.
The method according to any one of the foregoing, wherein the antibody composition, after being stored at 40°C for 4 weeks, the content of polysorbate 80 therein is reduced by no more than 10%, preferably no more than 5% or less.
The method according to any one of claims, wherein the mixture containing adalimumab is a monoclonal antibody collection solution recovered from a mammalian host cell culture that recombinantly expresses adalimumab.
A method of treating diseases in which TNFα activity is detrimental, the method comprising administering to a subject in need an antibody pharmaceutical composition according to any one of the preceding claims.