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WO2021139720A1 - 阿达木单抗纯化方法及其稳定组合物 - Google Patents

阿达木单抗纯化方法及其稳定组合物 Download PDF

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Publication number
WO2021139720A1
WO2021139720A1 PCT/CN2021/070654 CN2021070654W WO2021139720A1 WO 2021139720 A1 WO2021139720 A1 WO 2021139720A1 CN 2021070654 W CN2021070654 W CN 2021070654W WO 2021139720 A1 WO2021139720 A1 WO 2021139720A1
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Prior art keywords
adalimumab
antibody
composition
stability
purified
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PCT/CN2021/070654
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English (en)
French (fr)
Inventor
杨忠华
马一冬
邢振荣
蔺智勇
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信达生物制药(苏州)有限公司
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Priority to CN202180008565.2A priority Critical patent/CN114929725A/zh
Publication of WO2021139720A1 publication Critical patent/WO2021139720A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons

Definitions

  • 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.
  • impurities include, for example, process-related impurities and product-related impurities.
  • process-related impurities include, for example, process-related impurities and product-related impurities.
  • 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.
  • 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.
  • the detection of charge heterogeneity has attracted more and more attention from global pharmaceutical companies and regulatory agencies.
  • the control of charge heterogeneity is one of the important links.
  • the main modifications include deamidation and glycosylation of sialic acid and asparagine residues.
  • 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).
  • Some methods for stabilizing antibody products have been proposed, which mainly include adding a certain amount and type of excipients to the antibody product.
  • the addition of excipients may cause problems in product safety or efficacy.
  • 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).
  • 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
  • SEC SEC
  • RP HPLC RP HPLC
  • cIEF CE-SDS
  • CE-SDS CE-SDS
  • 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- ⁇ ) 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the method includes the following steps:
  • 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;
  • (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.
  • the purified adalimumab composition obtained by the method has one or more characteristics selected from the following:
  • 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%;
  • the monomer purity, as measured by SEC-HPLC is 97% or more, preferably 98% or 99% or more;
  • 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%.
  • 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.
  • 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 cation exchange chromatography step further comprises: washing with a cation washing solution containing a metal ion chelating agent before elution.
  • a 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 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.
  • 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.
  • the purified adalimumab composition obtained by the method has stability selected from the group consisting of oxidation stability, monomer stability, and charge variant stability.
  • 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)
  • 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.
  • the present invention provides an adalimumab composition
  • 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.
  • the adalimumab is purified by the method of any one of claims 1-8.
  • the composition exhibits stability selected from the group consisting of oxidation stability, monomer stability, and charge variant stability after storage.
  • the present invention provides the use of the antibody composition of the present invention in the treatment of diseases in which TNF ⁇ activity is detrimental.
  • 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.
  • the term “comprising” or “including” means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps.
  • the term “comprises” or “includes” is used, unless otherwise specified, it also encompasses the situation consisting of the stated elements, integers or steps.
  • CEX-HPLC refers to cation exchange high performance liquid chromatography.
  • SEC-HPLC refers to size exclusion high performance liquid chromatography
  • RH relative humidity
  • SV refers to the sample volume
  • PS80 is synonymous with “Tween 80” and “Polysorbate 80”.
  • EDTA refers to ethylenediaminetetraacetic acid
  • the EDTA described herein also includes its salts and other variants, such as disodium ethylenediaminetetraacetic acid.
  • CV refers to column volume
  • excipients refer to auxiliary materials used in the preparation of biological products, such as adjuvants, stabilizers, excipients, etc.
  • a drug substance also called bulk, refers to a uniform drug substance used to make a final formulation.
  • drug products also called final products, refer to products formed by filling drug substances into the final storage container of drugs.
  • the drug substance can be diluted and/or formulated as appropriate or not, such as adding excipients.
  • 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.
  • 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.
  • 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.
  • the composition 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • charge variant stability 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 is an antibody variant containing 0, 1, and 2 terminal lysines.
  • % 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • FLD-HPLC method high performance liquid chromatography-fluorescence detection method
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • the fermentation broth is centrifuged to clarify impurities such as cells to obtain a supernatant containing adalimumab.
  • affinity chromatography is performed on the Adalimumab mixture recovered from the producer cells.
  • affinity chromatography fillers such as protein A affinity fillers, are used for affinity chromatography.
  • a loading capacity of 10-45 g/L is used.
  • the sample flow rate is 100-250 cm/h.
  • 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.
  • 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.
  • 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.
  • the elution buffer has a pH of 3.3-3.7.
  • 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.
  • 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.
  • 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.
  • a column bed height of about 20 cm can be used.
  • 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 is performed in the affinity collection solution obtained by affinity chromatography.
  • 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.
  • 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.
  • an acidic solution such as citric acid, acetic acid, etc., is used to adjust the pH of the collection solution.
  • the protein concentration in the collection solution is ⁇ 25mg/ml.
  • the acid inactivation treatment is carried out at 18 to 26°C for 90 to 180 minutes.
  • an alkaline solution such as Tris, Tris-bis, histidine, etc., is preferably used to adjust the pH of the sample to neutral.
  • 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.
  • an acidic solution such as citric acid
  • 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.
  • 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.
  • affinity chromatography depth filtration is performed. It can be carried out using any depth filtration device known in the art.
  • a depth filtration membrane package such as X0HC or the like is used for filtration.
  • 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.
  • 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.
  • 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 may be implemented, including but not limited to ion exchange chromatography, hydrophobic interaction chromatography, and mixed mode chromatography.
  • 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.
  • a flushing solution containing a metal chelating agent can also be used to flush the chromatography column.
  • 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.
  • 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
  • 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.
  • 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 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.
  • a metal chelating agent 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.
  • 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.
  • anion exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and quaternary amine (Q) groups.
  • Cation exchange substituents include carboxymethyl (CM),
  • 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.
  • 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.
  • DEAE-, QAE-, CM- and SP-SEPHADEX and DEAE-, Q-, CM- and S-SEPHAROSE and SEPHAROSE Fast Flow can be obtained from multiple suppliers.
  • synthetic polymethacrylate resins such as EMD "tentacles" ion exchange media can also be used in the present invention.
  • anion exchange chromatography uses Q Sepharose Fast Flow, for example, Q Sepharose Fast Flow from GE Company in the United States.
  • 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).
  • 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.
  • anion exchange packing is used.
  • Q Sepharose Fast Flow anion exchange packing is used.
  • 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.
  • the collected solution of the depth filtration is diluted with an anion balance buffer.
  • the chromatography column 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.
  • a suitable equilibration buffer is Tris/NaCl, pH approximately 7.
  • the loading capacity is ⁇ 30g/L.
  • the absorption peak at 280 nm is monitored by ultraviolet after the start of sample loading.
  • the penetrating fluid is collected when the absorption value rises to greater than or equal to 25 mAU/mm optical path.
  • the collecting liquid is adjusted to pH 6.0 using an acidic solution, for example, a 2mol/L citric acid solution.
  • 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.
  • the method of the present invention includes: after anion exchange chromatography, cation exchange chromatography is used to purify adalimumab in an adsorption-elution mode.
  • cation 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.
  • a linear gradient wash is performed prior to elution.
  • 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.
  • a metal chelating agent such as EDTA or DTPA or other metal ion chelating agents
  • the chromatography column is equilibrated with a cation balance solution.
  • 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.
  • 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.
  • the antibody collection solution obtained by ion exchange chromatography is subjected to virus removal filtration.
  • a virus-removing filter is used to filter the sample, for example, at a working pressure ⁇ 4 bar.
  • multiple filters can be connected in series for sample filtration. For example, you can change Prefilter and Pro filters are connected in series.
  • 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.
  • the method of the present invention further includes subjecting the antibody collection solution obtained by ion exchange chromatography to ultrafiltration and diafiltration.
  • an ultrafiltration membrane is used for ultrafiltration and concentration.
  • the sample is diafiltered after being concentrated by ultrafiltration.
  • the diafiltration buffer can be used for continuous liquid exchange.
  • the diafiltration buffer is used in an amount of 5-12 times the volume after ultrafiltration and concentration.
  • 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.
  • the diafiltration buffer used in the present invention may include buffers, salts, and polyols.
  • 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.
  • 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).
  • 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.
  • the stability of the purified antibody composition produced by the process during storage is related to the quality attributes of the product.
  • 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.
  • 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.
  • cIEF capillary isoelectric focusing electrophoresis
  • iCIEF imaging capillary isoelectric focusing electrophoresis
  • IEX Ion exchange chromatography
  • 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.
  • 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.
  • 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 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.
  • 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).
  • HMMS high molecular weight form
  • LMMS low molecular weight form
  • Antibody purity can be calculated as the percentage of the main peak area on the chromatogram to the sum of all peak areas.
  • 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.
  • the monomer purity, as measured by SEC-HPLC is 97% or more, preferably 98% or 99% the above.
  • 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.
  • CE capillary electrophoresis
  • C EX cation exchange chromatography
  • 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).
  • the charge variant of the antibody in the antibody preparation is determined by cation exchange high performance liquid chromatography (CEX-HPLC).
  • CEX-HPLC cation exchange high performance liquid chromatography
  • 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.
  • the acid charge variant does not increase by more than 40%, preferably No more than 30%, 25%, 20%, 15%, 10%.
  • Product-related impurities include, but are not limited to, oxidation products, deamidation products, or other structurally incomplete molecules.
  • 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.
  • the product-related impurities include adalimuma antioxidant products, especially oxidation products at position M256.
  • 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.
  • the present invention provides a stable adalimumab composition, especially a stable adalimumab composition produced by the antibody purification method of the present invention.
  • 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.
  • the present invention provides an adalimumab composition
  • 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.
  • the metal ion chelating agent is selected from: EDTA, DTPA, EGTA, BAPTA, DMPS, DMSA, ALA, or a combination thereof; more preferably, EDTA or DTPA.
  • the present invention provides a stable liquid adalimumab composition
  • adalimumab especially an adalimumab composition produced by the purification method of the present invention
  • a buffer e.g., a buffer for adalimumab
  • surfactant e.g., sodium chloride.
  • the pH of the antibody composition is about 5.0-7.0.
  • 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.
  • 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.
  • the present invention relates to antibody drug substances produced at a concentration of 10-50 mg/mL.
  • the present invention relates to a drug product formed from the drug substance in combination with pharmaceutically acceptable excipients.
  • the adalimumab composition of the present invention comprises:
  • metal chelating agent such as EDTA or DTPA
  • the composition of the present invention has a pH of about 5-7, such as 6.0 ⁇ 0.5, such as about 6.0.
  • the adalimumab composition of the present invention further comprises:
  • 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)".
  • the composition of the invention has stability.
  • the composition has stability selected from one or more of the following: oxidation stability, monomer stability, and charge variant stability, and polysorbate 80 stability.
  • 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%.
  • the monomer purity, as measured by SEC-HPLC is 97% or more, preferably 98% or 99% or more.
  • 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%.
  • the content of polysorbate 80 is reduced by no more than 10%, for example no more than 5%.
  • 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.
  • the present invention provides a method of treating a disease in which TNF ⁇ 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.
  • the disease in which TNF ⁇ activity is detrimental is selected from autoimmune diseases, intestinal diseases, and skin diseases.
  • the disease is selected from rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis and chronic plaque psoriasis disease.
  • 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.
  • another therapeutic agent for example, with methotrexate, DMARDS, glucocorticoids, non-steroidal anti-inflammatory drugs (NSAID), and/or Analgesics.
  • 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 TNF ⁇ activity is harmful.
  • 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.
  • Reversed-phase high performance liquid chromatography (RP-HPLC method) was used to detect the residual amount of EDTA in the purified composition.
  • RP-HPLC method Reversed-phase high performance liquid chromatography
  • 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.
  • Figure 1 exemplarily shows a typical map of the EDTA residue test.
  • the FLD-HPLC method (High Performance Liquid Chromatography Fluorescence Detection Method) was used to detect the polysorbate 80 content of the samples.
  • 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, 5.0 ⁇ mol/L NPN, 15ppm Brij, flow rate: 1.5ml/min; collection time: 3 minutes; injection volume: 10 ⁇ l; column temperature: 30°C; detection wavelength: excitation light 350nm, emission light 420nm. Calculate the polysorbate 80 content
  • Cation exchange chromatography (CEX-HPLC method) was used to detect the charge heterogeneity of the samples.
  • 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.
  • the acidic components, main components (main peak + alkali peak 1 + alkali peak 2) and basic components of the sample were calculated.
  • 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.
  • Size exclusion chromatography (SEC-HPLC method) was used to detect the molecular size variants of the samples.
  • TSK-gel G3000SWxl manufactured by TSK-gel G3000SWxl
  • the mobile phase is 20mmol/L phosphate buffer +200mmol/L sodium chloride
  • the flow rate is 0.5ml/min
  • the detection wavelength is 280nm
  • the column temperature is 25°C.
  • This example describes an unoptimized purification process.
  • the process includes the following steps:
  • 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.
  • 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°C for 90 ⁇ 180min, then use 2mol/L Tris Adjust the pH of the sample to neutral.
  • 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.
  • 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.
  • 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.
  • diafiltration buffer pH 3.9g/L L-histidine, 12.0g/L sorbitol, 6.2g/L sodium chloride, pH 6.0
  • This example describes the optimized purification process.
  • the process includes the following steps:
  • 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.
  • 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.
  • 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.
  • 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°C for 90 ⁇ 180min, then use 2mol/L Tris Adjust the pH of the sample to neutral.
  • 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.
  • 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.
  • 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.
  • 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.
  • diafiltration buffer 3.9g/L L-histidine, 12.0g/L sorbitol, 6.2g/L sodium chloride, pH 6.0
  • 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.
  • 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.
  • 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.
  • 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.
  • LC-MS/MS liquid chromatography tandem mass spectrometry
  • Example 2 The process conditions of Example 2, wherein the cation exchange chromatography eluent contains 20mM EDTA, and ultrafiltration/diafiltration dialysis 6SV (sample volume)
  • Example 2 The process conditions of Example 2, wherein the cation exchange chromatography eluent contains 20mM EDTA, and the ultrafiltration/diafiltration dialysis 10SV
  • Example 3 The process conditions of Example 1, wherein the cation exchange chromatography eluent does not contain EDTA, and the ultrafiltration/diafiltration dialysis 10SV
  • R is 0 without interception, so generally Cc>e ⁇ (-n) ⁇ Co
  • Example 2 obtained by using different ultrafiltration/diafiltration dialysis volumes (SV), namely the aforementioned sample 1 and sample 2, were determined in duplicate.
  • SV ultrafiltration/diafiltration dialysis volumes
  • This example describes the product produced by the optimized process (Example 2), and the result of the forced stability test at 40°C.
  • Example 2 The process conditions of Example 2, in which only EDTA was added to the washing solution of cation exchange chromatography.
  • 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.
  • 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.
  • 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%).
  • 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.

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Abstract

一种阿达木单抗的纯化方法，以及由此纯化方法获得的稳定阿达木单抗组合物，和该组合物的医药用途。

Description

阿达木单抗纯化方法及其稳定组合物 技术领域
本发明涉及抗体领域。具体地本发明涉及阿达木单抗的纯化方法，以及由此纯化方法获得的稳定阿达木单抗组合物，和该组合物的医药用途。
背景技术
抗体制品的稳定性问题
在整个的生物药物生产过程中，从蛋白药物的制备到纯化、配制、封装中，都存在向药物成品中引入杂质的可能性。这些杂质包括例如工艺相关杂质和产品相关杂质。由制剂中目的蛋白药物的失稳定而形成的杂质，在本领域中称作产品相关杂质。这些产品相关杂质是目的产物的分子变体，但往往不具备与目的产品相当的活性、功效和安全性，例如，在生产、纯化和/或存储过程中形成的各种降解产物、电荷变异体等。因此，需要控制这些产物相关杂质在药物产品中的存在和含量。
单克隆抗体通常展示几种异质性，包括例如，大小异质性、电荷异质性、糖基化模式异质性。这些异质性可以由多种不同的因素引起。这些因素可以包括，但不限于，可能在药物的制备、纯化、和存储的任一阶段中引入药物产品中的杂质、以及杂质之间的相互作用、和杂质与目的蛋白之间的相互作用。另一方面，抗体药物作为大分子，其自身在结构和性质上的复杂性，也进一步加剧了抗体制剂中失稳定因素分析的困难性。因此，单克隆抗体的稳定性问题具有复杂性，往往呈现出与具体蛋白质的相关性，并与具体抗体生产工艺的相关性。
电荷异质性的产生是由于抗体药物在生产过程中存在一系列翻译后修饰和降解导致其空间电荷分布等表面电荷特性发生了改变。研究发现，电荷异质性的形成有可能对抗体药物的结合力、药代动力学、免疫原性、生物学活性及稳定性等体内体外特性有影响，进而影响临床应用时的有效性和安全性，是抗体的一类重要的质量属性。近来，电荷异质性检测越来越受到全球制药企业和监管机构的关注，尤其是在生物类似药生产工艺开发过程中，电荷异质性的控制是重要环节之一。然而，电荷异质性的来源众多，形成机制复杂，对电荷异质性的研究一直是抗体药物开发过程中的难题之一。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。
已经鉴定了多种抗体修饰可以成为引起电荷变异体酸性或碱性成分形成的原因。对于酸性成分，主要的修饰包括唾液酸、天冬酰胺残基的脱酰胺和糖基化。对于碱性成分，主要的修饰包括部分前导序列的留存，N端谷氨酰胺的开环，C端酰胺化，天冬氨酸异构化导致的琥珀酰亚胺，和C端赖氨酸。参见于传飞等(药物分析杂质,2014,34(7)，p1212，通过成像毛细管等点聚焦电泳法对单抗制品进行电荷异质性分析)。此外有报告，Fc区24位甲硫氨酸氧化的抗体在阳离子交换层析中表现为碱性组分。参见Chumsae,C.；Gaza-Bulseco,G.；Sun,J.；Liu,H.J.Chromatogr.B.2007,850,285-294。
吴霖萍等(阿达木单抗理化特性的关键质量属性分析，中国医学工业杂质，2018,49(3))，对美国艾伯维公司生产的阿达木单抗注射液的稳定性进行了研究。对于电荷异质性的表征结 果表明，阿达木单抗注射液发生的主要修饰是N脱酰胺和末端K修饰，其中N脱酰胺修饰是酸性变异性的主要来源；而末端的K修饰，即包含0个，1个和2个K残基的变异体是主要的碱性变异性的主要来源。
已经提出了一些用于稳定抗体产品的方法，主要包括在抗体产品中加入一定量和种类的赋形剂。但是，赋形剂的加入可能会引起产品在安全性或功效上的问题。例如，SHUXIA ZHOU等人公开了在单克隆抗体制剂中加入0.05mg-1mg/ml金属螯合物可以提供制剂的稳定性(Comparative Evaluation of Disodium Edetate and Diethylenetriaminepentaacetic Acid as Iron Chelators to Prevent Metal-Catalyzed Destabilization of a Therapeutic Monoclonal Antibody,JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.99,NO.10,2010,pp4239-4250)。但是，并非所有的制剂都适用于加入金属螯合剂。一方面，相当量的赋形剂(例如金属螯合剂)的加入会增加临床不良反应的可能性。例如，已经发现，在生物药Leukine(重组GM集落刺激因子)的制剂中加入少量EDTA会导致临床不良反应。另一方面，由于抗体稳定性问题的复杂性，并不存在通用适用于任何抗体制剂的赋形剂解决方案。例如，CN 104768576A中使用SEC,RP HPLC,cIEF和CE-SDS方法，通过在40℃保持一周或在25℃保持两周，研究了多种辅料和赋形剂在阿达木单抗水性药物组合物中对于抗体稳定性的影响，结果显示，EDTA对于阿达木单抗制剂更倾向于是一种去稳定剂；并提出金属离子(例如锌、镁和钙)可以用作阿达木抗体制剂的稳定剂。
因此，在单克隆抗体领域，优化抗体的生产工艺来最小化存在于抗体药品中的失稳定性因素，是本领域的一个富于挑战性的方向。
阿达木单抗
阿达木单抗(adalimumab)是由1330个氨基酸组成并具有大约148千道尔顿分子量的全人源IgG1单克隆抗体。该抗体作用于肿瘤坏死因子α(TNF-α)相关疾病，主要用于治疗免疫系统介导的疾病，包括类风湿关节炎、银屑病关节炎、强直性脊柱炎及克罗恩病等。通常阿达木单抗通过重组DNA技术在哺乳动物细胞表达系统，尤其是中国仓鼠卵巢CHO细胞中表达产生。以商品名Humira(修美乐)销售的阿达木单抗注射液于2003年1月在美国首次上市，其组成为：甘露醇、柠檬酸一水合物、柠檬酸钠、磷酸二氢钠二水合物、磷酸氢二钠二水合物、氯化钠、聚山梨酯80、氢氧化钠、注射用水，和40mg/0.8ml阿达木单抗。之后，一些生物类似药也相继被批准上市，包括Exemptia,Adfrar,Amjevita和Cyltezo。
为保证单抗药物的安全、有效，仍需要可以提供质量属性稳定的阿达木单抗药物的工艺方法。
发明内容
本发明人发现，采用蛋白A亲和层析和离子交换层析法纯化的阿达木单抗组合物在存储过程中存在稳定性下降的问题，主要体现为SEC-HPLC纯度下降、产品相关性杂质——酸性电荷异构体的上升、以及赋形剂吐温80含量的下降。为了最小化失稳定性因素对阿达木抗体的影响，本发明人进行了生产工艺优化研究。在这些深入研究的基础上，提出了本发明的技术方案，建立了一种成本有效且实际的改善纯化阿达木单抗组合物稳定性(尤其是改善的电荷变异体稳定性)的方法。
因此，在一个方面，本发明提供一种通过改进的纯化工艺来制备具有改善的稳定性的阿达木单抗组合物的方法，其中所述稳定性优选为电荷变异体稳定性，任选地还包括单体稳定性、氧化稳定性、或其组合。在一个实施方案中，所述方法包括以下步骤：
(a)亲和层析:将包含阿达木单抗的混合物施加到亲和层析树脂，以洗脱缓冲液洗脱，收集包含所述抗体的收集液；
(b)阴离子交换层析：将包含所述抗体的收集液施加到阴离子交换树脂，收集包含所述抗体的穿透液；和
(c)阳离子交换层析:将包含所述抗体的收集液施加到阳离子交换树脂，以洗脱缓冲液洗脱，收集包含所述抗体的收集液，其中，所述洗脱液含有金属离子螯合剂，例如选自但不限于以下的金属离子螯合剂：EDTA，DTPA,EGTA,BAPTA,DMPS,DMSA,ALA，或其组合；优选EDTA或DTPA。
优选地，通过所述方法获得的纯化阿达木单抗组合物具有选自以下的一个或多个特征：
-在存储后，例如40℃存储4周后，酸性电荷变异体增加不超过40％，优选不超过30％，25％，20％，15％，10％；且任选地，电荷主成分下降不超过40％，优选不超过30％，25％，20％，15％，10％；
-在存储后，例如40℃存储4周后，单体纯度，如通过SEC-HPLC测量，为97％以上，优选98％或99％以上；
-具有氧化稳定性，在存储后，例如25℃存储4周后，如通过肽作图测量，抗体256位甲硫氨酸残基(M256)的氧化比率不超过40％，或更优选地不超过30％，20％或10％，5％。
在一个优选的实施方案中，阳离子交换层析步骤包括：使用含有金属离子螯合剂的阳离子洗脱液洗脱，其中洗脱液中金属螯合剂的量导致通过所述方法获得的纯化阿达木单抗组合物中具有0.00026-0.05mg/ml的金属螯合剂，优选0.0005-0.03mg/ml，更优选0.001-0.02mg/ml，例如大约0.002,0.004，0.008，0.01mg/ml。在另一优选实施方案中，洗脱液中金属离子螯合剂的量为5-40mmol/L,例如10-30mmol/L，例如，20mmol/l。
在再一优选实施方案中，阳离子交换层析步骤还包括：在洗脱之前，用含有金属离子螯合剂的阳离子冲洗液冲洗。优选，冲洗液中金属离子螯合剂为5-40mmol/L,例如10-30mmol/L，例如，10mmol/l。
在再一方面，本发明也提供了一种用于改善纯化阿达木单抗水性组合物的稳定性的方法，其中所述稳定性优选地选自电荷变异体稳定性、单体稳定性、氧化稳定性、或其组合。
在再一方面，本发明也提供一种从包含阿达木单抗的混合物中纯化阿达木单抗的方法，所述方法包括：亲和层析、阴离子交换层析和阳离子交换层析，其中阳离子交换层析使用包含金属离子螯合剂的洗脱剂进行洗脱。优选地，通过所述方法获得的纯化阿达木单抗组合物具有选自以下的稳定性：氧化稳定性、单体稳定性、和电荷变异体稳定性。
在一个优选实施方案中，本发明的方法包括步骤：(1)从重组表达阿达木单抗的细胞培养物回收阿达木单抗，获得包含阿达木单抗的混合物；(2)使用亲和层析(例如蛋白A柱)捕获抗 体；(3)进行病毒灭活；(4)进行阴离子交换和阳离子交换层析；(5)病毒过滤(使病毒滴度降低例如4log10以上)；(6)超滤/渗滤(例如，用于将抗体蛋白置换于利于其稳定的制剂缓冲液中并浓缩至合适的浓度)；和任选地(7)纯化组合物的配制(用于加入合适的辅料)；其中所述方法的特征在于，在阳离子交换层析中，使用含有金属离子螯合剂(例如，EDTA或DTPA)的洗脱缓冲液洗脱，以收集包含阿达木单抗抗体的收集液。
在再一方面，本发明提供了一种阿达木单抗组合物，其包含阿达木单抗和金属螯合剂，优选地0.00026-0.05mg/ml的金属螯合剂，优选0.0005-0.03mg/ml，更优选0.001-0.02mg/ml，例如大约0.002,0.004，0.008，0.01mg/ml的金属螯合剂。优选地，所述阿达木单抗为通过权利要求1-8任一项的方法纯化获得。优选地，所述组合物在存储后表现出选自以下的稳定性：氧化稳定性、单体稳定性、和电荷变异体稳定性。
在再一方面，本发明提供了本发明抗体组合物在治疗其中TNFα活性有害的疾病中的用途。
附图说明
图1显示高效液相色谱法检测EDAT残留量的一个典型图谱。
图2显示FLD-HPLC法检测样品的聚山梨酯80含量的一个典型图谱。
图3显示CEX-HPLC法检测抗体电荷异质性的一个典型图谱。
图4显示工艺优化对阿达木单抗电荷异构体主成分的影响。
图5显示工艺优化对纯化组合物中吐温80含量的影响。
发明详述
在详细描述本发明前，应了解，本发明不受限于所述的特定方法及实验条件，因为所述方法以及条件是可以改变的。另外，本文所用术语仅是供说明特定实施方案之用，而不意欲为限制性的。
定义：
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
术语“和/或”当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项的任意两项或多项。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。
如本文所用，短语“CEX-HPLC”是指阳离子交换高效液相色谱。
如本文所用，短语“SEC-HPLC”是指体积排阻高效液相色谱
如本文所用，短语“RH”是指相对湿度。
如本文所用，短语“SV”是指样品体积。
如本文所用，术语“PS80”，与“吐温80”、“聚山梨酯80”同义。
如本文所用，术语“EDTA”是指乙二胺四乙酸，本文所述EDTA也包含其盐类和其他变体，如乙二胺四乙酸二钠。
如本文所用，短语“CV”是指柱体积。
如本文所用，辅料(excipients)指生物制品在配制过程中所使用的辅助材料，如佐剂、稳定剂、赋形剂等。
如本文所用，药物物质(DS)，也称作原液(bulk)，指用于制造最终配制物(Final Formulation)的均一药物物质。
如本文所用，药物产品(DP)，也称作成品(Final products)，指将药物物质灌装到药品最终的储存容器中后形成的制品。在此过程中，药物物质可以酌情进行或不进行稀释和/或配制，例如加入辅料。
如本文所用，表述“稳定”的抗体组合物旨在涵盖，组合物中的抗体在储存于特定条件下之后保有可接受程度的物理稳定性和/或化学稳定性。例如，在储存特定时间之后，如果抗体组合物中所含的抗体仍维持了约90％、约95％、约96％、约97％、约98％或约99％的抗体结构或功能，则通常可以认为该抗体组合物是“稳定的”。在一些实施方案中，本发明的抗体组合物在储存后，基本上保留其物理和化学稳定性。本领域已知多种分析技术可以用于测定抗体组合物的稳定性，参见例如Peptide and Protein Drug Delivery,247-301,Vincent Lee Ed.,Marcel Dekker,Inc.,New York,N.Y.,Pubs(1991)and Jones,A.Adv.Drug Delivery Rev.10:29-90(1993)。可以在选定的温度和选定的储存时间测量稳定性。例如，可以基于预期的制剂货架期来选择储存时间。或者可以使用加速或强制稳定性试验。在一些实施方案中，对抗体组合物进行强制稳定性测试。
如本文所用，表述抗体组合物的“单体稳定性”是指，通过测定在特定温度下储存特定时间之后组合物中的抗体单体的百分比来量度的抗体组合物稳定性，其中组合物中的抗体单体的百分比越高，则组合物的稳定性越高。如本文所用，当于特定温度下储存特定时间之后，在组合物中检测到至少约95％，优选至少96％、97％、98％或99％或更高的抗体单体，则该组合物可以视为具有改善的单体稳定性。在一些实施方案中，在特定温度储存至少2周、至少4周、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月或更久后，“改善的单体稳定性”表示组合物中保持至少约95％、96％、97％、98％、99％的抗体单体。在评估单体稳定性时，用于组合物储存的特定温度可为约-80℃至约45℃的任一温度，例如约-80℃、约-30℃、约-20℃、约0℃、约4℃-8℃、约5℃、约 25℃、约35℃、约37℃、约40℃、约42℃或约45℃。在一个优选的实施方案中，若组合物储存于约40℃±2℃1个月之后，检测到至少约95％、96％、97％、98％、99％的抗体单体，则该组合物视为是具有改善的单体稳定性的。抗体的单体百分比测量可以采用例如本领域已知的多种方式进行，例如SEC-HPLC。在优选的实施方案中，通过本发明方法生产的抗体组合物，相比于使用对照方法生产的抗体组合物，具有改善的单体稳定性。优选地，对照方法与本发明方法的差别仅在于缺乏本发明方法的EDTA使用步骤。
如本文所用，表述抗体组合物的“电荷变异体稳定性”是指，通过测定在特定温度下储存特定时间之后组合物中的抗体电荷变异体(例如，酸性电荷变异体、碱性电荷变异体、电荷变异体主成分、或其组合)的百分比来量度的抗体组合物稳定性，其中相比于存储前，组合物中的电荷变异体的变化百分比越低，则组合物的稳定性越高。在一个优选实施方案中，电荷变异体主成分为含有0、1、和2个末端赖氨酸的抗体变异体。如本文所用，当于特定温度下储存特定时间之后，在组合物中检测到不超过约30％，优选不超过25％,24％,23％,22％,21％，更优选地不超过20％的电荷变异体变化(例如酸性电荷变异体变化)，则该组合物可以视为具有改善的电荷变异体稳定性(或更具体地，酸性电荷变异体稳定性)。在评估电荷变异体稳定性时，组合物可以在特定温度储存至少2周、至少4周、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月或更久。用于组合物储存的特定温度可为约-80℃至约45℃的任一温度，例如约-80℃、约-30℃、约-20℃、约0℃、约4℃-8℃、约5℃、约25℃、约35℃、约37℃、约40℃、约42℃或约45℃。在一个优选的实施方案中，若组合物储存于约40℃±2℃1个月之后，检测到不超过约30％，优选不超过25％,24％,23％,22％,21％，更优选地不超过20％的酸性变异体变化，则该组合物视为是具有改善的电荷变异体稳定性或具有改善的酸性电荷变异性稳定性。在另一优选的实施方案中，若组合物储存于约40℃±2℃1个月之后，检测到不超过约30％，优选不超过25％,24％,23％,22％,21％，更优选地不超过20％的电荷变异体主成分变化，则该组合物视为是具有改善的电荷变异体稳定性。抗体的电荷变异体百分比测量可以采用例如本领域已知的多种方式进行，例如CEX-HPLC法。在优选的实施方案中，通过本发明方法生产的抗体组合物，相比于使用对照方法生产的抗体组合物，具有改善的电荷变异体稳定性。优选地，对照方法与本发明方法的差别仅在于缺乏本发明方法的EDTA使用步骤。
如本文所用，表述抗体组合物的“氧化稳定性”是指，通过测定在特定温度下储存特定时间之后组合物中抗体256位甲硫氨酸残基的氧化比率(即，氧化残基/(氧化残基+未氧化残基))来量度的抗体组合物稳定性，其中相比于存储前，组合物中的氧化比率越低，则组合物的稳定性越高。抗体M256位的氧化比率测量可以采用例如本领域已知的多种方式进行，例如肽作图法。在一个实施方案中，通过本发明方法生产的抗体组合物，相比于使用对照方法生产的抗体组合物，具有改善的氧化稳定性。优选地，对照方法与本发明方法的差别仅在于缺乏本发明的EDTA使用步骤。
如本文所用，表述“聚山梨醇80稳定性”是指，通过测定在特定温度下储存特定时间之后组合物中的赋形剂聚山梨醇80的含量来量度的抗体组合物稳定性，其中相比于存储前，赋形剂聚山梨醇80的含量降低得越少，则聚山梨醇80稳定性越高。聚山梨醇80的含量可以采用例如本领域已知的多种方式进行，例如高效液相色谱-荧光检测法(FLD-HPLC法)。在一个 实施方案中，通过本发明方法生产的抗体组合物，相比于使用对照方法生产的抗体组合物，具有改善的聚山梨醇80稳定性。优选地，对照方法与本发明方法的差别仅在于缺乏本发明的EDTA使用步骤。
I.本发明方法
本发明人发现，通过优化阿达木单抗纯化工艺，可以有效地解决生产的纯化抗体组合物在储存中的稳定性问题，尤其是电荷异质性问题。因此，在深入研究的基础上，本发明人提出了本发明的纯化方法。通过本发明方法获得的阿达木单抗组合物具有改善的阿达木单抗纯度、和电荷异构体稳定性(例如，在40℃强制稳定性4周主成分下降低于26％)、且组合物中聚山梨醇酯80含量更稳定(40℃强制稳定性4周下降低于10％)。
因此，在一个方面中，本发明提供了一种通过纯化工艺来改善阿达木单抗组合物稳定性的方法，其中所述方法包括步骤：亲和层析；阴离子交换层析；和阳离子交换层析；其中，在阳离子交换层析步骤中，使用含有金属离子螯合剂(例如，EDTA或DTPA)的阳离子洗脱液进行洗脱。优选地，本发明方法还包括：病毒灭活步骤，超滤和洗滤步骤，以及纯化的阿达木单抗组合物配制步骤。
以下就本发明方法的各方面做进一步描述。
阿达木单抗
在本文中，术语“阿达木单抗”是修美乐中的活性药物成分，并旨在涵盖在修美乐中使用的阿达木单抗蛋白质的生物仿制或生物改良变体。为了本发明的目的，该术语也涵盖氨基酸序列结构稍作修饰但不显著影响抗体功能的阿达木单抗蛋白质。阿达木单抗的氨基酸序列及其药理学和治疗性质已经在例如WO97/029131中公开，该文献特此并入本文作为参考。如下显示了阿达木单抗轻链和重链全长序列：
阿达木单抗轻链全长序列:
Figure PCTCN2021070654-appb-000001
阿达木单抗重链全长序列：
EVQLVESGGGLVQPGRSLRLSCAASGFTFD DYAMHWVRQAPGKGLEWVS AITWNSG HIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAK VSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:2，斜体表示重链恒定区，下划线表示HCDR1-3，和位于Fc区中 的256位Met)
在本发明的一些实施方案中，可以通过基因重组方法在宿主细胞(例如哺乳动物细胞，尤其是CHO细胞)中产生阿达木单抗。阿达木单抗可以在细胞中产生，或优选地被分泌至培养基中。可以通过本领域常规的方法，从细胞或培养基中回收阿达木单抗。该回收步骤可以包括例如一个或多个离心或深度过滤步骤，由此提供可以用于本发明纯化方法中的包含阿达木单抗的混合物。在一个实施方案中，在发酵结束后将发酵液离心澄清去除细胞等杂质以获得包含阿达木单抗的上清。
亲和层析：
在一些实施方案中，对从生产细胞回收的阿达木单抗混合物，实施亲和层析。在一个实施方案中，采用亲和层析填料，例如蛋白A亲和填料，进行亲和层析。在一个实施方案中，采用载量10～45g/L。在一个实施方案中，上样流速为100～250cm/h。在一个实施方案中，在上样前，采用中性缓冲液进行平衡；且在完成上样后，再次用中性缓冲液进行平衡。合适的中性缓冲液的一个实例是Tris/NaCl缓冲液，pH为例如约7.2。在一个实施方案中，用于平衡的中性缓冲液包含低浓度的盐，例如浓度50-200mmol/L，例如150mmol/L的NaCl。在一个实施方案中，在洗脱前，用含高盐的冲液溶液，进行冲洗，优选地，冲液溶液包含300mmol/L-1000mmol/L，例如500mmol/L的NaCl。合适的冲液溶液的一个实例为包含500mM NaCl的中性Tris缓冲液。在上样和冲洗后，可以使用适当的洗脱缓冲液，洗脱蛋白A柱。在一个实施方案中，洗脱缓冲液具有3.3-3.7的pH。合适的洗脱缓冲液的一个实例是枸橼酸/NaCl缓冲液，例如20mmol枸橼酸+100mmol/L NaCl。可以使用本领域技术人员已知的技术，监测洗脱液。例如，可以跟踪紫外280nm处的吸光度。在一个实施方案中，在洗脱后，收集紫外280nm处主峰。
在一个优选的实施方案中，亲和层析步骤包括：采用蛋白A亲和填料，在中性缓冲液平衡后上样，载量10～45g/L，上样流速100～250cm/h；在完成上样后用中性缓冲液平衡3CV，再用含高盐的冲洗溶液冲洗3CV，并用中性缓冲液平衡3CV；然后开始洗脱，洗脱缓冲液pH范围为3.3～3.7，并收集紫外280nm处主峰。
可用于本发明的蛋白A亲和填料并无限制。已知蛋白A树脂存在几种商业来源，包括、但不限于：来自GE Healthcare的MabSelect和来自Millipore的ProSep Ultra Plus。在一个优选的实施方案中，在本发明方法中使用的蛋白A亲和填料为耐强碱的层析介质，例如Mabselect SuRe亲和填料。
可用于本发明亲和层析步骤的亲和柱并无限制。例如，可以使用大约20cm的柱床高度。例如，填充了MabSelect的合适柱的一个非限制性实例是约1.0cm直径x约21.6cm长的柱(～17mL柱床体积)。该尺寸的柱可以用于小规模纯化。本发明也可以使用更大的柱尺寸，例如，20cm x 21cm柱(其柱床体积是约6.6L)，或更大。
病毒灭活：
在一个实施方案中，在亲和层析获得的亲和收集液中，进行病毒灭活。优选地，对病毒采用酸灭活方法。病毒的酸灭活取决于蛋白质浓度、pH和灭活时间，可以通过常规实验选择这些参数。在一些实施方案中，对收集的包含阿达木单抗的蛋白A洗脱液进行pH调节，以 达到约3-4的pH，例如pH3.2～3.7。在一个实施方案中，用酸性溶液，例如枸橼酸、醋酸等，调节收集液的pH。优选地，收集液中的蛋白浓度≤25mg/ml。优选地，酸灭活处理于18～26℃下进行90～180min。在酸灭活处理后，优选地用碱性溶液，例如Tris、Tris-bis、组氨酸等，调节样品pH至中性。
在一个优选的实施方案中，病毒灭活步骤包括：将亲和层析收集液用酸性溶液，例如枸橼酸，调节pH至3.2～3.7，蛋白浓度≤25mg/ml，于18～26℃下静置90～180min，然后用碱性溶液，例如2M Tris，调节样品pH至中性，例如pH约6.5-8.5。
深层过滤：
深层过滤可以通过吸附作用从重组单克隆抗体工艺流中去除宿主细胞蛋白质杂质。深层过滤可以在蛋白A层析捕获之前进行，也可以在亲和层析捕获后，例如在病毒灭活后进行。在一个优选实施方案中，在亲和层析之后，进行深层过滤。可以采用本领域已知的任何深层过滤装置进行。在一个优选实施方案中，采用深层过滤膜包如X0HC等，进行过滤。在一个优选的实施方案中，深层过滤步骤包括：采用X0HC膜包，以通量≤150LMH，载量为65～160L/m2，进行过滤。
离子交换层析：
在一些实施方案中，本发明的阿达木单抗纯化方法包括，在亲和层析步骤后，进行离子交换层析。本发明人经过深入的研究发现，在纯化工艺的此离子交换层析步骤中，采用包含金属螯合剂例如EDTA或DTPA的洗脱液进行抗体洗脱，可以很好地克服纯化的阿达木单抗组合物的稳定性问题，尤其是抗体组合物在储存过程中的电荷异质性问题。
因此，在一个实施方案中，对来自之前纯化步骤的包含抗体的收集液，实施至少一个离子交换分离步骤，其中采用包含金属螯合剂的洗脱液，自离子交换层析获得包含阿达木单抗的洗脱收集液。任选地，在所述离子交换分离步骤前，可以实施其他的层析分离步骤，包括不但限于，离子交换层析、疏水相互作用层析、混合模式层析。
在优选的实施方案中，本发明方法涉及序贯组合使用阴离子和阳离子交换层析，并在阳离子交换层析中使用包含金属螯合剂的洗脱液进行抗体的洗脱。在一个更优选的实施方案中，在离子交换层析中，还可以使用包含金属螯合剂的冲洗液进行层析柱的冲洗。
本领域已知多种临床可用的金属螯合剂，这些金属离子螯合剂均可以在本发明中使用。优选地，金属螯合剂选自：EDTA(乙二胺四乙酸)，DTPA(二乙基三胺五乙酸),EGTA(乙二醇双(2-氨基乙基醚)四乙酸),BAPTA(1,2-双(2-氨基苯氧基)-乙烷-N,N,N',N'-四乙酸),DMPS(二巯基丙磺酸),DMSA(二巯基丁二酸),ALA(阿尔法硫辛酸)，或其组合。更优选，金属螯合剂为EDTA或DTPA。用于洗脱液中的金属螯合剂的量可以由本领域技术人员根据本说明书的教导进行确定。例如，本领域技术人员可以根据用于纯化抗体组合物浓缩和换液的超滤/洗滤透析体积，推算用于洗脱液中的金属螯合剂的量。在一个优选实施方案中，洗脱液中金属离子螯合剂为5-40mmol/L,例如10-30mmol/L，例如，20mmol/l。优选地，洗脱液中的金属螯合剂的量，导致纯化得到的抗体组合物，例如药物物质或药物产品中具有0.00026-0.05mg/ml的金属螯合剂，优选0.0005-0.03mg/ml，更优选0.001-0.02mg/ml，例如大约0.002,0.004，0.008，0.01mg/ml。在再一优选实施方案中，除了使用包含金属螯合剂的洗脱液，还可以在用于离子 交换层析的冲洗液中加入金属螯合剂。在一个优选的实施方案中，冲洗液中包含5-40mmol/L,例如10-30mmol/L，例如，10mmol/l的金属离子螯合剂，尤其是EDTA或DTPA。
可以用于本发明的离子交换分离填料并无特别限制。本领域已知，阴离子或阳离子取代基可以与基质附着，以形成阴离子或阳离子支持体用于层析。阴离子交换取代基的非限制性实例包括二乙氨乙基(DEAE)、季氨乙基(QAE)和季胺(Q)基团。阳离子交换取代基包括羧甲基(CM)、磺乙基(SE)、磺丙基(SP)、磷酸基(P)和磺酸基(S)。可以使用层析膜，例如可从Sartorius获得的离子交换膜，进行膜层析。可用的层析膜例子包括但不限于，例如Sartobind Q、Sartobind S(Sartorius AG),Mustang Q(Pall)，ChromaSorb、和
Figure PCTCN2021070654-appb-000002
HD-Q(millipore)。也可以使用多糖基离子交换树脂。例如，纤维素离子交换树脂例如DE23、DE32、DE52、CM-23、CM-32和CM-52，可从Whatman Ltd.Maidstone，Kent，U.K获得。基于葡聚糖凝胶的数值，例如基于SEPHADEX和SEPHAROSE的离子交换树脂也是已知的。例如，DEAE-、QAE-、CM-和SP-SEPHADEX以及DEAE-、Q-、CM-和S-SEPHAROSE和SEPHAROSE Fast Flow可以从多个供应商获得。此外，合成的聚甲基丙烯酸酯树脂例如
Figure PCTCN2021070654-appb-000003
EMD“触角型”离子交换介质也可以用于本发明中。优选地，在一个实施方案中，在本发明方法中，阴离子交换层析使用Q Sepharose Fast Flow，例如，来自美国GE公司的Q Sepharose Fast Flow。在一个实施方案中，在本发明方法中，阳离子交换层析使用与磺酸基连接的聚甲基丙烯酸酯树脂Fractogel SO 3 -，例如，来自MERCK公司的Fractogel EMD SO 3 -(M)。
阴离子交换层析：
在优选的实施方案中，本发明方法包括：在亲和层析后，在阳离子交换层析前，采用阴离子交换填料，进行流通模式的抗体纯化。在一个优选实施方案中，采用Q Sepharose Fast Flow阴离子交换填料。在一个实施方案中，在进行阴离子交换层析上样前，将来自之前纯化步骤的含目的抗体的收集液，调节pH至6.8～7.4，电导率≤7mS/cm。优选地，在进行深层过滤的实施方案中，用阴离子平衡缓冲液稀释该深层过滤的收集液。在一个实施方案中，阴离子交换层析上样前，优选，先用高盐的缓冲液冲洗层析柱，再用阴离子平衡液平衡层析柱。合适的平衡缓冲液的一个例子是Tris/NaCl，pH约7。在一个实施方案中，上样载量为≤30g/L。在一个实施方案中，上样开始后，紫外监测280nm处吸收峰。在一个实施方案中，在吸收值上升至大于等于25mAU/mm光程时开始收集穿透液。在一个实施方案中，上样完毕后，用阴离子平衡缓冲液冲洗，继续收集紫外280nm处吸收峰，当吸收值下降至小于等于1000mAU/mm光程时停止收集。优选地，收集液使用酸性溶液，例如2mol/L枸橼酸溶液调节至pH 6.0。
在一个优选实施方案中，阴离子交换层析步骤包括：用阴离子平衡缓冲液稀释深层过滤收集液，用碱性溶液调节pH至6.8～7.4，电导率≤7mS/cm；上样前，阴离子交换层析柱先用含高盐的缓冲液冲洗，再用阴离子平衡液平衡；上样并收集穿透液，例如吸收值上升至大于等于25mAU/mm光程时开始收集穿透液；上样完毕后，用阴离子平衡缓冲液冲洗，继续收集紫外280nm处吸收峰，优选地当吸收值下降至小于等于1000mAU/mm光程时停止收集；优选地，收集液使用2mol/L枸橼酸溶液调节至pH 6.0。
阳离子交换层析：
在优选的实施方案中，本发明方法包括：在阴离子交换层析后，利用阳离子交换层析，采用吸附-洗脱模式，进行阿达木单抗纯化。优选地，在阳离子离子交换层析中，使用含有金属螯合剂例如EDTA或DTPA的阳离子洗脱液。更优选地，使用金属离子螯合剂用于阳离子交换层析的冲洗液及洗脱液。在一个实施方案中，在洗脱前，进行线性梯度冲洗。例如，使用冲洗A液和冲洗B液进行线性梯度冲洗，其中，冲洗液A为阳离子平衡液(例如，20mmol/L磷酸缓冲液，pH6.0)；冲洗液B具有高于阳离子平衡液的离子强度和/或pH(例如，70mmol/L磷酸缓冲液，pH7.5)。在一个优选实施方案中，冲液也包含步骤：用包含金属螯合剂例如EDTA或DTPA或其他金属离子螯合剂的冲洗液，冲洗层析柱；优选地，在线性梯度冲洗前进行。优选地，在上样前和洗脱前，用阳离子平衡液平衡层析柱。在一个实施方案中，紫外监测层析洗脱液，当紫外280nm处吸收峰上升至大于等于50mAU/mm光程时开始收集洗脱峰，下降至小于等于1000mAU/mm光程时停止收集。
在一个优选的实施方案中，阳离子交换层析步骤包括：用阳离子平衡液平衡层析柱后进行上样；用含有EDTA(或其他金属离子螯合剂)的阳离子冲洗液冲洗；用阳离子平衡液平衡后，使用冲洗液A和B进行线性梯度冲洗，其中冲洗A液为阳离子平衡液，冲洗B液为离子强度和/或pH提高的阳离子平衡液；冲洗后用阳离子平衡液平衡层析柱，用含有EDTA(或其他金属离子螯合剂)的阳离子洗脱液开始洗脱；优选地，当紫外280nm处吸收峰上升至大于等于50mAU/mm光程时开始收集洗脱峰，且在吸收峰下降至小于等于1000mAU/mm光程时停止收集。
除病毒过滤：
在本发明方法中，在一个可选实施方案中，将由离子交换层析获得的抗体收集液，进行除病毒过滤。优选地，采用除病毒滤器对样品进行过滤，例如在工作压力≤4bar。在一个优选的实施方案中，可以多个滤器串联用于样品过滤。例如，可以将
Figure PCTCN2021070654-appb-000004
Prefilter预过滤器与
Figure PCTCN2021070654-appb-000005
Pro滤器串联。
超滤及洗滤：
在一个优选实施方案中，本发明方法还包括超滤及洗滤步骤，用于例如将抗体蛋白置换于利于其稳定的制剂缓冲液中并浓缩至合适的浓度。
因此，在本发明方法中，在一个实施方案中，还包括将离子交换层析获得的抗体收集液，进行超滤和洗滤。在一个实施方案中，采用超滤膜进行超滤浓缩。在一个实施方案中，样品在超滤浓缩后进行洗滤。可以用洗滤缓冲液连续换液，优选，洗滤缓冲液使用量为5～12倍的超滤浓缩后体积。优选地，通过超滤/洗滤步骤获得的抗体物组合物具有10-150mg/ml，优选10-100mg/ml,更优选，10-60mg/ml,例如40-50mg/ml阿达木抗体。
在一个实施方案中，用于本发明的洗滤缓冲液可以包含缓冲液、盐、多元醇。在一个优选的实施方案中，洗滤缓冲液包含：约10-30mM L-组氨酸，约50-100mM氯化钠，约1-2％(w/v)山梨醇，pH6.±0.5。更优选地，洗滤缓冲液包含：约25mM L-组氨酸，约100mM氯化钠，约1.2％(w/v)山梨醇，pH 6。
纯化抗体组合物的配制
在一些实施方案中，本发明方法还包括步骤：将上述纯化步骤收获的纯化阿达木单抗进行除菌过滤，形成DS(药物物质)。在药物物质中可以进一步加入或不加入辅料，例如稳定剂或赋形剂(例如聚山梨醇酯表面活性剂，例如聚山梨醇酯80)。此外，在一些实施方案中，本发明方法还包括，将药物物质分装于无菌终容器中，形成药物产品DP。可以对药物物质和药物产品进行检测，以进行纯化工艺的验证和产品相关杂质的分析，和产品稳定性的表征。
II.纯化组合物的稳定性表征
由工艺生产的纯化抗体组合物在储存过程中的稳定性与产品的质量属性相关。例如，抗体的聚集、降解或化学修饰，会导致抗体异质性，包括大小异质性和电荷异质性等，从而影响抗体药物产品的质量。因此，有必要进行工艺生产的纯化抗体组合物的稳定性的监测。在本领域中已知多种方法可以用于检测抗体组合物的稳定性。例如，可以通过非还原型CE-SDS和SEC-HPLC等方法，分析抗体组合物在存储过程中的纯度变化；可以通过毛细管等电聚焦电泳(cIEF)、成像毛细管等电聚焦电泳(iCIEF)和离子交换色谱(IEX)等，分析抗体组合物在存储过程中的电荷变异体成分变化。此外，可以采用本领域技术人员已知的方法，进行抗体组合物在存储过程中的其他产品相关变体的监测。
稳定性研究一般包括实际贮存条件下的长期稳定性研究、加速稳定性研究和强制条件试验研究。在加速和强制条件下，降解被加速，降解物可以在更早的时间点、以更大的量被监测到。因此，加速和强制条件试验可以用于阐明目标抗体产品的降解途径，有助于理解目的抗体产品降解的性质(机制)；提供产品在偏离最佳保存条件和极端情况下的稳定性情况。在本发明的一个优选实施方案中，将抗体组合物样品放置于升高的温度，例如约40℃±2℃、25℃±2℃条件下进行强制或加速稳定性研究。
纯度的检测
用于监测抗体组合物的纯度变化的方法可以包括但不限于，非还原型或还原型SDS-PAGE凝胶电泳、或CE-SDS(十二烷基硫酸钠-聚丙烯酰胺毛细管电泳)、分子排阻色谱法(SEC-HPLC)等方法。这些方法可以用于对抗体的单体、聚合体或片段进行定量分析。
尺寸排阻高效液相色谱法，即SEC-HPLC法，是用于单克隆抗体纯度分析的一个重要方法。该方法主要依据分子的尺寸大小或流体动力学半径差异来进行分子的分离。通过SEC-HPLC，抗体可以分离出三种主要形式：高分子量形式(HMMS)、主峰(主要是抗体单体)、和低分子量形式(LMMS)。抗体纯度可以计算为色谱图上主峰面积占所有峰面积之和的百分比。通过SEC-HPLC法，可以测量制剂产品中抗体单体的百分数，给出可溶性聚集物和剪切物的含量信息。关于SEC-HPLC法的进一步描述，可以参见例如，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)。
优选地，通过本发明方法获得的纯化阿达木单抗组合物在存储后，例如40℃存储4周后，单体纯度，如通过SEC-HPLC测量，为97％以上，优选98％或99％以上。
电荷变异体的检测
电荷变异体是重组单克隆抗体的主要质量属性之一，在生产过程中被严密监测和控制。 通常用于分析电荷变体的方法包括：阳离子交换色谱、阴离子交换色谱、等电聚焦电泳和毛细管聚焦电泳，例如但不限于，cIEF，IEX-HPLC，疏水高效液相色谱(HIC-HPLC)、反相高效液相色谱(RP-HPLC)等。参见例如，Kahle J等，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(CEX)for the analysis of monoclonal antibodies。
电荷变异体被分类为相对于主成分的酸性或碱性成分。从阳离子交换色谱早期洗脱的电荷变体或从阴离子交换色谱晚期洗脱的电荷变体通常称作酸性成分(酸性电荷变体)。从阳离子交换色谱较晚洗脱的电荷变体或从阴离子交换色谱早期洗脱的电荷变体通常称作碱性成分(碱性电荷变体)。
在本发明的一个优选方案中，通过阳离子交换高效液相色谱法(CEX-HPLC)测定抗体制剂中抗体的电荷变异体。在该测定法中，以比主峰的保留时间更早从CEX-HPLC柱洗脱出的峰被标记为“酸性峰”，而那些以比主峰的保留时间更晚从CEX-HPLC柱洗脱出的峰被标记为“碱性峰”。优选地，采用CEX-HPLC测量方法，通过本发明方法获得的纯化阿达木单抗组合物在存储后，例如40℃存储4周后，电荷主成分下降不超过40％，优选不超过30％，25％，20％，15％，10％，其中主成分定义为通过CEX-HPLC检测到的主峰与碱性峰1和2的和，其中所述主峰、碱性峰1、碱性峰2分别为含有0、1、2个末端赖氨酸的抗体变异体。在另一优选方案中，采用CEX-HPLC测量方法，通过本发明方法获得的纯化阿达木单抗组合物在存储后，例如40℃存储4周后，酸性电荷变异体增加不超过40％，优选不超过30％，25％，20％，15％，10％。
产品相关杂质的检测
产品相关杂质包括但不限于，氧化产物，脱酰胺产物或其他结构不完整分子。例如，可以使用肽作图(peptide mapping)方法，对抗体的相关变体，例如脱酰胺、二硫键错配和氧化等进行检测。参见例如，Li X等J Chromatogr A.2016 Aug 19；1460:51-60，High throughput peptide mapping method for analysis of site specific monoclonal antibody oxidation。在一个优选实施方案中，产品相关杂质包括阿达木单抗氧化产物，尤其是M256位的氧化产物。在进一步优选的实施方案中，相对于未使用本发明纯化工艺得到的组合物，本发明纯化抗体组合物中阿达木单抗氧化产物的量降低。
III.本发明的抗体组合物
在一方面，本发明提供稳定的阿达木单抗组合物，尤其是通过本发明的抗体纯化方法生产的稳定阿达木单抗组合物。在再一方面，本发明也提供包含包装材料、本发明阿达木单抗组合物和标签或包装插页的制造品。
在一个实施方案中，本发明提供了一种阿达木单抗组合物，其包含阿达木单抗和金属螯合剂，优选地0.00026-0.05mg/ml的金属螯合剂，优选0.0005-0.03mg/ml，更优选0.001-0.02mg/ml，例如大约0.002,0.004，0.008，0.01mg/ml的金属螯合剂。优选地，金属离子螯合剂选自：EDTA，DTPA,EGTA,BAPTA,DMPS,DMSA,ALA，或其组合；更优选EDTA或DTPA。
在一个实施方案中，本发明提供稳定的液体阿达木单抗组合物，其包含(i)阿达木单抗，尤其是通过本发明纯化方法生产的阿达木单抗组合物；(ii)缓冲剂，(iii)多元醇，和(iv) 表面活性剂，所述抗体组合物的pH为约5.0-7.0。任选地，本发明组合物还包含盐，如氯化钠。
本发明的抗体组合物中所包含的抗体的量可随着组合物的特定目的特性、特定环境、和使用组合物的特定目的而改变。在一些实施方案中，抗体组合物为液体，其可含有约1-150mg/mL，优选地为约10-100mg/mL，例如，约15、20、25、30、35、40、45、50、55、60mg/mL抗体。在一个实施方案中，本发明涉及以10-50mg/mL的浓度生产的抗体药物物质。在另一实施方案中，本发明涉及由所述药物物质组合可药用辅料形成的药物产品。
在一个优选的实施方案中，本发明的阿达木单抗组合物包含：
(i)10-150mg/ml，例如20-50mg/ml,如大约40mg/ml量的阿达木单抗；
(ii)0.001-0.02mg/ml的金属螯合剂，例如EDTA或DTPA；
(iii)0.8-1.5mg/ml；更优选1.0-1.2mg/ml的聚山梨酯类非表面活性剂，例如聚山梨酯80；
(iv)10-50mM组氨酸缓冲液，如大约25mM组氨酸，且
优选地，本发明的组合物具有pH为大约5-7，例如6.0±0.5，如大约6.0。
在再一优选实施方案中，本发明阿达木单抗组合物还包含：
(vi)50-100mM氯化钠，如100mM氯化钠；和
(vii)约1-2％(w/v)山梨醇，如约1.2％(w/v)山梨醇。
本发明的抗体液体组合物中可以包含或不包含其它赋形剂。所述赋形剂包括，例如，调味剂、抗微生物剂、甜味剂、抗静电剂、抗氧化剂、明胶等等。这些和另外已知的药物赋形剂和/或适用于本发明制剂的添加剂是本领域公知的，例如，列出于“The Handbook of Pharmaceutical Excipients，第4版，Rowe等人编，American Pharmaceuticals Association(2003)；和Remington:the Science and Practice of Pharmacy，第21版，Gennaro编，Lippincott Williams&Wilkins(2005)”。
在优选的实施方案中，本发明的组合物具有稳定性。优选地，所述组合物具有选自以下之一项或多项的稳定性：氧化稳定性、单体稳定性、和电荷变异体稳定性、和聚山梨醇酯80稳定性。更优选地，本发明组合物在存储后，例如40℃存储4周后，电荷主成分下降不超过40％，优选不超过30％，25％，20％，15％，10％；优选地，酸性电荷变异体增加不超过40％，优选不超过30％，25％，20％，15％，10％。更优选地，本发明组合物在存储后，例如40℃存储4周后，单体纯度，如通过SEC-HPLC测量，为97％以上，优选98％或99％以上。优选地，本发明组合物在存储后，例如25℃存储4周后，不具有显著增加的M265氧化产物，例如该残基的氧化比率低于40％，更优选地低于20％，更优选低于10％，更优选低于5％。优选地，本发明组合物在存储后，例如40℃存储4周后，聚山梨醇酯80含量的降低不超过10％，例如不超过5％。
IV.本发明抗体组合物的用途
再一方面，本发明提供本发明抗体组合物的医药用途、和使用本发明抗体组合物的治疗方法。
因此，在一个实施方案中，本发明提供治疗其中TNFα活性有害的疾病的方法，所述方法包括将包含用本发明任何方法获得的抗体的药物组合物给予人受试者。在一个实施方案中， 该其中TNFα活性有害的疾病选自自身免疫疾病、肠道疾病和皮肤疾病。在一个实施方案中，所述的疾病选自类风湿性关节炎、幼年特发性关节炎、牛皮癣关节炎、强直性脊柱炎、克罗恩病、溃疡性结肠炎和慢性斑块型银屑病。在一个实施方案中，本发明的组合物与另外的治疗剂组合施用于有需要的个体，例如与甲氨蝶呤、DMARDS、糖皮质激素、非甾体抗炎药(NSAID)，和/或镇痛药。
在再一实施方案中，本发明涉及本发明抗体组合物，尤其是通过本发明方法制备的抗体组合物，在制备用于治疗TNFα活性有害的疾病的药物组合物中的用途。
本发明由下面的实施例作进一步详细说明，实施例不应被认为是限制性的。本申请通篇引用的所有的参考文献、专利和公布的专利申请，其内容通过引用结合到本文中。
实施例
材料
阿达木单抗
在以下实施例中进行纯化的阿达木单抗为在中国仓鼠卵巢CHO细胞中表达的阿达木单抗。抗体的重链和轻链序列如SEQ ID NO:1和2所示。
工艺用原材料：
Figure PCTCN2021070654-appb-000006
一般方法描述
EDTA残留测试方法
采用反相高效液相色谱(RP-HPLC法)，进行纯化组合物中EDTA残留量的检测。使用Agilent1260高效液相色谱仪。取7.5g/L EDTA溶液(厂家sigma，货号E4884-100G)，用超纯水依次稀释至1.0g/L、0.5g/L、0.1g/L、0.05g/L、0.01g/L进行标准曲线样品制备，并建立标准曲线。取样品100μl，加入30μl 10％三氯乙酸(厂家Aladdin，货号T104257-500g)，混匀，13000r/min离心5min，取50μl上清液，加入0.5μl 20g/L CuSO 4溶液(厂家Aladdin，货号C112401-100g)，混匀。色谱分析条件：采用Symmetry C18色谱柱(厂家Waters，货号WAT054275)，流速：0.4ml/min，进样体积：10μl，柱温：25℃，检测波长：254nm，流动相A为pH4.0、含10％四丁基氢氧化铵的50mM甲酸铵水溶液，流动相B为甲醇，比例：A:B＝90:10，运行时间：20分钟。根据标准曲线计算EDTA含量。采用以下EDTA含量计算 公式：
EDTA含量(g/L)＝样品EDTA检测含量值(μg/μl)×1.3(稀释倍数)
图1示例性显示了EDTA残留测试的一个典型图谱。
聚山梨酯80含量测试方法
采用FLD-HPLC法(高效液相色谱荧光检测法)检测样品的聚山梨酯80含量。使用Agilent1260高效液相色谱仪。取2.5％PS80储备液，用不含PS80的缓冲液分别稀释至1.6mg/ml、0.8mg/ml、0.4mg/ml、0.2mg/ml、0.1mg/ml进行标准曲线的制备，并建立标准曲线，色谱分析条件：采用Knitted Reactor Coil色谱柱(厂家SUPELCO，货号57405)，流动相：0.15mol/L氯化钠，0.05mol/L Tris，pH 8.0，5％乙腈，5.0μmol/L NPN，15ppm Brij，流速：1.5ml/min；采集时间：3分钟；进样量：10μl；柱温：30℃；检测波长：激发光350nm、发射光420nm。根据标准曲线计算聚山梨酯80含量。图2示例性显示了一个典型图谱。
电荷异质性测定方法(CEX-HPLC法)
采用阳离子交换色谱(CEX-HPLC法)检测样品的电荷异质性。使用ProPac TM WCX-10离子交换柱(厂家Thermo，货号054993)分离，流动相A为10mmol/L磷酸盐缓冲液、流动相B为10mmol/L磷酸盐+200mmol/L氯化钠缓冲液，流速1.0ml/min，梯度洗脱，检测波长280nm，柱温35℃。用超纯水将样品稀释至2.0mg/ml，作为供试品溶液,取供试品溶液50μl注入液相色谱仪，进样器温度8℃，运行时间35min。按照面积归一化法计算样品的酸性组分、主成分(主峰+碱峰1+碱峰2)和碱性组分。与本领域的研究结果一致，主峰、碱性峰1、碱性峰2分别为含有0、1、2个末端赖氨酸的变异体(参见例如，吴霖萍等，阿达木单抗理化特性的关键质量属性分析，中国医学工业杂质，2018,49(3))。图3示例性显示了一个典型图谱。
CEX-HPLC法洗脱梯度
Figure PCTCN2021070654-appb-000007
抗体纯度测定方法(SEC-HPLC法)
采用体积排阻色谱(SEC-HPLC法)法检测样品的分子大小变异体。使用TSK-gel G3000SWxl(厂家TOSOH，货号0008541)分离，流动相为20mmol/L磷酸盐缓冲液+200mmol/L氯化钠，流速0.5ml/min，检测波长280nm，柱温25℃。用超纯水将供试品稀释至2.0mg/ml，作为供试品溶液；供试品溶液50μl注入液相色谱仪，进样器温度8℃，运行时间 30min。按照面积归一化法计算样品的聚合体、单体和片段。
金属元素含量检测方法
使用标准的IPC-MS法进行金属元素含量检测。所述方法的操作方案可以参见例如《金属离子分析技术》化学工业出版社，余自力、程光磊主编。2004，ISBN 750256148。
稳定性实验
将供试样品在超净台(苏州苏净安泰，型号：SW-CJ-2FD)中进行过滤，分装，加塞和轧盖。之后，将样品置于恒温恒湿箱(德国binder，型号：KBF P 720)RH 60％±5％，避光条件下设置用于加速稳定性实验的温度(例如，大约25℃或40℃)、或用于长期稳定性实验的温度(例如，大约5℃)。在选取的时间点(例如，t＝0,1周，2周，3周，4周)，取样检测。检测包括但不限于，CEX-HPLC法检测电荷变异体，SEC-HPLC检测抗体纯度，和/或FLD-HPLC法检测聚山梨酯80含量。
实施例1：
本实施例描述未优化的纯化工艺。工艺包括如下步骤：
a)亲和层析：采用Mabselect SuRe(美国GE，货号17-5438-03、17-5438-04、17-5438-05)亲和填料。可以根据工艺需要，使用不同的填料pack size。例如，可以使用1-10升pack size，例如1升，5升和10升。使用平衡缓冲液20mmol/L Tris+150mmol/L NaCl，pH7.2平衡后上样，载量10～45g/L，上样流速100～250cm/h。完成上样后，用20mmol/L Tris+150mmol/L NaCl，pH7.2平衡3CV，再用20mmol/L Tris+0.5mol/L NaCl，pH7.2冲洗3CV，20mmol/L Tris+150mmol/L NaCl，pH7.2平衡3CV。然后开始洗脱，洗脱缓冲液(20mmol枸橼酸+100mmol/L NaCl)pH范围为3.3～3.7。收集紫外280nm处主峰。
b)病毒灭活：亲和收集液用2mol/L枸橼酸，调节pH至3.2～3.7，蛋白浓度≤25mg/ml，于18～26℃下静置90～180min，然后用2mol/L Tris调节样品pH至中性。
c)深层过滤：采用X0HC(millipore公司产品，货号：MX0HC10FS1)膜包，通量≤150LMH，载量为65～160L/m2，进行过滤。
d)阴离子交换层析：采用Q Sepharose Fast Flow(美国GE，货号17-0510-04、17-0510-05、17-0510-60)阴离子交换填料。用阴离子平衡缓冲液20mmol/L Tris-HCl，pH7.1稀释深层过滤收集液，用2mol/L Tris调节pH至6.8～7.4，完全混匀后取样检测，电导率≤7mS/cm。先用20mmol/L Tris-HCl+1mol/L NaCl，pH7.0冲洗层析柱，再用阴离子平衡液平衡层析柱。开始上样，上样载量≤30g/L，紫外280nm处吸收值上升至大于等于25mAU/mm光程时开始收集穿透液。上样完毕后，用阴离子平衡缓冲液冲洗，继续收集紫外280nm处吸收峰，当吸收值下降至小于等于1000mAU/mm光程时停止收集。收集液使用2mol/L枸橼酸溶液调节至pH 6.0。
e)阳离子交换层析：采用阳离子交换填料Fractogel EMD SO3-(M)(德国Merck，1.16882.5000)填料。用阳离子平衡液20mmol/L磷酸缓冲液，pH6.0平衡层析柱后，开始上样。上样完毕后用阳离子平衡液平衡，然后开始线性梯度冲洗，冲洗A液为阳离子平衡液，冲洗B液 为70mmol/L磷酸缓冲液，pH7.5，线性梯度冲洗10CV。冲洗后再次用阳离子平衡液平衡层析柱，用阳离子洗脱液20mmol/L磷酸缓冲液，200mmol/L NaCl，pH7.0开始洗脱。当紫外280nm处吸收峰上升至大于等于50mAU/mm光程时开始收集洗脱峰，下降至小于等于1000mAU/mm光程时停止收集。
f)除病毒过滤：采用
Figure PCTCN2021070654-appb-000008
Prefilter(美国Merck Millipore，货号：MSPV05FS1)预过滤器与
Figure PCTCN2021070654-appb-000009
Pro(美国Merck Millipore，货号：VPMG201NB1)滤器串联对样品进行过滤。工作压力≤4bar。
g)超滤及洗滤：采用Pellicon2(30kD)(美国Merck Millipore，货号：P2B030A05)超滤膜包进行超滤浓缩。样品浓缩后开始洗滤，用洗滤缓冲液(3.9g/L L-组氨酸，12.0g/L山梨醇，6.2g/L氯化钠，pH6.0)连续换液5～12倍浓缩后体积，得到洗滤收集液。
h)原液配制：添加聚山梨酯80(德国Merck KGaA，货号：8.17061.1000)及除菌过滤。
实施例2
本实施例描述经优化的纯化工艺。工艺包括如下步骤：
a)亲和层析：采用Mabselect SuRe(美国GE，货号17-5438-03、17-5438-04、17-5438-05)亲和填料。采用20mmol/L Tris+150mmol/L NaCl，pH7.2平衡后上样，载量10～45g/L，上样流速100～250cm/h。完成上样后，用20mmol/L Tris+150mmol/L NaCl，pH7.2平衡3CV，再用20mmol/L Tris+0.5mol/L NaCl，pH7.2冲洗3CV，20mmol/L Tris+150mmol/L NaCl，pH7.2平衡3CV。然后开始洗脱，洗脱缓冲液(20mmol枸橼酸+100mmol/L NaCl)pH范围为3.3～3.7。收集紫外280nm处主峰。
b)病毒灭活：亲和收集液用2mol/L枸橼酸，调节pH至3.2～3.7，蛋白浓度≤25mg/ml，于18～26℃下静置90～180min，然后用2mol/L Tris调节样品pH至中性。
c)深层过滤：采用X0HC(millipore公司产品，货号：MX0HC10FS1)膜包，通量≤150LMH，载量为65～160L/m2，进行过滤。
d)阴离子交换层析：采用Q Sepharose Fast Flow(美国GE，货号17-0510-04、17-0510-05、17-0510-60)阴离子交换填料。用阴离子平衡缓冲液20mmol/L Tris-HCl，pH7.1稀释深层过滤收集液，用2mol/L Tris调节pH至6.8～7.4，完全混匀后取样检测，电导率≤7mS/cm。先用20mmol/L Tris-HCl+1mol/L NaCl，pH7.0冲洗层析柱，再用阴离子平衡液平衡层析柱。开始上样，上样载量≤30g/L，紫外280nm处吸收值上升至大于等于25mAU/mm光程时开始收集穿透液。上样完毕后，用阴离子平衡缓冲液冲洗，继续收集紫外280nm处吸收峰，当吸收值下降至小于等于1000mAU/mm光程时停止收集。收集液使用2mol/L枸橼酸溶液调节至pH 6.0。
e)阳离子交换层析：采用阳离子交换填料Fractogel EMD SO3-(M)(德国Merck，1.16882.5000)填料。用阳离子平衡液20mmol/L磷酸缓冲液，pH6.0平衡层析柱后，开始上样。上样完毕后用阳离子冲洗液20mmol/L磷酸缓冲液，10mmol/L依地酸二钠，pH6.0冲洗3CV。用阳离子平衡液平衡，然后如实施例1中所述，开始线性梯度冲洗，冲洗A液为阳离子平衡液，冲洗B液为70mmol/L磷酸缓冲液，pH7.5，线性梯度冲洗10CV。冲洗后再次用 阳离子平衡液平衡层析柱，用阳离子洗脱液20mmol/L磷酸缓冲液，160mmol/L NaCl，20mmol/L EDTA·2Na，pH6.8开始洗脱。当紫外280nm处吸收峰上升至大于等于50mAU/mm光程时开始收集洗脱峰，下降至小于等于1000mAU/mm光程时停止收集。
f)除病毒过滤：采用
Figure PCTCN2021070654-appb-000010
Prefilter(美国Merck Millipore，货号：MSPV05FS1)预过滤器与
Figure PCTCN2021070654-appb-000011
Pro(美国Merck Millipore，货号：VPMG201NB1)滤器串联对样品进行过滤，工作压力≤4bar。
g)超滤及洗滤：采用Pellicon2(30kD)(美国Merck Millipore，货号：P2B030A05)超滤膜包，进行超滤浓缩。样品浓缩后开始洗滤，用洗滤缓冲液(3.9g/L L-组氨酸，12.0g/L山梨醇，6.2g/L氯化钠，pH6.0)，连续换液5～12倍浓缩后体积，得到洗滤收集液。
h)原液配制：添加聚山梨酯80(德国Merck KGaA，货号：8.17061.1000)及除菌过滤。
通过上述优化工艺获得的原液包含：40mg/ml阿达木单抗，3.9g/L L-组氨酸，12.0g/L山梨醇，6.2g/L氯化钠，1mg/mL聚山梨酯-80，pH6.0。
实施例3：
抗体自身的结构、残留杂质、生产操作过程、包材的浸出物和制剂处方等都会影响制剂的稳定性，包括化学和物理性质的影响，例如脱酰胺、异构化、水解、消旋、末端修饰、糖化、氧化、二硫键变化、变性、聚集、形成沉淀或颗粒、吸附等。因此抗体制剂对诸如温度变化、氧化、光照、离子含量及切割力等环境因素较为敏感。
本实施例描述以实施例1为生产工艺生产的原液样品的稳定性实验及结果，以考察生产工艺对抗体制剂稳定性的影响。
在超净台(苏州苏净安泰，型号：SW-CJ-2FD)中进行过滤，分装，加塞和轧盖，置于恒温恒湿箱(德国binder，型号：KBF P 720)RH 60％±5％，避光条件下设置加速、长期条件下的温度。在表1所示的时间点取样检测。通过CEX-HPLC法检测样品中的电荷变异体主成分。实验结果详见表1。实验中使用了来自相同工艺的不同批次抗体进行。
表1.工艺优化前稳定性实验结果
Figure PCTCN2021070654-appb-000012
Figure PCTCN2021070654-appb-000013
从稳定性实验结果可以看出，使用优化前工艺生产的纯化阿达木单抗组合物是不稳定的，电荷变异体主成分的量在储存中出现降低，抗体组合物表现出增加的电荷异质性。
实施例4：
在本实施例中，使用肽段比对方法，识别在实施例1工艺生产的阿达木单抗组合物中引起电荷异质性不稳定性的主要因素。
肽段比对方法：
为确认组合物原液和成品(即，将原液灌装到药品最终的储存容器中后形成的成品)在加速条件下CEX-HPLC主成分下降的原因，将批次1的原液和两个成品在长期(5℃)和加速(25℃)条件下储存一个月的样品分别用胰蛋白酶(trypsin)酶解成肽段，再利用液相色谱串联质谱(LC-MS/MS)技术比较样品中的翻译后修饰及二硫键配对等差异。结果表明两个成品中的二硫键配对一致，没有发生变化，脱酰胺含量无明显变化，而氧化含量有明显变化，如组合物重链肽段(253-259)DTLMISR中M(256位)的氧化含量有较大差异，详见表2，由此确认M氧化是造成在加速条件下CEX-HPLC主成分下降的原因。
表2.组合物原液和成品肽图分析结果
Figure PCTCN2021070654-appb-000014
实施例5：
在生产工艺中，金属离子是引起抗体氧化的一个可能因素。因此，在本实施例中，对以实施例1工艺生产的两个批次的阿达木单抗组合物中的金属元素含量进行了检测。
检测结果见下表3.
Figure PCTCN2021070654-appb-000015
实施例6：
在本实施例中，针对去除金属离子氧化物进行了后续的工艺优化研究，以考察工艺优化对纯化的阿达木单抗组合物的稳定性影响。
对不同优化条件生产的产品，进行40℃强制稳定性实验。
实验样品：使用以下不同工艺条件生产的阿达木单抗原液
①实施例2的工艺条件，其中阳离子交换层析洗脱液含20mM EDTA，且超滤/洗滤透析6SV(样品体积)
②实施例2的工艺条件，其中阳离子交换层析洗脱液含20mM EDTA，且超滤/洗滤透析10SV
③实施例1的工艺条件，其中阳离子交换层析洗脱液不含EDTA，且超滤/洗滤透析10SV
④50％样品②+50％样品③的混合
样品中EDTA含量的测定
根据超滤/洗滤连续操作模式下的理论方程：
Figure PCTCN2021070654-appb-000016
推算原液中的EDTA浓度；
其中，
n＝透析体积倍数
Cc＝回流液中EDTA浓度
Co＝原料EDTA浓度
R＝杂质截流率
R在无截留情况下为0，因此一般Cc>e^(-n)×Co
e＝2.72，当n＝10，Cc>4.5×10 -5×Co，实施例2的阳离子洗脱液含有20mM EDTA，因此可以推算制剂中Cc EDTA含量大于9×10 -4mM，即大于0.00026mg/ml。
对使用不同超滤/洗滤透析体积(SV)得到的实施例2工艺样品，即前述样品①和样品②，中的EDTA含量，进行一式两份测定。结果如下表4：
表4 EDTA含量测定
Figure PCTCN2021070654-appb-000017
将实验样品置于40℃进行稳定性实验。在实验开始(T＝0)时、以及在存储1周、2周和4周时取样，测定抗体电荷变异体和组合物中聚山梨酯80的含量。结果显示在图4和图5中。
结果表明，EDTA含量大于0.00026g/L的阿达木单抗组合物，阿达木单抗纯度和电荷异构体更稳定(40℃强制稳定性4周主成分下降低于26％)、聚山梨酯80含量更稳定(40℃强制稳定性4周下降低于10％)。
实施例7：
本实施例描述以优化后的工艺(实施例2)生产的产品，40℃强制稳定性实验结果。
实验样品：使用以下不同工艺条件生产的阿达木单抗原液
①实施例2的工艺条件，其中EDTA加入阳离子交换层析的冲洗液和洗脱液中；
②实施例2的工艺条件，其中仅将EDTA加入阳离子交换层析的冲洗液。
将实验样品置于40℃进行稳定性实验。在实验开始(T＝0)时、以及在存储1周、2周和4周时取样，测定抗体电荷变异体(CEX-HPLC法)和纯度(SEC-HPLC法)，并测定组合物中聚山梨酯80的含量。结果见下表5。
表5.工艺优化前后40℃强制稳定性实验结果
Figure PCTCN2021070654-appb-000018
结果表明，相比于仅在冲洗液中加入EDTA，在冲洗液和洗脱液中加入EDTA，可以导致显著改善纯化的阿达木单抗组合物的稳定性，尤其是改善的电荷异质性；同时经SEC-HPLC检测的抗体纯度也表现出一些增加。此外，EDTA在洗脱液中的加入也增加了组合物中表面活性剂聚山梨酯80的稳定剂，显著减少了在组合物存储期间聚山梨酯80的降解。
实施例8
为了进一步展示本发明优化工艺对抗体组合物的影响，将优化工艺得到的产品与
Figure PCTCN2021070654-appb-000019
Figure PCTCN2021070654-appb-000020
原研药(美国AbbVie公司，规格40mg:0.8ml,批号45024LX01)进行稳定性比较。
实验样品：
①根据实施例2的优化工艺条件生产的两批阿达木单抗原液。
②阿达木单抗
Figure PCTCN2021070654-appb-000021
将实验样品置于40℃进行稳定性实验。在实验开始(T＝0)时、以及在存储1周、2周和4周时取样，测定抗体电荷变异体(CEX-HPLC法)和纯度(SEC-HPLC法)，并测定组合物中聚山梨酯80的含量。结果见下表6。
表6.原研药与工艺优化后的本发明产品的40℃强制稳定性实验结果
Figure PCTCN2021070654-appb-000022
Figure PCTCN2021070654-appb-000023
结果表明，采用本发明优化工艺得到的纯化阿达木单抗组合物，在稳定性方面与市售的阿达木单抗制剂(修美乐)表现相当或更好。在40℃强制稳定性实验中，存储4周，本发明产品与市售制剂在电荷变异体主成分和酸性成分的变化上基本相当(主成分：约23.3％vs.约20％；酸性成分：约21％vs.约19％)；而通过SEC-HPLC检测，本发明产品在纯度上较市售制剂更好(约99％vs.约92％)。
进一步，对本发明工艺优化后组合物的成品进行了肽图分析以检查产品的氧化率。检测方法同如上所述。
表7.工艺优化后组合物成品肽图分析结果
Figure PCTCN2021070654-appb-000024
如上肽图分析结果证实，采用本发明优化工艺可以显著地改进阿达木单抗组合物的氧化稳定性。

Claims (17)

  1. 一种用于制备具有改善的稳定性的阿达木单抗组合物的方法，所述稳定性优选为电荷变异体稳定性，任选地还包括单体稳定性、氧化稳定性、或其组合；其中所述方法包括以下步骤：
    (a)亲和层析:将包含阿达木单抗的混合物施加到亲和层析树脂，以洗脱缓冲液洗脱，收集包含所述抗体的收集液；
    (b)阴离子交换层析：将包含所述抗体的收集液施加到阴离子交换树脂，收集包含所述抗体的穿透液；和
    (c)阳离子交换层析:将包含所述抗体的收集液施加到阳离子交换树脂，以洗脱缓冲液洗脱，收集包含所述抗体的收集液，其中，所述洗脱液含有金属离子螯合剂(例如，EDTA或DTPA)，
    优选地，通过所述方法获得的纯化阿达木单抗组合物在存储后，例如40℃存储4周后，酸性电荷变异体增加不超过40％，优选不超过30％，25％，20％，15％，10％；且任选地，电荷主成分下降不超过40％，优选不超过30％，25％，20％，15％，10％；
    更优选地，通过所述方法获得的纯化阿达木单抗组合物在存储后，例如40℃存储4周后，单体纯度，如通过SEC-HPLC测量，为97％以上，优选98％或99％以上；
    更优选地，通过所述方法获得的纯化阿达木单抗组合物具有氧化稳定性，在存储后，例如25℃存储4周后，如通过肽作图测量，抗体256位甲硫氨酸残基(M256)的氧化比率不超过40％，或更优选地不超过30％，20％或10％，5％。
  2. 权利要求1的方法，其中，在亲和层析之后，在进行阴离子交换层析之前，进行病毒灭活和任选地深层过滤。
  3. 权利要求1的方法，其中亲和层析采用蛋白A亲和填料进行，载量10～45g/L；优选地，上样流速为100-250cm/h；优选地，洗脱缓冲液的pH范围为3.3～3.7；更优选地，在洗脱前，用含高盐的冲洗溶液进行冲洗，并使用中性缓冲液进行平衡。
  4. 前述权利要求之任一项的方法，其中，阴离子交换层析步骤包括：在进行阴离子交换层析前，调节待进行层析的含阿达木单抗收集液的pH至6.8～7.4；且电导率≤7mS/cm。
  5. 前述权利要求之任一项的方法，其中，阳离子交换层析步骤包括：使用含有金属离子螯合剂的阳离子洗脱液洗脱，其中洗脱液中金属螯合剂的量导致通过所述方法获得的纯化阿达木单抗组合物中具有0.00026-0.05mg/ml的金属螯合剂，优选0.0005-0.03mg/ml，更优选0.001-0.02mg/ml，例如大约0.002,0.004，0.008，0.01mg/ml；
    优选，洗脱液中金属离子螯合剂的量为5-40mmol/L,例如10-30mmol/L，例如，20mmol/l。
  6. 前述权利要求之任一项的方法，其中，阳离子交换层析步骤还包括：在洗脱之前，用含有金属离子螯合剂的阳离子冲洗液冲洗，
    优选，冲洗液中金属离子螯合剂为5-40mmol/L,例如10-30mmol/L，例如，10mmol/l。
  7. 前述权利要求之任一项的方法，其中金属离子螯合剂选自：EDTA，DTPA,EGTA,BAPTA,DMPS,DMSA,ALA，或其组合；优选EDTA或DTPA。
  8. 前述权利要求之任一项的方法，其中所述方法还包括：对纯化的阿达木单抗进行超滤及洗滤的步骤和纯化抗体组合物配制步骤；优选地，使用5～12倍超滤浓缩后体积的洗滤缓冲液，得到洗滤收集液；优选地，在纯化抗体组合物配制步骤，向组合物中加入稳定剂聚山梨醇酯(优选聚山梨醇酯80)。
  9. 一种阿达木单抗组合物，其包含阿达木单抗和金属螯合剂，优选地0.00026-0.05mg/ml的金属螯合剂，优选0.0005-0.03mg/ml，更优选0.001-0.02mg/ml，例如大约0.002,0.004，0.008，0.01mg/ml的金属螯合剂。
  10. 权利要求9的阿达木单抗组合物，其中阿达木单抗为通过权利要求1-8任一项的方法纯化获得。
  11. 权利要求9-10的阿达木单抗组合物，其中所述组合物为水性组合物，其包含：
    阿达木单抗，例如10-150mg/ml，例如20-50mg/ml,如大约40mg/ml量的阿达木单抗；
    金属螯合剂，例如0.001-0.02mg/ml，例如0.002,0.004，0.008，0.01mg/ml的EDTA或DTPA；
    聚山梨酯类非表面活性剂，例如聚山梨酯80或20，例如0.8-1.5mg/ml；更优选1.0-1.2mg/ml聚山梨酯80；
    缓冲液，例如组氨酸缓冲液，例如10-30mM组氨酸，如大约25mM组氨酸；
    多元醇，例如山梨醇，例如约1-2％(w/v)山梨醇，如约1.2％(w/v)山梨醇；
    氯化钠，例如约50-100mM氯化钠，如约100mM氯化钠，
    优选地，pH为大约5-7，例如大约6.0。
  12. 权利要求9-11任一项的阿达木单抗组合物，其中，
    所述组合物在存储后，例如40℃存储4周后，酸性电荷变异体增加不超过40％，优选不超过30％，25％，20％，15％，10％；优选地，电荷主成分下降不超过40％，优选不超过30％，25％，20％，15％，10％；
    优选地，所述组合物在存储后，例如40℃存储4周后，单体纯度，如通过SEC-HPLC测量，为97％以上，优选98％或99％以上；
    优选地，所述组合物具有氧化稳定性。
  13. 一种用于改善纯化阿达木单抗水性组合物的稳定性的方法，其中所述稳定性优选地选自电荷变异体稳定性、单体稳定性、氧化稳定性、或其组合，
    其中所述方法包括：
    (a)亲和层析:将包含阿达木单抗的混合物施加到亲和层析树脂，以洗脱缓冲液洗脱，收集包含所述抗体的收集液；
    (b)阴离子交换层析：将包含所述抗体的收集液施加到阴离子交换树脂，收集包含所述抗体的穿透液；和
    (c)阳离子交换层析:将包含所述抗体的收集液施加到阳离子交换树脂，以洗脱缓冲液 洗脱，收集包含所述抗体的收集液，其中，所述洗脱液含有金属离子螯合剂(例如，EDTA或DTPA)，
    优选地，通过所述方法获得的纯化阿达木组合物在存储后，例如40℃存储4周后，电荷主成分下降不超过40％，优选不超过30％，25％，20％，15％，10％；优选地，酸性电荷变异体增加不超过40％，优选不超过30％，25％，20％，15％，10％；
    优选地，通过所述方法获得的纯化阿达木单抗组合物在存储后，例如40℃存储4周后，单体纯度，如通过SEC-HPLC测量，为97％以上，优选98％或99％以上；
    优选地，通过所述方法获得的纯化阿达木单抗组合物具有氧化稳定性。
  14. 一种从包含阿达木单抗的混合物中纯化阿达木单抗的方法，所述方法包括：
    (a)亲和层析:将包含阿达木单抗的混合物施加到亲和层析树脂，以洗脱缓冲液洗脱，收集包含所述抗体的收集液；
    (b)阴离子交换层析：将包含所述抗体的收集液施加到阴离子交换树脂，收集包含所述抗体的穿透液；和
    (c)阳离子交换层析:将包含所述抗体的收集液施加到阳离子交换树脂，以洗脱缓冲液洗脱，收集包含所述抗体的收集液，其中，所述洗脱液含有金属离子螯合剂(例如，EDTA或DTPA)，
    其中，优选地，通过所述方法获得的纯化阿达木单抗组合物具有选自以下的稳定性：氧化稳定性、单体稳定性、和电荷变异体稳定性；
    优选地，通过所述方法获得的纯化阿达木组合物在存储后，例如40℃存储4周后，电荷主成分下降不超过40％，优选不超过30％，25％，20％，15％，10％；优选地，酸性电荷变异体增加不超过40％，优选不超过30％，25％，20％，15％，10％；
    优选地，通过所述方法获得的纯化阿达木单抗组合物在存储后，例如40℃存储4周后，单体纯度，如通过SEC-HPLC测量，为97％以上，优选98％或99％以上；
    优选地，通过所述方法获得的纯化阿达木单抗组合物具有氧化稳定性。
  15. 根据前述任一项的方法，其中抗体组合物，在40℃存储4周后，其中的聚山梨醇80含量降低不超过10％，优选地不超过5％或更少。
  16. 根据权利要求任一项的方法，其中包含阿达木单抗的混合物为自重组表达阿达木单抗的哺乳动物宿主细胞培养物回收的单抗收集液。
  17. 一种治疗其中TNFα活性有害的疾病的方法，所述方法包括向有需要的受试者施用根据前述权利要求之任一项的抗体药物组合物。
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