WO2023284073A1 - 降低单克隆抗体生产中宿主细胞蛋白含量的亲和纯化方法、抗人ifnar1单克隆抗体浓缩溶液的制备方法以及液体制剂 - Google Patents

降低单克隆抗体生产中宿主细胞蛋白含量的亲和纯化方法、抗人ifnar1单克隆抗体浓缩溶液的制备方法以及液体制剂 Download PDF

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WO2023284073A1
WO2023284073A1 PCT/CN2021/114952 CN2021114952W WO2023284073A1 WO 2023284073 A1 WO2023284073 A1 WO 2023284073A1 CN 2021114952 W CN2021114952 W CN 2021114952W WO 2023284073 A1 WO2023284073 A1 WO 2023284073A1
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amino acid
buffer
cdr
seq
monoclonal antibody
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PCT/CN2021/114952
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English (en)
French (fr)
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薛刚
戴长松
李帅
朱华杰
黄文俊
何勇梅
吴亦亮
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江苏荃信生物医药股份有限公司
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Priority claimed from CN202110790532.XA external-priority patent/CN113527490B/zh
Priority claimed from CN202110790531.5A external-priority patent/CN113521276B/zh
Priority claimed from CN202110882251.7A external-priority patent/CN113621063A/zh
Application filed by 江苏荃信生物医药股份有限公司 filed Critical 江苏荃信生物医药股份有限公司
Publication of WO2023284073A1 publication Critical patent/WO2023284073A1/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
    • 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
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

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  • the invention relates to the field of biotechnology, in particular to an affinity purification method for reducing host cell protein content in the production of anti-human interferon alpha receptor 1 monoclonal antibody, a preparation method of concentrated solution of anti-human IFNAR1 monoclonal antibody and a liquid preparation.
  • Affinity purification is a very critical process step in the production process of antibody drugs. This process captures and concentrates the antibodies in the fermentation broth to realize the first step of rough purification of antibodies.
  • HCP host cell protein
  • HCP refers to protein components derived from host cells, including host cell structural proteins and transforming proteins (growth-promoting proteins secreted by cells). HCP may not only induce the body to produce anti-HCP antibodies and cause allergic reactions, but may also have an "adjuvant effect" that may cause the body to produce antibodies to protein drugs and affect the therapeutic effect of the drug.
  • Antibody binding forms a precipitate, while HCP is not prone to precipitation due to its low isoelectric point.
  • the HCP content of the precipitated antibody sample was significantly reduced, but this process is not suitable for process scale-up, and the precipitation may have a certain impact on the activity of the antibody. Therefore, there are many methods for removing HCP. Due to the difference in the sample fermentation process and antibody properties of each project antibody, it is necessary to comprehensively consider the selection process.
  • the biologics market has numerous R&D pipelines and brings more innovative treatments to patients. For some diseases that are difficult to treat with traditional chemical drugs, biologics targeted drugs provide more choices.
  • the dosage forms of biological agents have gradually changed from freeze-dried dosage forms to aqueous injection dosage forms, and from intravenous administration to subcutaneous injection dosage forms. Since the dosage of monoclonal antibody injection is usually in the range of 100mg to 600mg, and the volume of subcutaneous injection is generally limited to less than 2ml, in such cases, highly concentrated protein preparations must be prepared, and the protein content can usually reach 100mg/ ml or greater concentration.
  • the high concentration of monoclonal antibody injection brings many challenges to the manufacturability of the production process, process scale-up, and final patient administration.
  • One of the main challenges is the ultra-high viscosity. Due to the biopolymer properties of monoclonal antibodies and the interaction between protein molecules (such as hydrophobicity, charge interaction, etc.) under high concentration conditions, it tends to form a highly viscous solution. In some extreme cases, a gel-like substance may even form, which brings great challenges to the ultrafiltration membrane and the ultrafiltration equipment itself, such as the decrease in the tangential flow rate caused by the rapid increase in the pressure difference during the final concentration, and the concentration difference Polarization gets out of control until protein precipitation clogs the membrane, which inevitably leads to poor recovery or process failure.
  • the present invention provides a new method for effectively reducing CHO host cell protein (HCP) in antibody purification production, which can be widely used in antibody affinity purification processes, and the The cost of the materials used is relatively low, and the process is easy to enlarge.
  • the HCP content is effectively reduced mainly through intermediate pre-elution and elution of antibody affinity chromatography, which meets the requirements for large-scale, high-quality purification and preparation of antibody drugs, and ensures the safety of clinical use of antibody drugs.
  • the present invention provides an anti-human interferon alpha receptor 1 (IFNAR1) monoclonal
  • IFNAR1 anti-human interferon alpha receptor 1
  • a method for preparing an antibody concentrated solution the method prepares an ultrafiltration concentrated liquid containing an anti-human IFNAR1 monoclonal antibody through ultrafiltration concentration, and can effectively reduce the viscosity of the ultrafiltration concentrated solution, reduce the aggregation of the anti-human IFNAR1 monoclonal antibody and increase its concentration. stability.
  • the present invention also provides a liquid preparation comprising anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody, said liquid preparation comprising anti-human interferon alpha receptor 1 with a protein concentration of 100-200 mg/ml (IFNAR1) monoclonal antibody and basic amino acid with an amino acid concentration of 5-300mM, the liquid preparation can effectively reduce the viscosity of the ultrafiltration concentrate, reduce the aggregation of anti-human IFNAR1 monoclonal antibody and improve its stability, and can be used for Injections, especially hypodermic injections.
  • IFNAR1 anti-human interferon alpha receptor 1
  • Anti-human interferon ⁇ receptor 1 (IFNAR1) monoclonal antibody (QX006N) is a recombinant humanized monoclonal antibody targeting human type I interferon ⁇ / ⁇ receptor 1 independently developed, blocking IFN ⁇ , IFN ⁇
  • the activity of all type I interferons including IFN- ⁇ is intended to be used for the treatment of systemic lupus erythematosus (SLE), and it is intended to be administered by intravenous or subcutaneous injection.
  • SLE systemic lupus erythematosus
  • Anifrolumab developed by AstraZeneca, has the fastest progress and is currently undergoing phase 3 clinical research.
  • An affinity purification method for reducing host cell protein content in monoclonal antibody production comprising the steps of:
  • the monoclonal antibody is an isolated anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chains Complementarity determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein:
  • IFNAR1 isolated anti-human interferon alpha receptor 1
  • the anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein,
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7;
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.
  • the affinity chromatography medium is a ligand cross-linked to agarose, polyvinyl ether, hydroxylated polyether resin, polyacrylic acid resin , polystyrene divinylphenyl resin, polymethacrylic acid resin, polystyrene resin, hydroxyapatite or a chromatographic medium on a glass matrix, preferably, the affinity chromatographic medium is a ligand cross-linked to Chromatographic medium of polyvinyl ether;
  • the ligand is Protein A, Protein G or Protein L, preferably Protein A.
  • the affinity purification method according to any one of items 1-4, wherein the first equilibrium buffer is phosphate buffer, Tris-HCl buffer or boric acid-borax buffer, and the first The salt concentration in the equilibration buffer is 5mM-0.25M, and the pH is 5.5-8.0.
  • the intermediate pre-elution buffer is a neutral buffer and/acid buffer; preferably, the neutral buffer is phosphate buffer, Tris buffer or glycine buffer; the acidic buffer is citric acid-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer or citric acid-trisodium citrate buffer.
  • a pre-elution active agent is added in the intermediate pre-elution buffer, preferably, the pre-elution active agent is guanidine hydrochloride, polysorbate 80 Or sodium chloride, further preferably, the pre-elution active agent is guanidine hydrochloride.
  • the second equilibration buffer is used for equilibration, preferably, the second equilibration buffer is phosphate buffer, Tris-HCl buffer or boric acid-borax buffer.
  • the final elution buffer is selected from citric acid-disodium hydrogen phosphate buffer, acetate buffer, glycine-HCl buffer and citric acid - one or more of sodium citrate buffers, preferably citric acid-disodium hydrogen phosphate buffer.
  • a method for preparing an anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody concentrated solution comprising the steps of:
  • the first ultrafiltration concentration under the condition that the flow rate is 120-300L/m 2 h and the transmembrane pressure difference (TMP) is 0.6-1.5bar, the anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody will be
  • TMP transmembrane pressure difference
  • IFNAR1 anti-human interferon alpha receptor 1
  • Ultrafiltration replacement the concentrated sample is replaced with a replacement buffer, and an ultrafiltration replacement solution is obtained when the amount of the replacement buffer used is 6-10 times the weight of the concentrated sample;
  • the second ultrafiltration concentration mixing the ultrafiltration replacement solution and the basic amino acid mother liquor so that the concentration of the amino acid is 100-150mM, and performing ultrafiltration concentration to obtain a concentrated solution with a protein concentration of 100-200mg/ml;
  • the anti-IFNAR1 monoclonal antibody comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2 and CDR-L3), in:
  • the anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein,
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7;
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.
  • the concentration of the replacement buffer is 5-50 mM, preferably 20 mM; preferably, the pH of the replacement buffer is 6.0- 7.0, preferably 6.2-6.8.
  • the replacement buffer is histidine-hydrochloric acid buffer, citrate buffer, phosphate buffer or sodium acetate-acetic acid buffer, preferably histidine-hydrochloric acid buffer. HCl buffer.
  • the basic amino acid is arginine hydrochloride, lysine and/or proline, preferably Arginine Hydrochloride.
  • a liquid preparation comprising an anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody, said liquid preparation comprising an anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody having a protein concentration of 100-200 mg/ml Antibodies and basic amino acids with an amino acid concentration of 5-300mM;
  • IFNAR1 anti-human interferon alpha receptor 1
  • the anti-IFNAR1 monoclonal antibody comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2 and CDR-L3), in:
  • liquid formulation according to item 23 wherein the anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein,
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7;
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.
  • liquid formulation according to item 26 wherein the basic amino acid comprises 100-200 mM lysine and 5-50 mM histidine.
  • liquid formulation according to item 26 wherein the basic amino acid comprises 100-200 mM proline and 5-50 mM histidine.
  • liquid formulation according to any one of items 23-29, wherein the liquid formulation further comprises 20-150 mg/ml sucrose, preferably 50-100 mg/ml sucrose.
  • liquid formulation according to any one of items 23-26, wherein the liquid formulation further comprises 0.5-50 mg/ml sorbitol, preferably 1-20 mg/ml sorbitol.
  • liquid formulation according to any one of items 23-26, wherein the liquid formulation further comprises 20-300 mg/ml sodium chloride, preferably 100-200 mg/ml sodium chloride.
  • the affinity process described in the present invention is simple and easy, and can be enlarged and purified for production.
  • the cell fermentation supernatant does not need to be pretreated, and the yield of the eluted sample is high, while the HCP residue is also kept at a low level (residue control The amount is not higher than 0.1%), so as to reduce the pressure of HCP removal in the subsequent purification steps, so as to ensure that the residual amount of HCP in the final sample of the antibody is at an extremely low level.
  • the affinity purification process verification of different batches of fermentation supernatants in the invention proves that the affinity purification process of the present invention has good stability.
  • the preparation method of the concentrated solution of anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody of the present invention can reduce the viscosity of high-concentration antibody liquid medicine and improve stability, the method is simple and easy, and can be scaled up for production, and can Guarantee the high purity of the sample and obtain a high recovery rate.
  • the verification of high-concentration antibodies of different subtypes in the invention proves that the method of adding basic amino acids to reduce viscosity in the invention has strong applicability and good stability.
  • the liquid preparation of the present invention comprises anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody, and the viscosity of the liquid preparation is low, and can be easily injected with a syringe, so it can be used as an injection, especially hypodermic injection.
  • IFNAR1 anti-human interferon alpha receptor 1
  • the anti-human interferon ⁇ receptor 1 (IFNAR1) monoclonal antibody compared with the existing anti-human interferon ⁇ receptor 1 monoclonal antibody (Anifrolumab), has a similar affinity to IFNAR1, and has a similar affinity to IFNAR1 at the cellular level.
  • the neutralizing activity is comparable to that of Anifrolumab.
  • the monoclonal antibody has shown neutralizing activity comparable to that of Anifrolumab (expressed and prepared according to the patent disclosed sequence) at the cellular level, and it is expected to show good clinical effects in the prevention and treatment of related diseases.
  • Figure 1 is a graph showing the results of nucleic acid electrophoresis for the construction of the HZD1203-45 transient expression plasmid.
  • M Marker
  • Band 1 PCR product 362VH-Hu6
  • Band 2 pHZDCH, HindIII/NheI
  • Band 3 PCR product 362VK-Hu20
  • Band 4 pHZDCK, HindIII/BsiWI.
  • FIG. 2 is a flowchart of transient expression.
  • Figure 3 is the electrophoretic detection diagram of QX006N (HZD1203-45-IgG4.1).
  • the invention provides an affinity purification method for reducing the protein content of host cells in the production of monoclonal antibodies, which comprises the following steps:
  • the monoclonal antibody is an isolated anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chains Complementarity determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein:
  • IFNAR1 isolated anti-human interferon alpha receptor 1
  • CDR-H1 means heavy chain CDR1 in this description
  • SEQ ID NO: 1 The amino acid sequence of CDR-H1 (CDR-H1 means heavy chain CDR1 in this description) is shown in SEQ ID NO: 1;
  • CDR-H2 means heavy chain CDR2 in this description
  • SEQ ID NO: 2 The amino acid sequence of CDR-H2 (CDR-H2 means heavy chain CDR2 in this description) is shown in SEQ ID NO: 2;
  • CDR-H3 means heavy chain CDR3 in this description
  • SEQ ID NO: 3 The amino acid sequence of CDR-H3 (CDR-H3 means heavy chain CDR3 in this description) is shown in SEQ ID NO: 3;
  • CDR-L1 means light chain CDR1 in this specification
  • SEQ ID NO: 4 The amino acid sequence of CDR-L1 (CDR-L1 means light chain CDR1 in this specification) is shown in SEQ ID NO: 4;
  • CDR-L2 the amino acid sequence of CDR-L2 (in this description, CDR-L2 means light chain CDR2) is as shown in SEQ ID NO: 5;
  • CDR-L3 means light chain CDR3 in this specification
  • SEQ ID NO: 6 The amino acid sequence of CDR-L3 (CDR-L3 means light chain CDR3 in this specification) is shown in SEQ ID NO: 6.
  • amino acid sequence of SEQ ID NO: 1 is as follows:
  • amino acid sequence of SEQ ID NO: 2 is as follows:
  • amino acid sequence of SEQ ID NO: 3 is as follows:
  • amino acid sequence of SEQ ID NO: 4 is as follows:
  • amino acid sequence of SEQ ID NO: 5 is as follows:
  • amino acid sequence of SEQ ID NO: 6 is as follows:
  • monoclonal antibody an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies (e.g., containing native In addition to mutations that exist or arise during the production of monoclonal antibody preparations), such variants are usually present in minor amounts.
  • polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes)
  • monoclonal antibody preparations have each monoclonal antibody directed against a single determinant on the antigen.
  • monoclonal antibodies to be used in accordance with the present invention can be prepared by a variety of techniques including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and the use of all human immunoglobulin loci comprising human immunoglobulin loci. or portions of transgenic animals, such methods and other exemplary methods of making monoclonal antibodies are described herein.
  • the host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell.
  • Host cells include "transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom (regardless of passage number). Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity against the originally transformed cell are included in the specification.
  • HCPs The host cell proteins
  • mAbs monoclonal antibodies
  • ADCs antibody-drug conjugates
  • therapeutic proteins vaccines, and other protein-based biopharmaceuticals.
  • the anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody means a monoclonal antibody capable of binding to human interferon alpha receptor 1 with sufficient affinity such that the monoclonal antibody can be used as a target for human A diagnostic and/or therapeutic agent for interferon alpha receptor 1.
  • the anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody of the present invention does not bind to target-unrelated proteins.
  • unrelated protein refers to other proteins except human interferon alpha receptor 1 as the target; here, “non-binding” means: after the anti-human interferon alpha receptor 1 (IFNAR1) of the present invention
  • the binding ability of the monoclonal antibody to human interferon ⁇ receptor 1 as its target is 100%
  • the binding ability of the anti-human interferon ⁇ receptor 1 monoclonal antibody of the present invention to the irrelevant protein is less than 10% , such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.
  • the anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody of the present invention may not bind to interferon alpha receptor 1 of other animal species.
  • “other animal species” refers to animal species other than humans, such as marmoset monkeys, cynomolgus monkeys, pigs, dogs, rabbits, rats, mice, guinea pigs, etc.; : When the binding ability of the anti-human interferon ⁇ receptor 1 (IFNAR1) monoclonal antibody of the present invention to its target human interferon ⁇ receptor 1 is taken as 100%, the anti-human interferon ⁇ receptor 1 of the present invention Receptor 1 (IFNAR1) monoclonal antibody has less than 10% binding ability to interferon alpha receptor 1 of other animal species, such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.
  • the anti-human interferon alpha receptor 1 monoclonal antibody of the present invention has an equilibrium dissociation constant (KD) of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 50 nM, ⁇ 40 nM.
  • KD equilibrium dissociation constant
  • the anti-human interferon alpha receptor 1 (Human Interferon alpha/beta Receptor 1, IFNAR1) represents a kind of membrane protein derived from people, and its extracellular domain amino acid sequence is as shown in SEQ ID NO: 9, wherein, the underlined part Indicates signal peptide.
  • the residual amount of the host cell protein of the monoclonal antibody can be kept at an extremely low level.
  • the anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein,
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7;
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.
  • amino acid sequence of SEQ ID NO: 7 is as follows:
  • amino acid sequence of SEQ ID NO: 8 is as follows:
  • the amino acid sequence of the heavy chain of the anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody is shown in SEQ ID NO: 10; the amino acid sequence of the light chain is shown in SEQ ID NO: 11 .
  • amino acid sequence of SEQ ID NO: 10 is as follows:
  • amino acid sequence of SEQ ID NO: 11 is as follows:
  • SEQ ID NO: 10 and 11 are humanized sequences.
  • the affinity chromatography medium is ligand cross-linked to agarose, polyvinyl ether, hydroxylated polyether resin, polyacrylic acid resin, polystyrene divinylphenyl resin, polymethacrylic acid resin .
  • Chromatography medium on polystyrene resin, hydroxyapatite or glass matrix preferably, the affinity chromatography medium is a chromatography medium in which ligands are cross-linked to polyvinyl ether;
  • the ligand is Protein A, Protein G or Protein L, preferably Protein A.
  • the ligand can specifically bind to the monoclonal antibody.
  • the present invention is not limited to the affinity filler, which can be confirmed according to the needs of those skilled in the art, for example, the affinity filler can be Mabselect, Mabselect Sure of GE healthcare, Protein A Diamond of Bogeron Biotechnology Co., Ltd., MERCK
  • the first equilibration buffer is phosphate buffer, Tris-HCl buffer or boric acid-borax buffer.
  • the salt concentration in the first equilibrium buffer does not make any restrictions, those skilled in the art can select according to needs, for example, the salt concentration in the first equilibrium buffer is 5mM-0.25M, and the pH is 5.5-8.0 .
  • the salt concentration in the first equilibration buffer can be 5mM, 10mM, 20mM, 50mM, 0.1M, 0.15M, 0.2M, 0.25M, etc.; the pH can be 5.5, 6, 7, 8, etc.
  • the first equilibration buffer is phosphate buffer or Tris-hydrochloric acid buffer, more preferably, NaCl or Na 2 SO 4 is added to the first equilibration buffer to reduce the gap between the non-antibody protein and the filler. non-specific adsorption.
  • the salt concentration in the first equilibrium buffer is 5mM-0.15M, preferably 10mM-50mM, more preferably 20mM.
  • the pH value of the first equilibrium buffer does not make any limitation, those skilled in the art can select according to needs, for example, the pH of the first equilibrium buffer is 6.5-7.5, preferably 6.9.
  • the amount of NaCl or Na2SO4 added can be determined according to the needs of those skilled in the art, and the concentration of NaCl or Na2SO4 can be selected by those skilled in the art according to needs, for example, NaCl or Na2SO
  • the concentration of 4 may be 0-250 mM, preferably 150 mM.
  • the phosphate buffer can be, for example, a buffer of disodium hydrogen phosphate and sodium dihydrogen phosphate.
  • a first equilibration buffer is used for equilibration.
  • the intermediate pre-elution buffer is a neutral buffer and/or an acidic buffer; preferably, the neutral buffer is a phosphate buffer, a tris buffer or a glycine buffer ;
  • the acidic buffer is citric acid-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer or citric acid-trisodium citrate buffer.
  • the present invention does not make any limitation on the pH value of the intermediate pre-elution buffer, and those skilled in the art can select according to needs, for example, the pH of the intermediate pre-elution buffer is 5.0-7.5, preferably 5.8.
  • the pH of the intermediate pre-elution buffer can be 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, etc.
  • a pre-elution active agent is added to the intermediate pre-elution buffer, preferably, the pre-elution active agent is guanidine hydrochloride, polysorbate 80 or sodium chloride, further preferably, The pre-elution active agent is guanidine hydrochloride.
  • the present invention does not make any limitation on the concentration of guanidine hydrochloride, and those skilled in the art can choose according to needs, for example, the concentration of guanidine hydrochloride is 0.01-1M, preferably 0.05-0.15M, more preferably 0.1M.
  • the concentration of the guanidine hydrochloride can be 0.01M, 0.05M, 0.1M, 0.5M, 1M.
  • the salt concentration in the intermediate pre-elution buffer does not make any restrictions, those skilled in the art can select according to needs, for example, in one embodiment, the salt concentration in the described intermediate pre-elution buffer is 0 -0.5M, preferably 0.1M.
  • the salt concentration in the intermediate pre-elution buffer can be 0, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, etc.
  • the following steps are also included before the final elution:
  • the second equilibration buffer is used for equilibration, preferably, the second equilibration buffer is phosphate buffer, Tris-HCl buffer or boric acid-borax buffer.
  • the second equilibration buffer is phosphate buffer or Tris-HCl buffer, preferably, NaCl or Na 2 SO 4 is added to the second equilibration buffer to maintain conductance while maintaining partial buffer capacity.
  • the salt concentration in the second equilibration buffer is 5mM-0.15M, preferably 10mM-50mM, more preferably 20mM; preferably, the pH value is 5.5-8.0, preferably 6.5-7.5, more preferably 7.2 ;
  • the concentration of NaCl or Na 2 SO 4 is 0-250 mM, preferably 10 mM.
  • the phosphate buffer can be, for example, a buffer of disodium hydrogen phosphate and sodium dihydrogen phosphate.
  • the final elution buffer is selected from one or more of citric acid-sodium hydrogen phosphate buffer, acetate buffer, glycine-HCl buffer and citric acid-sodium citrate buffer, A citric acid-disodium hydrogen phosphate buffer is preferred.
  • the pH of the final elution buffer is 2.0-7.0, preferably 3.0 -4.0.
  • the pH of the final elution buffer can be 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, etc.
  • the salt concentration in the final elution buffer does not make any restrictions, those skilled in the art can select according to needs, for example, in one embodiment, the salt concentration in the described final elution buffer is 5-100mM , preferably 10-50 mM.
  • the salt concentration in the final elution buffer may be 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, etc.
  • the affinity medium can be regenerated after elution, preferably, the regeneration buffer includes but not limited to citric acid-disodium hydrogen phosphate buffer, hydrochloric acid, glycine, NaOH, preferably citric acid-hydrogen phosphate Disodium buffer and NaOH solution.
  • the regeneration buffer includes but not limited to citric acid-disodium hydrogen phosphate buffer, hydrochloric acid, glycine, NaOH, preferably citric acid-hydrogen phosphate Disodium buffer and NaOH solution.
  • the present invention uses the Protein A affinity purification process to be simple and easy, and can be enlarged and purified for production.
  • the cell fermentation supernatant does not need to be pre-treated, and the yield of the eluted sample is high.
  • the residual amount of HCP is kept at a low level ( The control amount is not higher than 0.1%), so as to reduce the pressure of removing HCP in the subsequent purification steps, so as to ensure that the residual amount of HCP in the final sample of the antibody is at an extremely low level.
  • the affinity purification process verification of different batches of fermentation supernatants in the invention proves that the affinity purification process of the present invention has good stability.
  • the mammals used for in vitro fermentative production of the monoclonal antibody include but are not limited to various hybridoma cells currently used, Chinese hamster ovary cells (CHO), preferably CHO cells.
  • CHO Chinese hamster ovary cells
  • the invention provides a method for preparing an anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody concentrated solution, which comprises the following steps:
  • the first ultrafiltration concentration under the condition that the flow rate is 120-300L/m 2 h and the transmembrane pressure difference (TMP) is 0.6-1.5bar, the anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody will be
  • TMP transmembrane pressure difference
  • IFNAR1 anti-human interferon alpha receptor 1
  • Ultrafiltration replacement the concentrated sample is replaced with a replacement buffer, and an ultrafiltration replacement solution is obtained when the amount of the buffer to be replaced is 6-10 times the weight of the concentrated sample;
  • the second ultrafiltration concentration mixing the ultrafiltration replacement solution and the basic amino acid mother liquor so that the concentration of the amino acid is 100-150mM, and performing ultrafiltration concentration to obtain a concentrated solution with a protein concentration of 100-200mg/ml;
  • the anti-IFNAR1 monoclonal antibody comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2 and CDR-L3), in:
  • CDR-H1 means heavy chain CDR1 in this description
  • SEQ ID NO: 1 The amino acid sequence of CDR-H1 (CDR-H1 means heavy chain CDR1 in this description) is shown in SEQ ID NO: 1;
  • CDR-H2 means heavy chain CDR2 in this specification
  • SEQ ID NO: 2 The amino acid sequence of CDR-H2 (CDR-H2 means heavy chain CDR2 in this specification) is shown in SEQ ID NO: 2;
  • CDR-H3 means heavy chain CDR3 in this description
  • SEQ ID NO: 3 The amino acid sequence of CDR-H3 (CDR-H3 means heavy chain CDR3 in this description) is shown in SEQ ID NO: 3;
  • CDR-L1 means light chain CDR1 in this specification
  • SEQ ID NO: 4 The amino acid sequence of CDR-L1 (CDR-L1 means light chain CDR1 in this specification) is shown in SEQ ID NO: 4;
  • CDR-L2 the amino acid sequence of CDR-L2 (in this description, CDR-L2 means light chain CDR2) is as shown in SEQ ID NO: 5;
  • CDR-L3 means light chain CDR3 in this specification
  • SEQ ID NO: 6 The amino acid sequence of CDR-L3 (CDR-L3 means light chain CDR3 in this specification) is shown in SEQ ID NO: 6.
  • the flow rate can be 120L/m 2 ⁇ h, 150L/m 2 ⁇ h, 180L/m 2 ⁇ h, 200L/m 2 ⁇ h, 250L/m 2 ⁇ h, 300L/m 2 ⁇ h, etc.;
  • Transmembrane pressure can be 0.6bar, 0.7bar, 0.8bar, 0.9bar, 1.0bar, 1.1bar, 1.2bar, 1.3bar, 1.4bar, 1.5bar, etc.;
  • the protein concentration of the concentrated sample can be, for example, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, etc.;
  • the ultrafiltration replacement solution is obtained when the usage amount of the replacement buffer is 6 times, 7 times, 8 times, 9 times, 10 times, etc. of the weight of the concentrated sample, and the usage amount is the volume usage amount.
  • the ultrafiltration replacement solution and the basic amino acid mother liquor are mixed so that the concentration of the amino acid can be, for example, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, etc.
  • the protein concentration is obtained such as It can be a concentrated solution of 100mg/ml, 110mg/ml, 120mg/ml, 130mg/ml, 140mg/ml, 150mg/ml, 160mg/ml, 170mg/ml, 180mg/ml, 190mg/ml, 200mg/ml, etc.
  • the solution containing the anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody is concentrated to a protein concentration of 30-50 mg/ml.
  • the present invention does not impose any limitation on the concentration of the replacement buffer, which can be selected by those skilled in the art according to needs, for example, the concentration of the replacement buffer is 5-50 mM, preferably 20 mM.
  • the present invention does not impose any limitation on the pH of the replacement buffer, as long as it is slightly acidic, for example, the pH of the replacement buffer is 6.0-7.0, preferably 6.2-6.8.
  • the concentration of the replacement buffer can be 5mM, 10mM, 20mM, 30mM, 40mM, 50mM, etc.
  • the pH of the replacement solution can be 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, etc. .
  • the replacement buffer is histidine-hydrochloric acid buffer, citrate buffer, phosphate buffer or sodium acetate-acetic acid buffer, preferably histidine-hydrochloric acid buffer.
  • the citric acid buffer is, for example, citric acid-sodium citrate buffer.
  • the phosphate buffer is, for example, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.
  • a peristaltic pump is used to pump the replacement buffer into the concentrated sample, and continue the ultrafiltration, while adjusting the pumping speed of the replacement buffer to be consistent with the permeation flow rate.
  • the pumping amount of the replacement buffer is 6% of the weight of the concentrated sample The replacement is completed at -10 times.
  • the basic amino acid is arginine hydrochloride, lysine and/or proline, preferably arginine hydrochloride.
  • the present invention does not make any limitation on the concentration of the basic amino acid mother liquor, and those skilled in the art can select according to needs.
  • the concentration of the basic amino acid mother liquor is 0.5-2.0mol/L, preferably 1 -1.5mol/L.
  • the concentration of the basic amino acid mother liquor can be 0.5 mol/L, 1.0 mol/L, 1.5 mol/L, 2.0 mol/L, etc.
  • the concentrated solution has a viscosity ⁇ 20 cP.
  • the viscosity of the concentrated solution may be, for example, 20 cP, 18 cP, 15 cP, 12 cP, 10 cP, 8 cP, 5 cP, 3 cP, 1 cP, etc.
  • the second ultrafiltration concentration calculate the protein concentration in the ultrafiltration replacement solution, and calculate the theoretical volume of the ultrafiltration concentrated solution, and add the basic amino acid mother liquor to mix, preferably, the volume of the basic amino acid mother liquor It is 1/9 of the theoretical volume of the ultrafiltration concentrated solution, so that the concentration of amino acids is 100-150mM, and after ultrafiltration and concentration, a concentrated solution with a protein concentration of 100-200mg/ml is obtained, and the viscosity of the concentrated solution is ⁇ 20cP.
  • the solution containing anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody is subjected to an affinity layer on a cell fermentation broth expressing anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody Analysis, low pH inactivation, anion chromatography, cation chromatography and nanofiltration.
  • IFNAR1 anti-human interferon alpha receptor 1
  • Mammals used in the production of the cell fermentation broth include, but are not limited to, various hybridoma cells and Chinese hamster ovary cells (CHO), preferably CHO cells.
  • the affinity chromatography uses Protein A for affinity chromatography.
  • the materials of the ultrafiltration membrane package used include but are not limited to improved polyethersulfone (PES), polyvinylidene fluoride (PVDF), cellulose acetate ( CA) etc., its pore size usually adopts 30kDa or 50kDa, preferably, the ultrafiltration membrane bag is the Pellicon2/Pellicon3 (A type screen mesh, 30kDa) of Merck Millipore, and the ultrafiltration membrane bag of Sartorius and Pall Company .
  • PES polyethersulfone
  • PVDF polyvinylidene fluoride
  • CA cellulose acetate
  • the ultrafiltration membrane bag is the Pellicon2/Pellicon3 (A type screen mesh, 30kDa) of Merck Millipore, and the ultrafiltration membrane bag of Sartorius and Pall Company .
  • the anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody is an IgG4 antibody, which blocks the activity of all type I interferons including IFN ⁇ , IFN ⁇ and IFN- ⁇ , and is intended to be used in For the treatment of systemic lupus erythematosus (SLE), it is intended to be administered intravenously or subcutaneously.
  • the protein concentration of this dosage form injection is as high as 150mg/ml.
  • the present invention uses the method described above to prepare a concentrated solution of anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody, which can reduce viscosity, avoid clogging, and at the same time inhibit protein aggregation and precipitation, and play the role of a protective agent.
  • IFNAR1 anti-human interferon alpha receptor 1
  • the method is simple and feasible, can be scaled up for production, can ensure high purity of the sample and obtain a high recovery rate.
  • the present invention provides a liquid preparation comprising anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody, said liquid preparation comprising anti-human interferon alpha receptor 1 (IFNAR1) with a protein concentration of 100-200 mg/ml Monoclonal antibodies and basic amino acids with an amino acid concentration of 5-300mM;
  • IFNAR1 anti-human interferon alpha receptor 1
  • the anti-IFNAR1 monoclonal antibody comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2 and CDR-L3), in:
  • CDR-H1 means heavy chain CDR1
  • SEQ ID NO: 1 The amino acid sequence of CDR-H1 (in this description, CDR-H1 means heavy chain CDR1) is shown in SEQ ID NO: 1;
  • CDR-H2 means heavy chain CDR2 in this description
  • SEQ ID NO: 2 The amino acid sequence of CDR-H2 (CDR-H2 means heavy chain CDR2 in this description) is shown in SEQ ID NO: 2;
  • CDR-L1 means light chain CDR1 in this specification
  • CDR-L2 means light chain CDR2 in this specification
  • SEQ ID NO:5 the amino acid sequence of CDR-L2 (CDR-L2 means light chain CDR2 in this specification) is as shown in SEQ ID NO:5;
  • CDR-L3 means light chain CDR3 in this description
  • SEQ ID NO:6 The amino acid sequence of CDR-L3 (CDR-L3 means light chain CDR3 in this description) is shown in SEQ ID NO:6.
  • the protein concentration can be 100 mg/ml, 110 mg/ml, 120 mg/ml, 130 mg/ml, 140 mg/ml, 150 mg/ml, 160 mg/ml, 170 mg/ml, 180 mg/ml, 190 mg/ml, 200 mg/ml, etc. ;
  • the amino acid concentration can be, for example, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, etc.
  • the viscosity of the liquid preparation can be controlled below 10cP, and can be easily injected with a syringe. Therefore, it can be used as an injection, especially a subcutaneous injection.
  • the basic amino acid is selected from one or more of arginine, lysine, histidine and proline.
  • the basic amino acid comprises 100-200mM arginine and 5-50mM histidine.
  • the basic amino acid may comprise arginine at 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, etc. and 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, Histidine at 40 mM, 45 mM, 50 mM, etc.
  • the basic amino acids are 20 mM histidine and 150 mM arginine.
  • the above-mentioned basic amino acid composition can greatly reduce the viscosity of the liquid preparation, for example, the viscosity is below 10 cp.
  • the basic amino acid comprises 100-200 mM lysine and 5-50 mM histidine.
  • the basic amino acid may comprise lysine at 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, etc. and 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, Histidine at 40 mM, 45 mM, 50 mM, etc.
  • the basic amino acids are 20 mM histidine and 150 mM lysine.
  • the above-mentioned basic amino acid composition can greatly reduce the viscosity of the liquid preparation, for example, the viscosity is below 10 cp.
  • the basic amino acid comprises 100-200 mM proline and 5-50 mM histidine.
  • the above-mentioned basic amino acid composition can greatly reduce the viscosity of the liquid preparation, for example, the viscosity is below 10 cp.
  • the basic amino acid may comprise 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM etc. proline and 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, Histidine at 40 mM, 45 mM, 50 mM, etc.
  • the basic amino acids are 20 mM histidine and 150 mM proline.
  • the above-mentioned basic amino acid composition can greatly reduce the viscosity of the liquid preparation, for example, the viscosity is below 10 cp.
  • the liquid formulation further comprises 20-150 mg/ml sucrose, preferably 50-100 mg/ml sucrose.
  • the liquid preparation may also contain 20mg/ml, 30mg/ml, 40mg/ml, 50mg/ml, 60mg/ml, 70mg/ml, 80mg/ml, 90mg/ml, 100mg/ml, 110mg/ml, 120mg /ml, 130mg/ml, 140mg/ml, 150mg/ml, etc. of sucrose.
  • the liquid formulation comprises 50 mg/ml sucrose.
  • the liquid formulation further comprises 0.5-50 mg/ml sorbitol, preferably 1-20 mg/ml sorbitol.
  • the liquid formulation may also contain 0.5 mg/ml, 1 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, etc. of sorbitol.
  • the liquid formulation comprises 70 mg/ml sucrose.
  • the liquid formulation further comprises 20-300 mg/ml sodium chloride, preferably 100-200 mg/ml sodium chloride.
  • the liquid preparation may also contain 20mg/ml, 30mg/ml, 40mg/ml, 50mg/ml, 60mg/ml, 70mg/ml, 80mg/ml, 90mg/ml, 100mg/ml, 110mg/ml, 120mg Sodium chloride of /ml, 130mg/ml, 140mg/ml, 150mg/ml, 160mg/ml, 170mg/ml, 180mg/ml, 190mg/ml, 200mg/ml, 250mg/ml, 300mg/ml, etc.
  • the liquid formulation comprises 150 mg/mL of sodium chloride, and the combination of the sodium chloride and histidine can control the viscosity of the liquid formulation below 10 cP.
  • the present invention does not impose any limitation on the pH of the liquid preparation, and those skilled in the art can choose according to needs.
  • the pH of the liquid preparation is 5.5-6.5.
  • the liquid preparation containing anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody is obtained by profiling cell fermentation broth expressing anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody And chromatography, low pH inactivation, anion chromatography, cation chromatography and ultrafiltration concentration, and then the concentrated solution obtained by ultrafiltration concentration is mixed with buffer solution or additive mother solution.
  • IFNAR1 anti-human interferon alpha receptor 1
  • Mammals used in the production of the cell fermentation broth include, but are not limited to, various hybridoma cells and Chinese hamster ovary cells (CHO), preferably CHO cells.
  • the affinity chromatography uses Protein A for affinity chromatography.
  • the anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody is an IgG4 antibody, which blocks the activity of all type I interferons including IFN ⁇ , IFN ⁇ and IFN- ⁇ , and is intended to be used in For the treatment of systemic lupus erythematosus (SLE), it is intended to be administered intravenously or subcutaneously.
  • the protein concentration of this dosage form injection is as high as 150mg/ml.
  • the liquid preparation of the present invention has low viscosity and can be easily injected by a syringe, so it is suitable for injection, especially subcutaneous injection.
  • the present invention generally and/or specifically describes the materials and test methods used in the test.
  • % means wt%, ie weight percentage.
  • the reagents or instruments used, whose manufacturers are not indicated, are all commercially available conventional reagent products.
  • CDR-H1 (SEQ ID No: 1), CDR-H2 (SEQ ID No: 2) and CDR-H3 (SEQ ID No: 3)) were grafted into the framework region of IGHV3-66*01; IGKV1-6*01 was selected as the light chain CDR grafting template, and the 1203#
  • the CDR region of the cloned light chain i.e.
  • CDR-L1 (SEQ ID No: 4), CDR-L2 (SEQ ID No: 5) and CDR-L3 (SEQ ID No: 6) was transplanted into IGKV1-6*01
  • the humanized heavy chain variable region sequence is shown in SEQ ID NO: 7; the humanized light chain variable region amino acid sequence is shown in SEQ ID NO: 8.
  • the above heavy chain variable region (SEQ ID NO: 7) gene and light chain variable region (SEQ ID NO: 8) gene were obtained by PCR amplification using the gene sequence of the 362# humanized antibody as a template.
  • the heavy chain expression plasmid pHZDCH was double-digested with HindIII and NheI; the light chain expression plasmid pHZDCK was double-digested with HindIII and BsiWI; the PCR amplified gene was inserted into the corresponding expression plasmid with Infusion recombinase to construct the heavy chain expression plasmid pHZDCH- 362VH-Hu6 and light chain expression plasmid pHZDCK-362VK-Hu20.
  • the gene of 1203# humanized antibody is numbered with 362, and the protein is numbered with 1203.
  • Figure 1 shows the result of the double enzyme digestion of the plasmid detected by nucleic acid electrophoresis. According to the results in Figure 1, it can be seen that the PCR amplification results of the variable region of the heavy chain and the variable region of the light chain of the antibody and the results of the expression plasmids of the heavy chain and the light chain of double digestion, wherein the plasmids of the heavy chain and the light chain have a size of about 10000bp, light chain variable region is about 447bp, heavy chain variable region is about 471bp.
  • Humanized antibody HZD1203-45 was obtained by humanizing 1203#. In order to reduce the ADCC effect of the antibody, the human IgG1 constant region of the HZD1203-45 heavy chain expression plasmid pHZDCH-362VH-Hu6 was replaced with human IgG4 to obtain the heavy chain expression plasmid pHZDCH-362VH-Hu6-IgG4.1.
  • the correct heavy chain expression plasmid pHZDCH-362VH-Hu6-IgG4.1 and the light chain expression plasmid pHZDCK-362VK-Hu20 were co-transfected into ExpiCHO-S cells.
  • ExpiCHO-S cells were diluted to 3 ⁇ 10 6 cells/ml for passage before transfection.
  • dilute the cell density to 6 ⁇ 10 6 cells/ml put 25ml cells in a 125ml shaker flask, and wait for transfection.
  • the transfection and expression process is shown in Figure 2.
  • the data in the table are: each sample was detected three times, and the average value was calculated.
  • HEK Blue IFN ⁇ / ⁇ reporter gene cell line to measure the phosphorylation activity of QX006N antagonizing interferon through IFNAR1-mediated intracellular signaling molecule STAT1/2: Add cells in the culture medium to 96 wells at 4 ⁇ 10 4 cells per well , and then cultured overnight at 37°C and 5% CO 2 . Serial dilutions of antibody concentrations ranging from 0 to 5 ⁇ g/ml were added to the cells along with 0.2 ng/ml of IFN ⁇ .2b.
  • the experimental results show that QX006N can inhibit the phosphorylation of STAT1/2 in HEK Blue IFN ⁇ / ⁇ cells induced by interferon, and inhibit 1/2 of interferon-induced HEK Blue IFN ⁇ / ⁇ cells
  • the IC 50 of phosphorylation activity is 5.23ng/ml
  • the IC 50 of Anifrolumab inhibits interferon-induced 1/2 phosphorylation activity in HEK Blue IFN ⁇ / ⁇ cells is 4.43ng/ml.
  • Daudi human lymphoma cell line to measure the cell proliferation activity of QX006N antagonizing interferon induced by IFNAR1: Add the cells in the culture medium to 96 wells at 4 ⁇ 10 4 cells per well, and then incubate at 37°C and 5% CO 2 Incubate overnight. Serial dilutions of antibody concentrations ranging from 0 to 20 ⁇ g/ml were added to the cells along with 0.8 ng/ml of IFN ⁇ .2b. Then cultured at 37°C and 5% CO 2 for 72 hours, collected cell cultures and used CellTiter-Glo to detect cell proliferation and draw a dose-effect curve, and then analyze the antagonistic activity of the antibody.
  • Example 5 QX006N and Anifrolumab neutralize human interferon-induced release of CXCL10/IP10 activity in whole blood.
  • Example 6 Different pre-elution buffers are compared for the removal effect of HCP in the fermentation broth of recombinant humanized anti-IFNAR1 monoclonal antibody (QX006N)
  • the antibody QX006N obtained in Example 1 was produced using CHO cells as host cells and Dynamis as the fermentation basal medium.
  • Use conventional cell culture technology for cell culture harvest when the cell viability is lower than 80% or cultured to 18 days, use primary filter MD0HC10FS1 and secondary filter MX0HC10FS1 to deep filter the harvested liquid, collect clarified cell culture supernatant, Recorded as fermentation broth intermediate.
  • the fermentation broth intermediate is subjected to affinity chromatography, and the method is as follows:
  • the composition of the intermediate pre-elution buffer is shown in Table 2, use the second equilibrium buffer (6mmol/L Na 2 HPO 4 , 4mmol/L NaH 2 PO 4 , pH7 .2) balancing;
  • the final elution is carried out to collect the samples, and the antibody concentration and host protein (HCP) residual content of the collected samples are determined, wherein the final elution buffer is 5mmol/L Na 2 HPO 4 , 6.5mmol/L citric acid , the pH was 3.6, and the chromatographic column was regenerated with a regeneration buffer of 100mmol/L NaOH and 1mol/L NaCl.
  • HCP host protein
  • the antibody concentration was determined as follows:
  • sample dilution factor is selected according to the estimated value of HCP in the sample, so that the final concentration of HCP falls within the range of the standard curve (generally 10ng/ml ⁇ 80ng/ml).
  • the dilution ratio of the sample in one step shall not exceed 10 times, and the minimum sampling volume shall not be less than 5 ⁇ l.
  • Sample loading add the standard, sample, and spiked sample in a certain arrangement (two replicate wells for each, and the standard product does not need multiple wells), 50 ⁇ l/well, and seal the plate. Place on a horizontal shaker at room temperature, 180 rpm, for 2 hours, protected from light.
  • wash the plate Discard the liquid in the wells, add 300 ⁇ l/well of washing solution with a multi-channel pipette, shake off the liquid after standing for 30 seconds, pat dry on absorbent paper, and wash the plate 4 times. After the last washing of the plate is completed, it is necessary to pat dry the residual washing solution in the well as much as possible.
  • CHO cell protein residue (%) sample average measured value (ng/ml) ⁇ dilution factor/undiluted sample protein content (mg/ml) amount obtains -4 (%), and its result is as shown in table 3 .
  • the residual amount of HCP in the fermentation broth intermediate is greater than 15%, and after the treatment of the affinity purification process, the yield is greater than 95%; for the removal of HCP, the method described above is adopted, after affinity chromatography
  • the residual HCP in the sample is less than 0.1%, and the HCP load in the subsequent purification process steps is significantly reduced, and the sample is adjusted by pH.
  • the sample is loaded in the subsequent chromatography process step (anion exchange chromatography)
  • the sample remains extremely clear and no other treatment is required. Direct sample injection enhances the simplicity of the process and saves process time.
  • the preparation of the fermentation broth intermediate and the affinity chromatography method were the same as in Example 6. Three batches of fermentation broth intermediates were prepared in total. The intermediate pre-elution and rinsing all used a pre-elution buffer containing guanidine hydrochloride: 0.1mol/ L sodium citrate, 0.1mol/L guanidine hydrochloride, 11mmol/L citric acid, pH5.8 rinse. Affinity process yield and HCP residual content testing methods are as described in Example 6, and the results are shown in Table 4.
  • the HCP residues in the intermediates of the QX006N fermentation broth of different batches were all above 15%, and the cell culture process was stable; the affinity process yields of the three batches of samples were all greater than 95%, and the yields met the process requirements;
  • the residues of HCP in the samples after chromatography and chromatography were all lower than 0.1%, and the removal of HCP by using guanidine hydrochloride for pre-elution and elution remained stable.
  • Example 1 Using CHO cells as host cells, fermentation was carried out in a 2L-scale bioreactor to produce the antibody QX006N obtained in Example 1. Obtain clarified fermentation broth through centrifuge or deep membrane bag filtration, use Protein A chromatography to capture the target protein, and then go through low pH virus inactivation, anion and cation chromatography to remove impurities, and perform nanofiltration to obtain anti-human interference IFNAR1 monoclonal antibody solution, the solution was concentrated using the following method:
  • the first ultrafiltration concentration the ultrafiltration equipment is the Labscale small ultrafiltration instrument of Merck Millipore (holding two 50cm 2 Pellicon XL membrane bags, the cut-off capacity is 30KD).
  • IFNAR1 anti-human interferon alpha receptor 1
  • the replacement solution is 20mM histidine-hydrochloric acid buffer solution, the pH value is 6.5, use a peristaltic pump to pump the histidine-hydrochloric acid buffer solution into the concentrated sample, continue ultrafiltration, and simultaneously Adjust the pumping speed of the histidine-hydrochloric acid buffer to be consistent with the permeation flow rate, that is, keep the weight of the sample constant, and when the volume of the histidine-hydrochloric acid buffer is 7 times the weight of the concentrated sample, the replacement is completed to obtain ultrafiltration Replacement solution.
  • the second ultrafiltration concentration take a sample to measure the concentration of protein in the ultrafiltration replacement solution, and calculate the theoretical volume of the ultrafiltration replacement solution at this time, and add 1/9 of the theoretical volume of the basic amino acid mother liquor, mix well, so that the buffer
  • the system is as shown in Table 5, and then use Merck Millipore's 30kDa ultrafiltration centrifuge tube to concentrate until the protein concentration is the concentration in Table 5 to obtain a concentrated solution.
  • the viscosity values of the concentrated solutions are measured respectively.
  • the viscosity value measurement method is as follows:
  • the viscosity of the anti-human IFNAR1 antibody increases exponentially. If no additives are added, the viscosity of the monoclonal antibody will reach 42.1cP when the concentration is 200mg/ml, which is so high The viscosity value cannot be achieved using conventional ultrafiltration membranes for concentration processes. Taking the protein concentration of 150mg/ml as an example, by adding 150mM basic amino acid or its combination, the antibody viscosity value under the same protein concentration can be reduced to below 50% of the original value, less than 20cP. Based on the above experimental results, the administration concentration of the anti-human IFNAR1 monoclonal antibody can be 100-150 mg/ml.
  • the second ultrafiltration concentration divide the ultrafiltration replacement solution into two parts and carry out the second step concentration process respectively:
  • the second part is directly concentrated by the first ultrafiltration concentration method, and the protein concentration is 150mg/ml, which is recorded as concentrated solution 2 (U2F2);
  • sample analysis method setting the flow rate is 1.0ml/min, the analysis time is 15min, the sample injection volume is 50mg; the detection wavelength is 280nm; the system suitability must be checked before sample injection (use the reference sample for continuous sample injection, the number of sample injections Not less than 5 times, take the results of the last 5 times for calculation), then analyze a blank sample, and then analyze the sample.
  • Example 8 was further scaled up to a 200L pilot scale, using an Merck Millipore Pellicon manual ultrafiltration system to clamp two 0.5m2 Pellicon 2 membrane cassettes.
  • the solution (UF0) containing the anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody after nanofiltration is controlled at 0.6-1.5bar at a flow rate of 120-300L/m 2 h, according to the method of Example 8.
  • Table 7 Summary of results of pilot scale-up confirmation of anti-human IFNAR1 monoclonal antibody ultrafiltration process
  • Embodiment 11 comprises the comparison of the liquid formulation viscosity of anti-human IFNAR1 monoclonal antibody and different basic amino acids
  • the antibody QX006N described in Example 1 was fermented and fermented in a 2L-scale bioreactor, and the clarified fermentation broth was obtained by centrifuge or deep membrane bag filtration, and the target protein was analyzed by Protein A chromatography. capture, and then go through low pH virus inactivation, anion and cation chromatography to remove impurities, and perform ultrafiltration and concentration to obtain a concentrated solution containing anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody. The obtained concentrated solution Contains 20mM His-HCL.
  • IFNAR1 anti-human interferon alpha receptor 1
  • the obtained concentrated solution is analyzed by SEC-HPLC method, and its operation steps are as follows:
  • sample analysis method setting the flow rate is 1.0ml/min, the analysis time is 15min, the sample injection volume is 50mg; the detection wavelength is 280nm; the system suitability must be checked before sample injection (use the reference sample for continuous sample injection, the number of sample injections Not less than 5 times, take the results of the last 5 times for calculation), then analyze a blank sample, and then analyze the sample.
  • the concentration of QX006N in the concentrated solution was determined to be 200 mg/mL.
  • the viscosity of the concentrated solution is measured by the ⁇ VISC viscometer of Rui Osen, and the operation steps are as follows:
  • the sample was filtered with a 0.2 ⁇ m filter membrane, it was mixed with different buffer solutions or additive mother solutions in Table 8 to make the protein concentration as shown in Table 8, and then the viscosity value was measured according to the above method, and the results are shown in Table 8.
  • liquid preparations with a viscosity of less than 30cP are suitable for subcutaneous injections.
  • the results in Table 8 show that for the liquid preparations of the present invention, the concentration of QX006N for subcutaneous injections can at least reach 100-180mg/mL or 150-180mg/mL. mL.
  • the present invention adopts the above-mentioned affinity purification method, and the residual amount of HCP in the obtained monoclonal antibody is kept at a low level, and the residual control amount is not higher than 0.1%, thereby reducing the subsequent purification steps
  • the pressure of removing HCP in the medium, and the affinity purification process of the present invention has good stability.
  • the method of the present invention can prepare the concentrated solution of high concentration, low viscosity anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody, when protein concentration is 100-200mg/ml, viscosity is controlled below 20cP, so The obtained concentrated solution can be used to prepare anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody subcutaneous injection.
  • IFNAR1 anti-human interferon alpha receptor 1
  • the liquid formulation of the present invention has low viscosity, and the liquid formulation is suitable for injection, especially subcutaneous injection.
  • the anti-human interferon alpha receptor 1 (IFNAR1) monoclonal antibody which is different from the existing Compared with human interferon ⁇ receptor 1 monoclonal antibody (Anifrolumab), the binding affinity to IFNAR1 is equivalent, and the neutralizing activity at the cellular level is equivalent to that of Anifrolumab.
  • the monoclonal antibody has shown neutralizing activity comparable to that of Anifrolumab (expressed and prepared according to the patent disclosed sequence) at the cellular level, and it is expected to show good clinical effects in the prevention and treatment of related diseases.

Abstract

提供了一种降低单克隆抗体生产中宿主细胞蛋白含量的亲和纯化方法以及一种抗人IFNAR1单克隆抗体浓缩溶液的制备方法和包含抗人干扰素α受体1（IFNAR1）单克隆抗体的液体制剂。所述亲和方法简单易行，能够进行放大纯化生产，细胞发酵上清液无需进行前预处理，洗脱样品得率较高，同时HCP残留量也保持在较低水平（残留控制量不高于0.1％），从而减轻之后纯化步骤中去除HCP的压力；所述的制备方法能够降低高浓度抗体药液的粘度并提高稳定性，方法简单易行，能够进行放大生产，可以保证样品的高纯度并获得较高的回收率；所述液体制剂粘度较低，可用注射器轻松推注，因此可以用作注射剂，尤其是皮下注射剂。

Description

降低单克隆抗体生产中宿主细胞蛋白含量的亲和纯化方法、抗人IFNAR1单克隆抗体浓缩溶液的制备方法以及液体制剂 技术领域
本发明涉及生物技术领域，尤其涉及一种降低抗人干扰素α受体1单克隆抗体生产中宿主细胞蛋白含量的亲和纯化方法、抗人IFNAR1单克隆抗体浓缩溶液的制备方法以及液体制剂。
背景技术
抗体药物生产过程中亲和纯化是非常关键的一个工艺步骤，该过程对发酵液中的抗体进行捕获浓缩，实现抗体粗纯化的第一步。在中国仓鼠卵巢细胞(CHO)等基因工程细胞株大批量发酵过程中，细胞在不同的生理周期会有凋亡裂解，释放宿主细胞蛋白(Host cell protein，HCP)。HCP是指来源于宿主细胞的蛋白成分，包括宿主细胞结构蛋白和转化蛋白(细胞分泌的促生长蛋白)。HCP不仅有可能诱导机体产生抗HCP抗体，引起过敏反应，还有可能有“佐剂效应”引起机体对蛋白质药物产生抗体，影响药物治疗效果，定量测定基因工程药物中残留的HCP是质量控制的一种重要手段，有助于保持纯化工艺的有效性和一致性。在抗体亲和纯化中，保证高效率的抗体回收率的同时，要对发酵过程中工程细胞所产生的HCP进行有效去除。因此，研究出一种能广泛应用于抗体大规模发酵后较为经济可行的抗体亲和纯化工艺，对于抗体药物的进一步产业化推广是极其有意义的。目前能够进行HCP残留去除的方法较多，各有特点：1)层析方式：包括Protein A亲和层析，阴、阳离子层析，三种层析工艺中对HCP的去除能力，Protein A亲和层析为基础，去除能力较强，是HCP去除的主要步骤，阴、阳离子层析则主要作为后续HCP的进一步再去除工艺。层析方式去除HCP的工艺在不断完善过程中，各种层析工艺在不断的提出应用实际生产，是HCP去除的主要手段；2)切向流超滤方式：对于HCP的去除能力有限，且不易控制残留量，工艺控制系数不高，只能作为去除HCP的辅助工艺；3)聚合物沉淀方式：PEG、聚丙烯酸等高聚合物在较广的pH范围内带正电荷，通过电荷作 用同抗体结合形成沉淀，而HCP由于等电点较低，不易发生沉淀。沉淀抗体样品的HCP含量明显降低，但该工艺不适于工艺放大，且沉淀对抗体的活性可能有一定影响。因此，去除HCP的工艺方法众多，由于各项目的抗体的样品发酵工艺及抗体性质的不同，需进行综合考虑选择工艺。
生物制剂市场作为一个快速增长的市场，拥有众多的研发管线，同时为患者带来更多创新疗法，针对一些传统化药较难治疗的疾病，生物制剂靶向药物提供了更多的选择。另一方面为了降低生物制剂的临床使用成本，并提高患者的依从性，生物制剂剂型逐渐由冻干剂型向水针剂型转变，由静脉给药方式向皮下注射剂型转变。由于单克隆抗体注射液的给药剂量通常在100mg～600mg范围，而皮下注射药液体积一般限制在2ml以下，在所述情况下，必须制备高度浓缩的蛋白制剂，通常蛋白含量可达到100mg/ml或者更大的浓度。
高浓度的单克隆抗体注射液给生产工艺的可制造、工艺放大、及最终的患者施用带来了许多挑战。最主要的一个挑战是超高的粘度，由于单克隆抗体的生物高分子特性，以及蛋白分子间相互作用力(如疏水、电荷作用等)在高浓度条件时增强，倾向于形成高粘性溶液。在某些极端情况下甚至会形成凝胶状物质，这对超滤膜和超滤设备本身带来不小的挑战，比如最终浓缩时压差的急速上升导致的切向流速减小，浓差极化逐渐失控，直至出现蛋白沉淀将膜堵塞的现象，如此必然会造成回收率降低或工艺失败。另一方面，即使通过改进设备或膜包类型获得最终的高浓度蛋白溶液，也难以将其投入到实际的临床应用中，因为皮下给药时需要使用一次性无菌注射器吸取或者采用预充针的最终包装形式，而过高的粘度将导致装药注射器的滑动性能降低，从而无法手动推注入皮下。超滤浓缩高浓度单克隆抗体药液的另一个难题是在高度浓缩时蛋白样品容易聚集形成可溶性聚体，进一步会聚集形成蛋白沉淀。
因此，针对高浓度的抗人IFNAR1单克隆抗体皮下注射剂的制备，需要开发一种能够有效降低超滤浓缩液的粘度、减少单克隆抗体聚集并提高其稳定性的超滤浓缩制备方法以及液体制剂。
发明内容
为了解决抗体生产中宿主细胞蛋白(HCP)残留问题，本发明提供了一种抗体纯化生产中有效降低CHO宿主细胞蛋白(HCP)的新方法，可以广泛适用于抗体亲和纯化工艺中，且所使用的材料***格较低，易于工艺放大。在本发明中，主要通过抗体亲和层析中间预洗脱淋洗来有效降低HCP含量，满足抗体药物的大规模高质量纯化制备要求，保证抗体药物的临床使用的安全。
为了解决现有技术中存在的超滤浓缩液的粘度、单克隆抗体易于聚集以及超滤浓缩液稳定性低的问题，本发明提供了一种抗人干扰素α受体1(IFNAR1)单克隆抗体浓缩溶液的制备方法，所述方法通过超滤浓缩制备包含抗人IFNAR1单克隆抗体的超滤浓缩液体，并能够有效降低超滤浓缩液的粘度、减少抗人IFNAR1单克隆抗体聚集并提高其稳定性。
此外，本发明还提供了一种包含抗人干扰素α受体1(IFNAR1)单克隆抗体的液体制剂，所述液体制剂包含蛋白质浓度为100-200mg/ml的抗人干扰素α受体1(IFNAR1)单克隆抗体以及氨基酸浓度为5-300mM的碱性氨基酸，所述的液体制剂能够有效降低超滤浓缩液的粘度、减少抗人IFNAR1单克隆抗体聚集并提高其稳定性，可以用于注射剂，尤其是皮下注射剂。
抗人干扰素α受体1(IFNAR1)单克隆抗体(QX006N)为自主研发的靶向人I型干扰素素α/β受体1的重组人源化单克隆抗体，阻断包括IFNα、IFNβ和IFN-ω在内的所有I型干扰素的活性，拟用于***性红斑狼疮(SLE)的治疗，拟采用静脉或皮下注射方式给药。目前国内外尚未有上市的靶向人干扰素α/β受体1(IFNAR1)单克隆抗体药物，阿斯利康开发的Anifrolumab进度最快，目前正在进行3期临床研究，其制剂规格为150mg/1ml，拟给药方式为静脉或皮下注射。体外药效学研究表明QX006N生物学活性与Anifrolumab相当。该新的抗人I型干扰素素α/β受体1单克隆抗体制备成皮下注射液，则该皮下注射液中的蛋白含量要高达100～150mg/mL。
本发明具体技术方案如下：
1.一种降低单克隆抗体生产中宿主细胞蛋白含量的亲和纯化方法，其包括下述步骤：
使用第一平衡缓冲液平衡亲和层析介质得到平衡好的亲和层析介质，并将单克隆抗体发酵液与所述平衡好的亲和层析介质结合；
然后进行中间预洗脱淋洗，接着进行终洗脱以除去宿主细胞蛋白得到单 克隆抗体；
所述单克隆抗体为分离的抗人干扰素α受体1(IFNAR1)单克隆抗体，其包含三个重链互补决定区(CDR-H1、CDR-H2和CDR-H3)和三个轻链互补决定区(CDR-L1、CDR-L2和CDR-L3)，其中：
(a)CDR-H1的氨基酸序列如SEQ ID NO：1所示；
(b)CDR-H2的氨基酸序列如SEQ ID NO：2所示；
(c)CDR-H3的氨基酸序列如SEQ ID NO：3所示；
(d)CDR-L1的氨基酸序列如SEQ ID NO：4所示；
(e)CDR-L2的氨基酸序列如SEQ ID NO：5所示；且
(f)CDR-L3的氨基酸序列如SEQ ID NO：6所示。
2.根据项1所述的亲和纯化方法，其中，所述抗人干扰素α受体1(IFNAR1)单克隆抗体包含重链可变区和轻链可变区，其中，
所述重链可变区的氨基酸序列如SEQ ID NO：7所示；且，
所述轻链可变区的氨基酸序列如SEQ ID NO：8所示。
3.根据项2所述的亲和纯化方法，其中，所述抗人干扰素α受体1(IFNAR1)单克隆抗体的重链的氨基酸序列如SEQ ID NO：10所示；轻链的氨基酸序列如SEQ ID NO：11所示。
4.根据项1-3中任一项所述的亲和纯化方法，其中，所述亲和层析介质为配基交联到琼脂糖、聚乙烯醚、羟基化聚醚树脂、聚丙烯酸树脂、聚苯乙烯二乙烯苯基树脂、聚甲基丙烯酸树脂、聚苯乙烯树脂、羟基磷灰石或玻璃基质上的层析介质，优选的，所述亲和层析介质为配基交联到聚乙烯醚的层析介质；
优选的，所述配基为Protein A、Protein G或Protein L，优选为Protein A。
5.根据项1-4中任一项所述的亲和纯化方法，其中，所述第一平衡缓冲液为磷酸盐缓冲液、Tris-HCl缓冲液或硼酸-硼砂缓冲液，所述第一平衡缓冲液中的盐浓度为5mM-0.25M，pH为5.5-8.0。
6.根据项1-5中任一项所述的亲和纯化方法，其中，中间预洗脱缓冲液为中性缓冲液和/酸性缓冲液；优选的，所述中性缓冲液为磷酸盐缓冲液、三羟甲基氨基甲烷缓冲液或甘氨酸缓冲液；所述酸性缓冲液为柠檬酸-磷酸氢二钠缓冲液、醋酸-醋酸钠缓冲液或柠檬酸-柠檬酸三钠缓冲液。
7.根据项6所述的亲和纯化方法，其中，所述中间预洗脱缓冲液的pH 为5.0-7.5。
8.根据项6所述的亲和纯化方法，其中，在所述中间预洗脱缓冲液中加入预洗脱活性剂，优选的，所述预洗脱活性剂为盐酸胍、聚山梨酯80或氯化钠，进一步优选的，所述预洗脱活性剂为盐酸胍。
9.根据项8所述的亲和纯化方法，其中，所述盐酸胍的浓度为0.01-1M。
10.根据项1-9中任一项所述的亲和纯化方法，其中，在中间预洗脱淋洗后，在进行终洗脱之前还包括下述步骤：
使用第二平衡缓冲液进行平衡，优选的，所述第二平衡缓冲液为磷酸盐缓冲液、Tris-HCl缓冲液或硼酸-硼砂缓冲液。
11.根据项1-10中任一项所述的亲和纯化方法，其中，终洗脱缓冲液选自柠檬酸-磷酸氢二钠缓冲液、醋酸缓冲液、甘氨酸-HCl缓冲液和柠檬酸-柠檬酸钠缓冲液中的一种或两种以上，优选为柠檬酸-磷酸氢二钠缓冲液。
12.根据项11所述的亲和纯化方法，其中，所述柠檬酸-磷酸氢二钠缓冲液的pH为2.0-7.0。
13.一种抗人干扰素α受体1(IFNAR1)单克隆抗体浓缩溶液的制备方法，其包括下述步骤：
第一超滤浓缩：在流速为120-300L/m 2·h和跨膜压差(TMP)为0.6-1.5bar的条件下将含有抗人干扰素α受体1(IFNAR1)单克隆抗体的溶液浓缩至蛋白质浓度为20-60mg/ml，得到浓缩样品；
超滤置换：采用置换缓冲液置换浓缩样品，当置换缓冲液的使用量为浓缩样品重量的6-10倍时得到超滤置换溶液；
第二超滤浓缩：将超滤置换溶液和碱性氨基酸母液混匀使得氨基酸的浓度为100-150mM，进行超滤浓缩后得到蛋白质浓度为100-200mg/ml的浓缩溶液；
所述抗IFNAR1单克隆抗体包含三个重链互补决定区(CDR-H1、CDR-H2和CDR-H3)和三个轻链互补决定区(CDR-L1、CDR-L2和CDR-L3)，其中：
(a)CDR-H1的氨基酸序列如SEQ ID NO：1所示；
(b)CDR-H2的氨基酸序列如SEQ ID NO：2所示；
(c)CDR-H3的氨基酸序列如SEQ ID NO：3所示；
(d)CDR-L1的氨基酸序列如SEQ ID NO：4所示；
(e)CDR-L2的氨基酸序列如SEQ ID NO：5所示；且
(f)CDR-L3的氨基酸序列如SEQ ID NO：6所示。
14.根据项13所述的制备方法，其中，所述抗人干扰素α受体1(IFNAR1)单克隆抗体包含重链可变区和轻链可变区，其中，
所述重链可变区的氨基酸序列如SEQ ID NO：7所示；且，
所述轻链可变区的氨基酸序列如SEQ ID NO：8所示。
15.根据项14所述的制备方法，其中，所述抗人干扰素α受体1(IFNAR1)单克隆抗体的重链的氨基酸序列如SEQ ID NO：10所示；轻链的氨基酸序列如SEQ ID NO：11所示。
16.根据项13-15中任一项所述的制备方法，其中，在第一超滤浓缩中，将含有抗人干扰素α受体1(IFNAR1)单克隆抗体的溶液浓缩至蛋白质浓度为30-50mg/ml。
17.根据项13-16中任一项所述的制备方法，其中，在超滤置换中，置换缓冲液的浓度为5-50mM，优选为20mM；优选的，置换缓冲液的pH为6.0-7.0，优选为6.2-6.8。
18.根据项17所述的制备方法，其中，所述置换缓冲液为组氨酸-盐酸缓冲液、柠檬酸缓冲液、磷酸盐缓冲液或醋酸钠-醋酸缓冲液，优选为组氨酸-盐酸缓冲液。
19.根据项13-18中任一项所述的制备方法，其中，在第二超滤浓缩中，所述碱性氨基酸为盐酸精氨酸、赖氨酸和/或脯氨酸，优选为盐酸精氨酸。
20.根据项13-19中任一项所述的制备方法，其中，所述碱性氨基酸母液的浓度为0.5-2.0mol/L，优选为1mol/L～1.5mol/L。
21.根据项13-20中任一项所述的制备方法，其中，所述浓缩溶液的粘度≤20cP。
22.根据项13-21中任一项所述的制备方法，其中，所述含有抗人干扰素α受体1(IFNAR1)单克隆抗体的溶液通过对表达抗人干扰素α受体1(IFNAR1)单克隆抗体的细胞发酵液进行亲和层析、低pH灭活、阴离子层析、阳离子层析和纳滤得到。
23.一种包含抗人干扰素α受体1(IFNAR1)单克隆抗体的液体制剂，所述液体制剂包含蛋白质浓度为100-200mg/ml的抗人干扰素α受体1(IFNAR1)单克隆抗体以及氨基酸浓度为5-300mM的碱性氨基酸；
所述抗IFNAR1单克隆抗体包含三个重链互补决定区(CDR-H1、CDR-H2和CDR-H3)和三个轻链互补决定区(CDR-L1、CDR-L2和CDR-L3)，其中：
(a)CDR-H1的氨基酸序列如SEQ ID NO：1所示；
(b)CDR-H2的氨基酸序列如SEQ ID NO：2所示；
(c)CDR-H3的氨基酸序列如SEQ ID NO：3所示；
(d)CDR-L1的氨基酸序列如SEQ ID NO：4所示；
(e)CDR-L2的氨基酸序列如SEQ ID NO：5所示；且
(f)CDR-L3的氨基酸序列如SEQ ID NO：6所示。
24.根据项23所述的液体制剂，其中，所述抗人干扰素α受体1(IFNAR1)单克隆抗体包含重链可变区和轻链可变区，其中，
所述重链可变区的氨基酸序列如SEQ ID NO：7所示；且，
所述轻链可变区的氨基酸序列如SEQ ID NO：8所示。
25.根据项24所述的液体制剂，其中，所述抗人干扰素α受体1(IFNAR1)单克隆抗体的重链的氨基酸序列如SEQ ID NO：10所示；轻链的氨基酸序列如SEQ ID NO：11所示。
26.根据项23-25中任一项所述的液体制剂，其中，所述碱性氨基酸选自精氨酸、赖氨酸、组氨酸和脯氨酸中的一种或两种以上。
27.根据项26所述的液体制剂，其中，所述碱性氨基酸包含100-200mM的精氨酸和5-50mM的组氨酸。
28.根据项26所述的液体制剂，其中，所述碱性氨基酸包含100-200mM的赖氨酸和5-50mM的组氨酸。
29.根据项26所述的液体制剂，其中，所述碱性氨基酸包含100-200mM的脯氨酸和5-50mM的组氨酸。
30.根据项23-29中任一项所述的液体制剂，其中，所述液体制剂还包含20-150mg/ml的蔗糖，优选为50-100mg/ml的蔗糖。
31.根据项23-26中任一项所述的液体制剂，其中，所述液体制剂还包含0.5-50mg/ml的山梨醇，优选为1-20mg/ml的山梨醇。
32.根据项23-26中任一项所述的液体制剂，其中，所述液体制剂还包含20-300mg/ml的氯化钠，优选为100-200mg/ml的氯化钠。
33.根据项23-32中任一项所述的液体制剂，其中，所述液体制剂的pH 为5.5～6.5。
发明的效果
本发明所述的亲和工艺简单易行，能够进行放大纯化生产，细胞发酵上清液无需进行前预处理，洗脱样品得率较高，同时HCP残留量也保持在较低水平(残留控制量不高于0.1％)，从而减轻之后纯化步骤中去除HCP的压力，从而保证抗体最终样品的HCP残留量处于极低水平。同时，发明中经过对不同批次发酵上清液进行亲和纯化工艺验证，证明本发明中的亲和纯化工艺具有良好的稳定性。
本发明所述的抗人干扰素α受体1(IFNAR1)单克隆抗体浓缩溶液的制备方法，能够降低高浓度抗体药液的粘度并提高稳定性，方法简单易行，能够进行放大生产，可以保证样品的高纯度并获得较高的回收率。同时，发明中经过对不同亚型的高浓度抗体进行验证，证明本发明中的添加碱性氨基酸降低粘度的方法具有较强的适用性及良好的稳定性。
本发明所述的液体制剂，其包含抗人干扰素α受体1(IFNAR1)单克隆抗体，并且所述的液体制剂的粘度较低，可用注射器轻松推注，因此可以用作注射剂，尤其是皮下注射剂。
所述的抗人干扰素α受体1(IFNAR1)单克隆抗体，其与现有的抗人干扰素α受体1单克隆抗体(Anifrolumab)相比，结合IFNAR1的亲和力相当，在细胞水平的中和活性与Anifrolumab相当。
所述的单克隆抗体在细胞水平显示出与Anifrolumab(根据专利公开序列表达制备)相当的中和活性，其有望在预防和治疗相关疾病方面展现出良好的临床效果。
附图说明
图1是显示构建HZD1203-45瞬转表达质粒的核酸电泳结果的图。其中，M：Marker；条带1：PCR产物362VH-Hu6；条带2：pHZDCH，HindIII/NheI；条带3：PCR产物362VK-Hu20；条带4：pHZDCK，HindIII/BsiWI。
图2是瞬转表达流程图。
图3是QX006N(HZD1203-45-IgG4.1)的电泳检测图。
具体实施方式
下面结合附图对本发明做以详细说明，其中所有附图中相同的数字表示相同的特征。虽然附图中显示了本发明的具体实施例，然而应当理解，可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
需要说明的是，在说明书及权利要求当中使用了某些词汇来指称特定组件。本领域技术人员应可以理解，技术人员可能会用不同名词来称呼同一个组件。本说明书及权利要求并不以名词的差异作为区分组件的方式，而是以组件在功能上的差异作为区分的准则。如在通篇说明书及权利要求当中所提及的“包含”或“包括”为开放式用语，故应解释成“包含但不限定于”。说明书后续描述为实施本发明的较佳实施方式，然而所述描述乃以说明书的一般原则为目的，并非用以限定本发明的范围。本发明的保护范围当视所附权利要求所界定者为准。
本发明提供了一种降低单克隆抗体生产中宿主细胞蛋白含量的亲和纯化方法，其包括下述步骤：
使用第一平衡缓冲液平衡亲和层析介质得到平衡好的亲和层析介质，并将单克隆抗体发酵液与所述平衡好的亲和层析介质结合；
然后进行中间预洗脱淋洗，接着进行终洗脱以除去宿主细胞蛋白得到单克隆抗体；
所述单克隆抗体为分离的抗人干扰素α受体1(IFNAR1)单克隆抗体，其包含三个重链互补决定区(CDR-H1、CDR-H2和CDR-H3)和三个轻链互补决定区(CDR-L1、CDR-L2和CDR-L3)，其中：
(a)CDR-H1(在本说明书中CDR-H1表示重链CDR1)的氨基酸序列如SEQ ID NO：1所示；
(b)CDR-H2(在本说明书中CDR-H2表示重链CDR2)的氨基酸序列如SEQ ID NO：2所示；
(c)CDR-H3(在本说明书中CDR-H3表示重链CDR3)的氨基酸序列如SEQ ID NO：3所示；
(d)CDR-L1(在本说明书中CDR-L1表示轻链CDR1)的氨基酸序列如SEQ ID NO：4所示；
(e)CDR-L2(在本说明书中CDR-L2表示轻链CDR2)的氨基酸序列如SEQ ID NO：5所示；且
(f)CDR-L3(在本说明书中CDR-L3表示轻链CDR3)的氨基酸序列如SEQ ID NO：6所示。
其中，SEQ ID NO：1的氨基酸序列如下所示：
SYYMT
SEQ ID NO：2的氨基酸序列如下所示：
VINVYGGTYYASWAKG
SEQ ID NO：3的氨基酸序列如下所示：
EDVAVYMAIDL
SEQ ID NO：4的氨基酸序列如下所示：
QASQSISNQLS
SEQ ID NO：5的氨基酸序列如下所示：
DASSLAS
SEQ ID NO：6的氨基酸序列如下所示：
LGIYGDGADDGIA
所述单克隆抗体表示得自基本上同源的抗体的群体的抗体，即，构成所述群体的各个抗体是相同的和/或结合相同表位，除了可能的变体抗体(例如，含有天然存在的突变或在单克隆抗体制品的生产过程中产生)以外，这样的变体通常以微量存在。与通常包括针对不同决定簇(表位)的不同抗体的多克隆抗体制品不同，单克隆抗体制品的每种单克隆抗体针对抗原上的单个决定簇。因而，修饰语“单克隆”指示所述抗体得自基本上同源的抗体群体的特征，并且不应解释为需要通过任何特定方法生产所述抗体。例如，要根据本发明使用的单克隆抗体可以通过多种技术来制备，所述技术包括、但不限于杂交瘤方法、重组DNA方法、噬菌体展示方法、和使用包含人免疫球蛋白基因座的全部或部分的转基因动物的方法，本文描述了这样的方法和其它示例性的制备单克隆抗体的方法。
所述宿主细胞表示其中已经引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括原代转化的细胞和由其来源的后代(不考虑传代数)。后代在核酸内容物方面可以与亲本细胞不完全相同，但是可以含有突变。针对最初转化的细胞筛选或选择的具有相同功能 或生物活性的突变体后代被包括在本说明书中。
所述宿主细胞蛋白(HCP)是与过程相关的杂质，由用于生产生物药物蛋白的宿主细胞表达。在纯化过程中，大部分的HCP被去除(>99％)，但残留的HCP量仍保留在分布的产品中，例如单克隆抗体(mAb)、抗体-药物结合物(ADC)、治疗性蛋白质、疫苗，以及其他基于蛋白质的生物制药。
所述抗人干扰素α受体1(IFNAR1)单克隆抗体表示这样的单克隆抗体：其能够以足够的亲和力结合人干扰素α受体1，使得所述单克隆抗体可用作靶向人干扰素α受体1的诊断剂和/或治疗剂。
本发明的抗人干扰素α受体1(IFNAR1)单克隆抗体与靶标无关的蛋白不结合。这里，“无关的蛋白”是指除作为靶标的人干扰素α受体1以外的其他蛋白；这里，“不结合”是指：在将本发明的抗人干扰素α受体1(IFNAR1)单克隆抗体与作为其靶标的人干扰素α受体1的结合能力作为100％的情况下，本发明的抗人干扰素α受体1单克隆抗体与所述无关蛋白的结合能力小于10％，例如9％、8％、7％、6％、5％、4％、3％、2％、1％或者0。
本发明所述的抗人干扰素α受体1(IFNAR1)单克隆抗体与其他动物种属的干扰素α受体1可以不结合。这里，“其他动物种属”是指除人以外的其他动物种属，例如狨猴、食蟹猴、猪、犬、兔、大鼠、小鼠、豚鼠等；这里，“不结合”是指：在将本发明的抗人干扰素α受体1(IFNAR1)单克隆抗体与作为其靶标的人干扰素α受体1的结合能力作为100％的情况下，本发明的抗人干扰素α受体1(IFNAR1)单克隆抗体与其他动物种属的干扰素α受体1的结合能力小于10％，例如9％、8％、7％、6％、5％、4％、3％、2％、1％或者0。
本发明所述的抗人干扰素α受体1单克隆抗体具有≤1μM、≤100nM、≤50nM、≤40nM的平衡解离常数(KD)。
所述抗人干扰素α受体1(Human Interferon alpha/beta Receptor 1，IFNAR1)表示一种源自人的膜蛋白，其胞外区氨基酸序列如SEQ ID NO：9所示，其中，下划线部分表示信号肽。
SEQ ID NO：9：
Figure PCTCN2021114952-appb-000001
Figure PCTCN2021114952-appb-000002
本发明通过使用上述方法进行亲和纯化，能够使单克隆抗体的宿主细胞蛋白的残留量处于极低的水平。
在一个实施方案中，所述抗人干扰素α受体1(IFNAR1)单克隆抗体包含重链可变区和轻链可变区，其中，
所述重链可变区的氨基酸序列如SEQ ID NO：7所示；且，
所述轻链可变区的氨基酸序列如SEQ ID NO：8所示。
其中，SEQ ID NO：7的氨基酸序列如下所示：
Figure PCTCN2021114952-appb-000003
SEQ ID NO：8的氨基酸序列如下所示：
Figure PCTCN2021114952-appb-000004
在一个实施方案中，所述抗人干扰素α受体1(IFNAR1)单克隆抗体的重链的氨基酸序列如SEQ ID NO：10所示；轻链的氨基酸序列如SEQ ID NO：11所示。
其中，SEQ ID NO：10的氨基酸序列如下所示：
Figure PCTCN2021114952-appb-000005
Figure PCTCN2021114952-appb-000006
SEQ ID NO：11的氨基酸序列如下所示：
Figure PCTCN2021114952-appb-000007
其中，SEQ ID NO：10和11均为经人源化的序列。
在一个实施方案中，所述亲和层析介质为配基交联到琼脂糖、聚乙烯醚、羟基化聚醚树脂、聚丙烯酸树脂、聚苯乙烯二乙烯苯基树脂、聚甲基丙烯酸树脂、聚苯乙烯树脂、羟基磷灰石或玻璃基质上的层析介质，优选的，所述亲和层析介质为配基交联到聚乙烯醚的层析介质；
优选的，所述配基为Protein A、Protein G或Protein L，优选为Protein A。
所述配基可与单克隆抗体特异性地结合。
本发明对亲和填料没有限制，其可以根据本领域技术人员的需要进行确认，例如，亲和填料可以为GE healthcare的Mabselect、Mabselect Sure，博格隆生物技术有限公司的Protein A Diamond、MERCK的
Figure PCTCN2021114952-appb-000008
在一个实施方案中，所述第一平衡缓冲液为磷酸盐缓冲液、Tris-HCl缓冲液或硼酸-硼砂缓冲液。
对于第一平衡缓冲液中的盐浓度，本发明不作任何限制，本领域技术人员可以根据需要进行选择，例如所述第一平衡缓冲液中的盐浓度为5mM-0.25M，pH为5.5-8.0。
例如，所述第一平衡缓冲液中的盐浓度可以为5mM、10mM、20mM、50mM、0.1M、0.15M、0.2M、0.25M等；pH可以为5.5、6、7、8等。
优选的，所述第一平衡缓冲液为磷酸盐缓冲液或Tris-盐酸缓冲液，更优选的，在所述第一平衡缓冲液中加入NaCl或Na 2SO 4来降低非抗体蛋白同填料间的非特异性吸附。
优选的，所述第一平衡缓冲液中的盐浓度为5mM-0.15M，优选为10mM-50mM，进一步优选为20mM。
对于第一平衡缓冲液的pH值，本发明不作任何限制，本领域技术人员 可以根据需要进行选择，例如所述第一平衡缓冲液的pH为6.5-7.5，优选为6.9。
对于NaCl或Na 2SO 4的加入量，其可以根据本领域技术人员的需要进行确定，对于NaCl或Na 2SO 4的浓度，本领域技术人员可以根据需要进行选择，例如，NaCl或Na 2SO 4的浓度可以为0-250mM，优选为150mM。
优选的，所述磷酸盐缓冲液例如可以为磷酸氢二钠和磷酸二氢钠的缓冲液。
在一个实施方案中，当将单克隆抗体发酵液与所述平衡好的亲和层析介质结合时，使用第一平衡缓冲液进行平衡。
在一个实施方案中，中间预洗脱缓冲液为中性缓冲液和/酸性缓冲液；优选的，所述中性缓冲液为磷酸盐缓冲液、三羟甲基氨基甲烷缓冲液或甘氨酸缓冲液；所述酸性缓冲液为柠檬酸-磷酸氢二钠缓冲液、醋酸-醋酸钠缓冲液或柠檬酸-柠檬酸三钠缓冲液。
对于中间预洗脱缓冲液的pH值，本发明不作任何限制，本领域技术人员可以根据需要进行选择，例如，所述中间预洗脱缓冲液的pH为5.0-7.5，优选为5.8.。
例如，所述中间预洗脱缓冲液的pH可以为5.0、5.5、6.0、6.5、7.0、7.5等。
在一个实施方案中，在所述中间预洗脱缓冲液中加入预洗脱活性剂，优选的，所述预洗脱活性剂为盐酸胍、聚山梨酯80或氯化钠，进一步优选的，所述预洗脱活性剂为盐酸胍。
对于盐酸胍的浓度，本发明不作任何限制，本领域技术人员可以根据需要进行选择，例如，所述盐酸胍的浓度为0.01-1M，优选为0.05-0.15M，进一步优选为0.1M。
例如，所述盐酸胍的浓度可以为0.01M、0.05M、0.1M、0.5M、1M。
对于中间预洗脱缓冲液中的盐浓度，本发明不作任何限制，本领域技术人员可以根据需要进行选择，例如，在一个实施方案中，所述中间预洗脱缓冲液中的盐浓度为0-0.5M，优选为0.1M。
例如，所述中间预洗脱缓冲液中的盐浓度可以为0、0.1M、0.2M、0.3M、0.4M、0.5M等。
在一个实施方案中，在中间预洗脱淋洗后，在进行终洗脱之前还包括下 述步骤：
使用第二平衡缓冲液进行平衡，优选的，所述第二平衡缓冲液为磷酸盐缓冲液、Tris-HCl缓冲液或硼酸-硼砂缓冲液。
优选的，所述第二平衡缓冲液为磷酸盐缓冲液或Tris-HCl缓冲液，优选的，在第二平衡缓冲液中加入NaCl或Na 2SO 4来维持电导同时保持部分缓冲能力。
对于第二平衡缓冲液中的盐浓度、第二平衡缓冲液的pH以及NaCl或Na 2SO 4的浓度，本发明不作任何限制，本领域技术人员可以根据需要进行选择，例如，在一个实施方案中，所述第二平衡缓冲液中的盐浓度为5mM-0.15M，优选为10mM-50mM，进一步优选为20mM；优选的，pH值为5.5-8.0，优选为6.5-7.5，进一步优选为7.2；优选的，NaCl或Na 2SO 4的浓度为0-250mM，优选为10mM。
所述磷酸盐缓冲液例如可以为磷酸氢二钠和磷酸二氢钠的缓冲液。
在一个实施方案中，终洗脱缓冲液选自柠檬酸-磷酸氢二钠缓冲液、醋酸缓冲液、甘氨酸-HCl缓冲液和柠檬酸-柠檬酸钠缓冲液中的一种或两种以上，优选为柠檬酸-磷酸氢二钠缓冲液。
对于终洗脱缓冲液的pH，本发明不作任何限制，本领域技术人员可以根据需要进行选择，例如，在一个实施方案中，所述终洗脱缓冲液的pH为2.0-7.0，优选为3.0-4.0。
例如，所述终洗脱缓冲液的pH可以为2.0、3.0、4.0、5.0、6.0、7.0等。
对于终洗脱缓冲液中的盐浓度，本发明不作任何限制，本领域技术人员可以根据需要进行选择，例如，在一个实施方案中，所述终洗脱缓冲液中的盐浓度为5-100mM，优选为10-50mM。
例如，所述终洗脱缓冲液中的盐浓度可以为5mM、10mM、20mM、30mM、40mM、50mM、60mM、70mM、80mM、90mM、100mM等。
在一个实施方案中，进行洗脱后可以将亲和介质再生，优选的，再生缓冲液包括但不限于柠檬酸-磷酸氢二钠缓冲液、盐酸、甘氨酸、NaOH，优先为柠檬酸-磷酸氢二钠缓冲液及NaOH溶液。
本发明使用Protein A亲和纯化工艺简单易行，能够进行放大纯化生产，细胞发酵上清液无需进行前预处理，洗脱样品得率较高，同时HCP残留量也保持在较低水平(残留控制量不高于0.1％)，从而减轻之后纯化步骤中 去除HCP的压力，从而保证抗体最终样品的HCP残留量处于极低水平。同时，发明中经过对不同批次发酵上清液进行亲和纯化工艺验证，证明本发明中的亲和纯化工艺具有良好的稳定性。
在一个实施方案中，所述单克隆抗体体外发酵生产使用的哺乳动物包括但不限于目前使用的各种杂交瘤细胞、中国仓鼠卵巢细胞(CHO)，优选的是CHO细胞。
本发明提供了一种抗人干扰素α受体1(IFNAR1)单克隆抗体浓缩溶液的制备方法，其包括下述步骤：
第一超滤浓缩：在流速为120-300L/m 2·h和跨膜压差(TMP)为0.6-1.5bar的条件下将含有抗人干扰素α受体1(IFNAR1)单克隆抗体的溶液浓缩至蛋白质浓度为20-60mg/ml，得到浓缩样品；
超滤置换：采用置换缓冲液置换浓缩样品，当待置换缓冲液的使用量为浓缩样品重量的6-10倍时得到超滤置换溶液；
第二超滤浓缩：将超滤置换溶液和碱性氨基酸母液混匀使得氨基酸的浓度为100-150mM，进行超滤浓缩后得到蛋白质浓度为100-200mg/ml的浓缩溶液；
所述抗IFNAR1单克隆抗体包含三个重链互补决定区(CDR-H1、CDR-H2和CDR-H3)和三个轻链互补决定区(CDR-L1、CDR-L2和CDR-L3)，其中：
(a)CDR-H1(在本说明书中CDR-H1表示重链CDR1)的氨基酸序列如SEQ ID NO：1所示；
(b)CDR-H2(在本说明书中CDR-H2表示重链CDR2)的氨基酸序列如SEQ ID NO：2所示；
(c)CDR-H3(在本说明书中CDR-H3表示重链CDR3)的氨基酸序列如SEQ ID NO：3所示；
(d)CDR-L1(在本说明书中CDR-L1表示轻链CDR1)的氨基酸序列如SEQ ID NO：4所示；
(e)CDR-L2(在本说明书中CDR-L2表示轻链CDR2)的氨基酸序列如SEQ ID NO：5所示；且
(f)CDR-L3(在本说明书中CDR-L3表示轻链CDR3)的氨基酸序列如SEQ ID NO：6所示。
例如，流速可以为120L/m 2·h、150L/m 2·h、180L/m 2·h、200L/m 2·h、250L/m 2·h、300L/m 2·h等；
跨膜压差(TMP)可以为0.6bar、0.7bar、0.8bar、0.9bar、1.0bar、1.1bar、1.2bar、1.3bar、1.4bar、1.5bar等；
在第一超滤浓缩中，浓缩样品的蛋白质浓度例如可以为20mg/ml、30mg/ml、40mg/ml、50mg/ml、60mg/ml等；
在超滤置换中，当置换缓冲液的使用量为浓缩样品重量的6倍、7倍、8倍、9倍、10倍等时得到超滤置换溶液，所述使用量为体积使用量。
在第二超滤浓缩中，将超滤置换溶液和碱性氨基酸母液混匀后使得氨基酸的浓度例如可以为100mM、110mM、120mM、130mM、140mM、150mM等，进行超滤浓缩后得到蛋白质浓度例如可以为100mg/ml、110mg/ml、120mg/ml、130mg/ml、140mg/ml、150mg/ml、160mg/ml、170mg/ml、180mg/ml、190mg/ml、200mg/ml等的浓缩溶液。
在一个实施方案中，在第一超滤浓缩中，将含有抗人干扰素α受体1(IFNAR1)单克隆抗体的溶液浓缩至蛋白质浓度为30-50mg/ml。
在一个实施方案中，在超滤置换中，对于置换缓冲液的浓度，本发明不作任何限制，本领域技术人员可以根据需要进行选择，例如置换缓冲液的浓度为5-50mM，优选为20mM。
对于置换缓冲液的pH，本发明不作任何限制，其只要为偏酸性即可，例如，置换缓冲液的pH为6.0-7.0，优选为6.2-6.8。
例如，置换缓冲液的浓度可以为5mM、10mM、20mM、30mM、40mM、50mM等；置换溶液的pH可以为6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0等。
在一个实施方案中，所述置换缓冲液为组氨酸-盐酸缓冲液、柠檬酸缓冲液、磷酸盐缓冲液或醋酸钠-醋酸缓冲液，优选为组氨酸-盐酸缓冲液。
所述柠檬酸缓冲液例如为柠檬酸-柠檬酸钠缓冲液。
所述磷酸盐缓冲液例如为磷酸氢二钠-磷酸二氢钠缓冲液。
优选的，使用蠕动泵将置换的缓冲液泵入浓缩样品中，继续超滤，同时调节置换缓冲液的泵入速度与透过流速一致，当置换缓冲液的泵入量为浓缩样品重量的6-10倍时即完成置换。
在一个实施方案中，在第二超滤浓缩中，所述碱性氨基酸为盐酸精氨酸、 赖氨酸和/或脯氨酸，优选为盐酸精氨酸。
对于碱性氨基酸母液的浓度，本发明不作任何限制，本领域技术人员可以根据需要进行选择，例如在一个实施方案中，所述碱性氨基酸母液的浓度为0.5-2.0mol/L，优选为1-1.5mol/L。
例如，所述碱性氨基酸母液的浓度可以为0.5mol/L、1.0mol/L、1.5mol/L、2.0mol/L等。
在一个实施方案中，所述浓缩溶液的粘度≤20cP。
例如，所述浓缩溶液的粘度例如可以为20cP、18cP、15cP、12cP、10cP、8cP、5cP、3cP、1cP等。
优选的，在第二超滤浓缩中，计算超滤置换溶液中的蛋白质浓度，并计算该超滤浓缩溶液的理论体积，并加入碱性氨基酸母液混匀，优选的，碱性氨基酸母液的体积是超滤浓缩溶液理论体积的1/9，使得氨基酸的浓度为100-150mM，进行超滤浓缩后得到蛋白质浓度为100-200mg/ml的浓缩溶液，所述浓缩溶液的粘度≤20cP。
在一个实施方案中，所述含有抗人干扰素α受体1(IFNAR1)单克隆抗体的溶液通过对表达抗人干扰素α受体1(IFNAR1)单克隆抗体的细胞发酵液进行亲和层析、低pH灭活、阴离子层析、阳离子层析和纳滤得到。
所述细胞发酵液生产使用的哺乳动物包括但不限于目前使用的各种杂交瘤细胞、中国仓鼠卵巢细胞(CHO)，优选的是CHO细胞。
所述亲和层析采用Protein A进行亲和层析。
在一个实施方案中，在第一超滤浓缩和超滤置换时，使用的超滤膜包的材质包括但不限于改良聚醚砜(PES)、聚偏氟乙烯(PVDF)、醋酸纤维素(CA)等，其孔径通常采用30kDa或50kDa，优选的，超滤膜包为默克密理博的Pellicon2/Pellicon3(A型筛网，30kDa)，以及赛多利斯和颇尔公司的超滤膜包。
在一个实施方案中，所述抗人干扰素α受体1(IFNAR1)单克隆抗体为IgG4型抗体，阻断包括IFNα、IFNβ和IFN-ω在内的所有I型干扰素的活性，拟用于***性红斑狼疮(SLE)的治疗，拟采用静脉或皮下注射方式给药。该剂型注射液的蛋白浓度高达150mg/ml。
本发明使用上述所述的方法来制备抗人干扰素α受体1(IFNAR1)单克隆抗体浓缩溶液，能够降低粘度，避免堵塞现象，同时抑制蛋白聚集沉淀, 起到保护剂作用。
并且所述的方法简单易行，能够进行放大生产，可以保证样品的高纯度并获得较高的回收率。
本发明提供了一种包含抗人干扰素α受体1(IFNAR1)单克隆抗体的液体制剂，所述液体制剂包含蛋白质浓度为100-200mg/ml的抗人干扰素α受体1(IFNAR1)单克隆抗体以及氨基酸浓度为5-300mM的碱性氨基酸；
所述抗IFNAR1单克隆抗体包含三个重链互补决定区(CDR-H1、CDR-H2和CDR-H3)和三个轻链互补决定区(CDR-L1、CDR-L2和CDR-L3)，其中：
(a)CDR-H1(在本说明书中CDR-H1表示重链CDR1)的氨基酸序列如SEQ ID NO:1所示；
(b)CDR-H2(在本说明书中CDR-H2表示重链CDR2)的氨基酸序列如SEQ ID NO:2所示；
(c)CDR-H3(在本说明书中CDR-H3表示重链CDR3)的氨基酸序列如SEQ ID NO:3所示；
(d)CDR-L1(在本说明书中CDR-L1表示轻链CDR1)的氨基酸序列如SEQ ID NO:4所示；
(e)CDR-L2(在本说明书中CDR-L2表示轻链CDR2)的氨基酸序列如SEQ ID NO:5所示；且
(f)CDR-L3(在本说明书中CDR-L3表示轻链CDR3)的氨基酸序列如SEQ ID NO:6所示。
例如，蛋白质浓度可以为100mg/ml、110mg/ml、120mg/ml、130mg/ml、140mg/ml、150mg/ml、160mg/ml、170mg/ml、180mg/ml、190mg/ml、200mg/ml等；
氨基酸浓度例如可以为5mM、10mM、20mM、30mM、40mM、50mM、60mM、70mM、80mM、90mM、100mM、150mM、200mM、250mM、300mM等。
使用上述所述的特定浓度的碱性氨基酸以及特定浓度的蛋白质，能够使液体制剂的粘度控制在10cP以下，可以用注射器轻松推注，因此，可以用作注射剂，尤其是皮下注射。
在一个实施方案中，所述碱性氨基酸选自精氨酸、赖氨酸、组氨酸和脯 氨酸中的一种或两种以上。
对于不同碱性氨基酸的浓度，本发明不作任何限制，本领域技术人员可以根据需要进行选择，例如，在一个实施方案中，所述碱性氨基酸包含100-200mM的精氨酸和5-50mM的组氨酸。
例如，所述碱性氨基酸可以包含100mM、110mM、120mM、130mM、140mM、150mM、160mM、170mM、180mM、190mM、200mM等的精氨酸和5mM、10mM、15mM、20mM、25mM、30mM、35mM、40mM、45mM、50mM等的组氨酸。
例如，所述碱性氨基酸为20mM的组氨酸和150mM的精氨酸。
使用上述所述的碱性氨基酸的组合物，能够使液体制剂的粘度大大降低，例如，粘度在10cp以下。
在一个实施方案中，其中，所述碱性氨基酸包含100-200mM的赖氨酸和5-50mM的组氨酸。
例如，所述碱性氨基酸可以包含100mM、110mM、120mM、130mM、140mM、150mM、160mM、170mM、180mM、190mM、200mM等的赖氨酸和5mM、10mM、15mM、20mM、25mM、30mM、35mM、40mM、45mM、50mM等的组氨酸。
例如，所述碱性氨基酸为20mM的组氨酸和150mM的赖氨酸。
使用上述所述的碱性氨基酸的组合物，能够使液体制剂的粘度大大降低，例如，粘度在10cp以下。
在一个实施方案中，所述碱性氨基酸包含100-200mM的脯氨酸和5-50mM的组氨酸。
使用上述所述的碱性氨基酸的组合物，能够使液体制剂的粘度大大降低，例如，粘度在10cp以下。
例如，所述碱性氨基酸可以包含100mM、110mM、120mM、130mM、140mM、150mM、160mM、170mM、180mM、190mM、200mM等的脯氨酸和5mM、10mM、15mM、20mM、25mM、30mM、35mM、40mM、45mM、50mM等的组氨酸。
例如，所述碱性氨基酸为20mM的组氨酸和150mM的脯氨酸。
使用上述所述的碱性氨基酸的组合物，能够使液体制剂的粘度大大降低，例如，粘度在10cp以下。
在一个实施方案中，所述液体制剂还包含20-150mg/ml的蔗糖，优选为50-100mg/ml的蔗糖。
例如，所述液体制剂还可以包含20mg/ml、30mg/ml、40mg/ml、50mg/ml、60mg/ml、70mg/ml、80mg/ml、90mg/ml、100mg/ml、110mg/ml、120mg/ml、130mg/ml、140mg/ml、150mg/ml等的蔗糖。
在一个实施方案中，所述液体制剂包含50mg/ml的蔗糖。
在一个实施方案中，所述液体制剂还包含0.5-50mg/ml的山梨醇，优选为1-20mg/ml的山梨醇。
例如，所述液体制剂还可以包含0.5mg/ml、1mg/ml、5mg/ml、10mg/ml、15mg/ml、20mg/ml、30mg/ml、40mg/ml、50mg/ml等的山梨醇。
在一个实施方案中，例如，所述液体制剂包含70mg/ml的蔗糖。
在一个实施方案中，所述液体制剂还包含20-300mg/ml的氯化钠，优选为100-200mg/ml的氯化钠。
例如，所述液体制剂还可以包含20mg/ml、30mg/ml、40mg/ml、50mg/ml、60mg/ml、70mg/ml、80mg/ml、90mg/ml、100mg/ml、110mg/ml、120mg/ml、130mg/ml、140mg/ml、150mg/ml、160mg/ml、170mg/ml、180mg/ml、190mg/ml、200mg/ml、250mg/ml、300mg/ml等的氯化钠。
在一个实施方案中，例如，所述液体制剂包含150mg/mL的氯化钠，使用所述的氯化钠与组氨酸组合，能够使液体制剂的粘度控制在10cP以下。
对于液体制剂的pH，本发明不作任何限制，本领域技术人员可以根据需要进行选择，例如，在一个实施方案中，所述液体制剂的pH为5.5-6.5。
在一个实施方案中，所述含有抗人干扰素α受体1(IFNAR1)单克隆抗体的液体制剂是通过对表达抗人干扰素α受体1(IFNAR1)单克隆抗体的细胞发酵液进行亲和层析、低pH灭活、阴离子层析、阳离子层析和超滤浓缩，然后将超滤浓缩所得到的浓缩液与缓冲液或添加剂母液进行混合得到。
所述细胞发酵液生产使用的哺乳动物包括但不限于目前使用的各种杂交瘤细胞、中国仓鼠卵巢细胞(CHO)，优选的是CHO细胞。
所述亲和层析采用Protein A进行亲和层析。
在一个实施方案中，所述抗人干扰素α受体1(IFNAR1)单克隆抗体为IgG4型抗体，阻断包括IFNα、IFNβ和IFN-ω在内的所有I型干扰素的活性，拟用于***性红斑狼疮(SLE)的治疗，拟采用静脉或皮下注射方式给 药。该剂型注射液的蛋白浓度高达150mg/ml。
本发明所述的液体制剂，具有较低的粘度，可用注射器轻松推注，因此适合用作注射剂、特别是皮下注射剂。
实施例
本发明对试验中所用到的材料以及试验方法进行一般性和/或具体的描述，在下面的实施例中，如果无其他特别的说明，％表示wt％，即重量百分数。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品。
实施例1抗人干扰素α受体1单克隆抗体QX006N的制备
从上海近岸科技有限公司采购人干扰素α受体1(IFNAR1)，用于免疫新西兰兔，运用B细胞克隆技术获得抗原结合特异性抗体克隆，进而筛选出结合人IFNAR1并具有人IFNAR1抑制活性的单克隆抗体。首先，用Binding ELISA检测细胞上清，挑选出与人IFNAR1结合的克隆；再用HEK Blue IFN α/β报告基因细胞法进行检测，挑选出具有人IFNAR1抑制活性的克隆。以上免疫和筛选过程委托给商业化公司完成。
先后挑选出37个克隆进行重组表达，并测序。经测定，362#与1203#的细胞中和活性最好，且两个克隆的序列很相似。因此，先对362#进行人源化改造，当筛选获得1203#、且发现1203#的活性更好时，在362#人源化的基础上对1203#克隆进行人源化改造。利用NCBI IgBlast进行人IgG胚系序列(Germline)同源性比对，选择IGHV3-66*01作为重链CDR移植模板，将1203#克隆重链的CDR区(即CDR-H1(SEQ ID No:1)、CDR-H2(SEQ ID No:2)和CDR-H3(SEQ ID No:3))移植入IGHV3-66*01的骨架区；选择IGKV1-6*01作为轻链CDR移植模板，将1203#克隆轻链的CDR区(即CDR-L1(SEQ ID No:4)、CDR-L2(SEQ ID No:5)和CDR-L3(SEQ ID No:6))移植入IGKV1-6*01的骨架区；对骨架区特定位点进行回复突变，获得本发明的单克隆抗体QX006N可变区。最终，人源化后的重链可变区序列如SEQ ID NO：7所示；人源化后的轻链可变区氨基酸序列如SEQ ID NO：8所示。
上述重链可变区(SEQ ID NO：7)的基因和轻链可变区(SEQ ID NO：8)的基因，以362#人源化抗体的基因序列为模板，利用PCR扩增获得。用 HindIII和NheI双酶切重链表达质粒pHZDCH；用HindIII和BsiWI双酶切轻链表达质粒pHZDCK；用Infusion重组酶将PCR扩增基因分别***对应的表达质粒中，构建重链表达质粒pHZDCH-362VH-Hu6和轻链表达质粒pHZDCK-362VK-Hu20。在人源化改造过程中，1203#人源化抗体的基因用362编号，蛋白用1203编号。
通过核酸电泳检测质粒的双酶切结果如图1所示。根据图1的结果可以看出，抗体重链可变区和轻链可变区PCR扩增结果以及双酶切重链和轻链表达质粒的结果，其中，重链和轻链的质粒大小约10000bp，轻链可变区约447bp，重链可变区约471bp。
通过对1203#进行人源化改造，获得人源化抗体HZD1203-45。为了降低抗体的ADCC效应，将HZD1203-45重链表达质粒pHZDCH-362VH-Hu6的人IgG1恒定区换成人IgG4，获得重链表达质粒pHZDCH-362VH-Hu6-IgG4.1。
将序列正确的重链表达质粒pHZDCH-362VH-Hu6-IgG4.1和轻链表达质粒pHZDCK-362VK-Hu20共转染ExpiCHO-S细胞。转染前一天，将ExpiCHO-S细胞稀释成3×10 6个细胞/ml进行转染前传代。转染当天，将细胞密度稀释成6×10 6个细胞/ml，125ml摇瓶装25ml细胞，等待转染。转染和表达过程如图2所示。
转染后第4-8天，收获培养上清，用ProteinA进行一步纯化。用SDS-PAGE电泳检测纯化的抗体，将其命名为QX006N(HZD1203-45-IgG4.1)，利用蛋白电泳检测该抗体的结果如图3所示。蛋白电泳用变性还原胶检测，图3的结果显示出有两条带，两个条带的大小分别约50kDa和25kDa，与重链(48.9kDa)和轻链(23.4kDa)理论分子量一致。
实施例2平衡解离常数(K D)的测定
用BiacoreT200检测QX006N(HZD1203-45-IgG4.1)与人IFNAR1的亲和力，所有过程都在25℃进行。采用商品化Protein A芯片，通过捕获法固定适量的抗体，使得Rmax在50RU左右，捕获流速是10μl/min。将抗原进行梯度稀释，仪器流速切换成30μl/min，按照浓度从低到高的顺序依次流过参比通道和固定抗体的通道，流过缓冲液作为阴性对照。每一个结合、解离完成后用pH1.5甘氨酸再生芯片。用仪器自带分析软件选择Kinetics选项中1:1 结合模型进行拟合，计算抗体的结合速率常数k a，解离速率常数k d以及解离平衡常数K D值。
除此之外，将QX006N(HZD1203-45-IgG4.1)与已进入临床III期的针对人IFNAR1的单克隆抗体，即Anifrolumab的亲和力进行比较，针对已知抗体的检测方法与对QX006N进行检测的方法相同，结果如表1所示。其中Anifrolumab根据专利WO2009100309A2提供的9D4序列，构建表达质粒，瞬转ExpiCHO-S细胞自制获得。
表1抗体结合人IFNAR1的亲和力
样品名称 k a(10 5M -1S -1) k d(10 -5S -1) K D(10 -10M)
HZD1203-45-IgG4.1 3.47 3.76 1.08
Anifrolumab 18.67 12.40 0.67
表中的数据为：每个样品检测三次，计算平均值的数据。
实施例3 QX006N和Anifrolumab中和人干扰素诱导的HEK Blue IFNα/β细胞STAT1/2磷酸化活性
利用HEK Blue IFNα/β报告基因细胞系测定QX006N拮抗干扰素通过IFNAR1介导的胞内信号分子STAT1/2磷酸化活性：将培养液中的细胞以每孔4×10 4细胞加入到96孔内，然后在37℃和5％CO 2条件下培养过夜。向细胞中加入抗体浓度范围为0至5μg/ml的系列稀释液，并加入0.2ng/ml的IFNα.2b。然后在37℃和5％CO 2条件下培养24小时，收集细胞培养上清加入10％的QUANTI-Blue TM检测试剂在37℃和5％CO 2条件下反应1小时，然后检测OD630nm值及绘制剂量效应曲线，进而分析抗体的拮抗活性，实验结果表明，QX006N能够抑制干扰素诱导HEK Blue IFNα/β细胞中STAT1/2磷酸化，其抑制干扰素诱导的HEK Blue IFNα/β细胞中1/2磷酸化活性的IC 50为5.23ng/ml，而Anifrolumab抑制干扰素诱导的HEK Blue IFNα/β细胞中1/2磷酸化活性的IC 50为4.43ng/ml。
实施例4 QX006N和Anifrolumab中和人干扰素抑制Daudi细胞增殖的活性
利用Daudi人淋巴瘤细胞系测定QX006N拮抗干扰素通过IFNAR1诱导的细胞增殖活性：将培养液中的细胞以每孔4×10 4细胞加入到96孔内，然 后在37℃和5％CO 2条件下培养过夜。向细胞中加入抗体浓度范围为0至20μg/ml的系列稀释液，并加入0.8ng/ml的IFNα.2b。然后在37℃和5％CO 2条件下培养72小时，收集细胞培养物采用CellTiter-Glo检测细胞增殖情况及绘制剂量效应曲线，进而分析抗体的拮抗活性，实验结果表明，QX006N能够抑制干扰素诱导的Daudi细胞增殖，其抑制干扰素诱导的Daudi细胞增殖活性的EC 50为29.9ng/ml，而Anifrolumab抑制干扰素诱导的Daudi细胞增殖活性的EC 50为31.7ng/ml。
实施例5 QX006N和Anifrolumab中和人干扰素诱导全血释放CXCL10/IP10活性.
利用人全血测定QX006N拮抗干扰素通过IFNAR1诱导的CXCL10/IP10释放活性：将全血按照100μl/孔加入到96孔板中，暂存于37℃和5％CO 2条件下，向全血中加入抗体浓度范围为0至40μg/ml的系列稀释液，并加入8ng/ml的IFNα.2b，40ng/ml的TNF-α。然后在37℃和5％CO 2条件下培养48小时，收集细胞培养上清采用夹心ELISA法检测上清中CXCL10/IP10的表达及绘制剂量效应曲线，进而分析抗体的拮抗活性，实验结果表明，QX006N能够抑制干扰素诱导的全血释放CXCL10/IP10，其抑制干扰素诱导的全血释放CXCL10/IP10活性的IC 50为698ng/ml，而Anifrolumab抑制干扰素诱导的全血释放CXCL10/IP10活性的IC 50为562ng/ml。
实施例6不同预洗脱缓冲液对于重组人源化抗IFNAR1单克隆抗体(QX006N)发酵液的HCP的清除效果比较
使用CHO细胞作为宿主细胞，Dynamis作为发酵基础培养基，生产实施例1所得到的抗体QX006N。使用常规的细胞培养工艺进行细胞培养，当细胞活率低于80％或培养至18天时开始收获，采用初级滤器MD0HC10FS1和次级滤器MX0HC10FS1对收获液进行深层过滤，收集澄清的细胞培养上清，记为发酵液中间体。将发酵液中间体进行亲和层析，方法如下：
采用第一平衡缓冲液(12mmol/L Na 2HPO 4、8mmol/L NaH 2PO 4和0.15mol/L NaCl)平衡Protein A层析柱(Merck
Figure PCTCN2021114952-appb-000009
0.15L)，并将QX006N发酵液中间体上样于平衡后的Protein A层析柱，以与其结合，然后再使用第一平衡缓冲液进行平衡，至发酵液完全流过层析柱；
然后进行中间预洗脱淋洗，中间预洗脱缓冲液的组成如表2所示，淋洗后使用第二平衡缓冲液(6mmol/L Na 2HPO 4、4mmol/L NaH 2PO 4，pH7.2)进行平衡；
接着进行终洗脱来收集样品，并对收集的样品进行抗体浓度和宿主蛋白(HCP)残留含量进行测定，其中，终洗脱缓冲液为5mmol/L Na 2HPO 4，6.5mmol/L柠檬酸，pH为3.6，并采用再生缓冲液100mmol/L NaOH，1mol/L NaCl对层析柱进行再生。
抗体浓度的测定方法如下：
1.分光光度计波长调至280nm，用第二平衡缓冲液作为对照，进行校零。
2.用第二平衡缓冲液对待测样品进行稀释，测定样品在280nm的吸光值(吸光值保证在0.5～1.5之间)，并按照之下公式计算样品浓度(QX006N消光系数为1.469)。
Figure PCTCN2021114952-appb-000010
所得到的结果如表3所示。
HCP含量的测定方法：
1.样品稀释：一般根据样品中HCP的估计值来进行稀释倍数选择，使最终HCP的浓度落在标准曲线范围内即可(一般10ng/ml～80ng/ml)。样品一步稀释倍数不超过10倍，最小取样量不低于5μl。
2.向取出板条每孔加入Anti-CHO HRP 100μl。
3.上样：按一定排列分别加入标准品、样品、加标样品(各两复孔，标准品不需复孔)，50μl/孔，封板。室温置于水平摇床，180rpm，2小时，避光。
4.洗板：弃去孔内液体，用多通道移液器加洗液300μl/孔，静置30秒后甩尽液体，在吸水纸上拍干，洗板4次。最后一次洗板完成后，需尽量拍干孔内残留洗液。
5.显色及终止读数：按100μl/孔加TMB试剂(TMB Substrate)，静置 显色30分钟，避光。30分钟后加终止液(Stop Solution)100μl/孔，酶标仪450nm读数，以650nm作为参考。
6.选择分析软件进行数据分析，以标准品OD值为纵坐标，浓度为横坐标，作四参数标准曲线。将样品测得的OD值代入标准品曲线，求得所加样品HCP的实测值。
7.CHO细胞蛋白残留量(％)＝样品平均实测值(ng/ml)×稀释倍数/未稀释样品蛋白含量(mg/ml)量求得 -4(％)，其结果如表3所示。
表2中间预洗脱缓冲液的组成
实验 预洗脱缓冲液
1 0.1mol/L柠檬酸钠，0.1mol/L盐酸胍，11mmol/L柠檬酸，pH5.8
2 12mmol/L Na 2HPO 4，8mmol/L NaH 2PO 4，0.15mol/L NaCl，0.5％Tween 80，pH7.0
3 0.1mol/L柠檬酸钠，0.5mol/L氯化钠，7mmol/L柠檬酸，pH5.8
表3 QX006N浓度及HCP残留含量结果
实验 细胞培养上清HCP含量％(w/w) 收率％ 亲和层析后HCP残留量％(w/w)
1 18.25 97.5 0.089
2 18.53 97.7 0.543
3 18.26 97.4 0.456
由表3结果可知，发酵液中间体中HCP残留量大于15％，经过亲和纯化工艺的处理，收率均大于95％；对于HCP的去除，采用上述所述的方法，亲和层析后样品HCP残留低于0.1％，后续纯化工艺步骤去除HCP负荷明显降低，且样品经pH调节，进行后续层析工艺步骤(阴离子交换层析)上样时，样品保持极为澄清，无需其他处理即可直接进样，增强了工艺的简便性，节省了工艺时间。
实施例7不同批次重组人源化抗IFNAR1单克隆抗体(QX006N)发酵液的HCP的清除效果比较
发酵液中间体的制备及亲和层析方法同实施例6，共制备三个批次的发酵液中间体，中间预洗脱淋洗均采用含盐酸胍的预洗脱缓冲液：0.1mol/L柠檬酸钠，0.1mol/L盐酸胍，11mmol/L柠檬酸，pH5.8淋洗。亲和工艺收率及HCP残留含量测试方法如实施例6所述，其结果如表4所示。
表4不同批次QX006N浓度及HCP残留含量结果
批次 细胞培养上清HCP含量％(w/w) 收率％ 亲和层析后HCP残留量％(w/w)
1 18.35 97.8 0.078
2 17.34 97.6 0.086
3 18.12 96.7 0.075
由表4结果所示，不同批次的QX006N发酵液中间体HCP残留均处于15％以上，细胞培养工艺稳定；三批样品的亲和工艺收率均大于95％，收率符合工艺要求；亲和层析后样品HCP残留均低于0.1％，采用盐酸胍进行预洗脱淋洗对于HCP的去除保持稳定。
实施例8抗人IFNAR1单克隆抗体高浓度超滤浓缩后粘度的比较
使用CHO细胞作为宿主细胞，在2L规模的生物反应器上进行发酵，生产实施例1所得到的抗体QX006N。通过离心机或深层膜包过滤获得澄清发酵液，采用Protein A层析进行目的蛋白的捕获，再经过低pH值病毒灭活、阴离子和阳离子层析去除杂质，并进行纳滤得到含有抗人干扰素α受体1(IFNAR1)单克隆抗体的溶液，将该溶液采用下述方法进行浓缩：
(1)第一超滤浓缩：超滤设备选用默克密理博的Labscale小型超滤仪(夹持两块50cm 2Pellicon XL膜包，截留量30KD)。以120-300L/m 2·h的流速，TMP维持在0.6～1.5bar条件下将含有抗人干扰素α受体1(IFNAR1)单克隆抗体的溶液浓缩至蛋白质浓度约为30～50mg/ml，得到浓缩样品。
(2)超滤置换：置换溶液为20mM的组氨酸-盐酸缓冲液，pH值为6.5，使用蠕动泵将组氨酸-盐酸缓冲液泵入至所述浓缩样品中，继续超滤，同时调节组氨酸-盐酸缓冲液的泵入速度与透过流速一致，即保持样品的重量恒定，待组氨酸-盐酸缓冲液的体积为浓缩样品重量的7倍时上即完成置换得到超滤置换溶液。
(3)第二超滤浓缩：取样测定超滤置换溶液中蛋白质的浓度，并计算此时超滤置换溶液的理论体积，并加入1/9理论体积的碱性氨基酸母液，混匀，使得缓冲体系如表5所示，然后使用默克密理博的30kDa的超滤离心管浓缩至蛋白质浓度为表5的浓度得到浓缩溶液，分别测定浓缩溶液的粘度值，粘度值测定方法如下：
1.取一次性专用注射器，抽取200～400微升浓缩溶液，排尽注射器内气泡，并用无尘纸轻擦残留液体；
2.将注射器安放至管槽中固定，点击仪器主机界面，设置剪切速率，设置完成后，点击“Run”进行检测，记录粘度值，一般多次测量取平均值；
3.对于多个样品的检测，前一个样品完成检测后，取出注射器，排空管内液体，无尘纸轻柔擦拭管口后及可进行下一个样品的吸取测量。
其粘度值结果如表5所示。
表5粘度值结果
编号 缓冲体系 蛋白浓度mg/mL 粘度值cP
A-1 20mM His-HCL 200 42.1
A-2 20mM His-HCL 180 29.3
A-3 20mM His-HCL 150 22.5
A-4 20mM His-HCL 120 14.4
A-5 20mM His-HCL+70mg/ml蔗糖 150 25.2
A-6 20mM His-HCL+45mg/ml山梨醇 150 25.6
A-7 20mM His-HCL+150mM NaCl 150 7.1
A-8 20mM His-HCL+150mM盐酸精氨酸 150 7.2
A-9 20mM His-HCL+150mM赖氨酸 150 7.3
A-10 20mM His-HCL+150mM脯氨酸 150 7.2
A-11 20mM His-HCL+50mg/ml蔗糖+50mM盐酸精氨酸 150 11.5
由表5结果可知，随着蛋白质浓度的提高，该抗人IFNAR1抗体的粘度值呈指数级增加，若无添加剂加入，该单抗在浓度为200mg/ml时粘度值会达到42.1cP，如此高的粘度值无法使用常规的超滤膜实现浓缩工艺。以150mg/ml蛋白浓度为例，通过添加150mM的碱性氨基酸或其组合可以将相同蛋白浓度条件下的抗体粘度值降至原来的50％以下，小于20cP。基于上述实验结果，可以实现该抗人IFNAR1单抗的给药浓度为100-150mg/ml。
实施例9不同方法的比较
含有抗人干扰素α受体1(IFNAR1)单克隆抗体QX006N的溶液(UF0)的制备、第一超滤浓缩和超滤置换的方法均与实施例6相同；
第二超滤浓缩：将超滤置换溶液分成两份分别进行第二步浓缩工艺：
A.从第一份中取样测定超滤置换溶液中蛋白质的浓度，并计算此时超滤置换溶液的理论体积，并加入1/9理论体积的盐酸精氨酸母液(1.5mol/L)混匀，然后使用默克密理博的30kDa的超滤离心管浓缩至蛋白质浓度为150mg/ml，记为浓缩溶液1(U1F2)；
B.将第二份直接采用第一超滤浓缩的方法进行浓缩，蛋白质浓度为150mg/ml，记为浓缩溶液2(U2F2)；
分别检测浓缩溶液1(U1F2)和2(U2F2)的粘度和纯度，其中粘度的 测定方法与实施例8的方法相同，纯度的测定方法如下：
1.采用高效液相色谱仪(Agilent 1260或等效仪器)、色谱柱(Waters，
Figure PCTCN2021114952-appb-000011
规格：3.5μm，7.8×300mm；或等效色谱柱)，流动相为PBS缓冲液；
2.拧开四元泵管路，在5ml/min流速条件下100％流动相冲洗管路3分钟，拧紧四元泵开关，接上色谱柱，在1.0ml/min流速条件下100％流动相冲洗色谱柱30min；
3.样品分析方法设置：流速为1.0ml/min，分析时间为15min，进样量为50mg；检测波长为280nm；进样前需先检查***适应性(使用参考品连续进样，进样次数不少于5次，取后5次结果计算)，然后分析1针空白样品，接着分析样品。
4.按照聚合体、主峰及降解产物等顺序依次积分每个样品的色谱峰，所有与空白样品保留时间不一致的色谱峰均应积分，以面积归一法计算各峰比例。
测定结果如表6所示。
表6不同方法处理的粘度和纯度结果
Figure PCTCN2021114952-appb-000012
由表6结果可知，超滤工艺的第二步浓缩在添加盐酸精氨酸后粘度值由原来的22.5cP下降至7.2cP，同时盐酸精氨酸也作为保护剂一定程度上抑制聚体形成。高浓度的抗人IFNAR1单抗加入盐酸精氨酸后其粘度值明显降低，不会引起过高的反压，有利于超滤工艺的流速和膜压力的控制，提高了工艺的可操作性。
实施例10放大实验
基于上述实验结果，将实施例8进一步放大至200L中试规模，采用默克密理博Pellicon手动超滤***，夹持两块0.5m2Pellicon 2型膜包。纳滤后的含有抗人干扰素α受体1(IFNAR1)单克隆抗体的溶液(UF0)以流速 120-300L/m 2·h，TMP控制在0.6-1.5bar，按照实施例8的方法先进行第一超滤浓缩(UF1)，将样品浓缩至30～50mg/ml；然后按照实施例8的超滤置换方法进行置换，置换结束后，加入盐酸精氨酸母液，浓缩至蛋白质浓度为150mg/ml，进一步浓缩得到蛋白质浓度为193mg/ml(UF2)，回收样品，其粘度和纯度结果如表7所示。
表7：抗人IFNAR1单抗超滤工艺中试放大确认结果汇总
Figure PCTCN2021114952-appb-000013
由表7结果可知，超滤工艺放大后，超滤的过程中流速和膜压力的控制参数和收率基本与小试规模一致，顺利实现放大。添加的盐酸精氨酸抑制聚体，超滤后样品的纯度与超滤前基本一致，且在190～200mg/ml范围时粘度值控制在20cP以下，若无精氨酸添加剂则无法通过上述的超滤***浓缩至如此高浓度。
实施例11包含抗人IFNAR1单克隆抗体和不同碱性氨基酸的液体制剂粘度的比较
使用CHO细胞作为宿主细胞，发酵生产实施例1所述的抗体QX006N，在2L规模的生物反应器上进行发酵，通过离心机或深层膜包过滤获得澄清发酵液，采用Protein A层析进行目的蛋白的捕获，再经过低pH值病毒灭活、阴离子和阳离子层析去除杂质，并进行超滤浓缩得到含有抗人干扰素α受体1(IFNAR1)单克隆抗体的浓缩液，所得到的浓缩液中含有20mM His-HCL。
将所得到的浓缩液，采用SEC-HPLC法进行分析，其操作步骤如下：
1.采用高效液相色谱仪(Agilent 1260或等效仪器)、色谱柱(Waters，
Figure PCTCN2021114952-appb-000014
规格：3.5μm，7.8×300mm；或等效色谱柱)，流动相为PBS缓冲液；
2.拧开四元泵管路，在5ml/min流速条件下100％流动相冲洗管路3分钟，拧紧四元泵开关，接上色谱柱，在1.0ml/min流速条件下100％流动相冲洗色谱柱30min；
3.样品分析方法设置：流速为1.0ml/min，分析时间为15min，进样量为50mg；检测波长为280nm；进样前需先检查***适应性(使用参考品连 续进样，进样次数不少于5次，取后5次结果计算)，然后分析1针空白样品，接着分析样品。
4.按照聚合体、主峰及降解产物等顺序依次积分每个样品的色谱峰，所有与空白样品保留时间不一致的色谱峰均应积分，以面积归一法计算各峰比例，确定该浓缩液中的QX006N单体大于99％，聚体小于1％，基本不含杂蛋白。
采用紫外分光度法测定该浓缩液中QX006N的浓度，测定方法如下：
1.分光光度计波长调至280nm，用空白缓冲液或水作为对照，进行校零。
2.待测用空白缓冲液或水对样品进行稀释，测定样品在280nm的吸光值(吸光值保证在0.5---1.5之间)，并按照之下公式计算样品浓度(QX006N的消光系数为1.469)。
Figure PCTCN2021114952-appb-000015
测定该浓缩液中QX006N的浓度为200mg/mL。
采用锐欧森的μVISC粘度计测定该浓缩液的粘度，其操作步骤如下：
1.取一次性专用注射器，抽取200～400微升待检样品，排尽注射器内气泡，并用无尘纸轻擦残留液体；
2.用将注射器安放至管槽中固定，点击仪器主机界面，设置剪切速率，设置完成后，点击“Run”进行检测，记录粘度值，一般多次测量取平均值；
3.对于多个样品的检测，前一个样品完成检测后，取出注射器，排空管内液体，无尘纸轻柔擦拭管口后及可进行下一个样品的吸取测量，测得该浓缩液的粘度为42.1cP。
使用0.2μm滤膜过滤样品后，与表8中不同的缓冲液或添加剂母液混合使得蛋白浓度如表8所示，然后按照上述方法测定粘度值，其结果如表8所示。
表8不同碱性氨基酸母液的加入的粘度结果
Figure PCTCN2021114952-appb-000016
Figure PCTCN2021114952-appb-000017
一般认为，粘度小于30cP的液体制剂适合用作皮下注射剂，由表8的结果表明，本发明所述的液体制剂，QX006N的皮下注射给药浓度至少可以达到100-180mg/mL或150-180mg/mL。
综上所述，本发明采用上述所述的亲和纯化方法，所得到的单克隆抗体中HCP的残留量保持在较低的水平，残留控制量不高于0.1％，从而减轻了之后纯化步骤中去除HCP的压力，并且本发明所述的亲和纯化工艺具有良好的稳定性。
本发明所述的方法能够制备出高浓度、低粘度的抗人干扰素α受体1(IFNAR1)单克隆抗体的浓缩溶液，蛋白质浓度为100-200mg/ml时，粘度控制在20cP以下，所得到的浓缩溶液可以用于制备抗人干扰素α受体1(IFNAR1)单克隆抗体皮下注射剂。
本发明所述的液体制剂粘度较低，所述的液体制剂适合用于注射剂，特别是皮下注射剂，所述的抗人干扰素α受体1(IFNAR1)单克隆抗体，其与现有的抗人干扰素α受体1单克隆抗体(Anifrolumab)相比，结合IFNAR1的亲和力相当，在细胞水平的中和活性与Anifrolumab相当。
所述的单克隆抗体在细胞水平显示出与Anifrolumab(根据专利公开序列表达制备)相当的中和活性，其有望在预防和治疗相关疾病方面展现出良好的临床效果。
以上所述，仅是本发明的较佳实施例而已，并非是对本发明作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本发明技术方案内容，依 据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本发明技术方案的保护范围。

Claims (30)

  1. 一种降低单克隆抗体生产中宿主细胞蛋白含量的亲和纯化方法，其包括下述步骤：
    使用第一平衡缓冲液平衡亲和层析介质得到平衡好的亲和层析介质，并将单克隆抗体发酵液与所述平衡好的亲和层析介质结合；
    然后进行中间预洗脱淋洗，接着进行终洗脱以除去宿主细胞蛋白得到单克隆抗体；
    所述单克隆抗体为分离的抗人干扰素α受体1(IFNAR1)单克隆抗体，其包含三个重链互补决定区(CDR-H1、CDR-H2和CDR-H3)和三个轻链互补决定区(CDR-L1、CDR-L2和CDR-L3)，其中：
    (a)CDR-H1的氨基酸序列如SEQ ID NO：1所示；
    (b)CDR-H2的氨基酸序列如SEQ ID NO：2所示；
    (c)CDR-H3的氨基酸序列如SEQ ID NO：3所示；
    (d)CDR-L1的氨基酸序列如SEQ ID NO：4所示；
    (e)CDR-L2的氨基酸序列如SEQ ID NO：5所示；且
    (f)CDR-L3的氨基酸序列如SEQ ID NO：6所示。
  2. 根据权利要求1所述的亲和纯化方法，其中，所述抗人干扰素α受体1(IFNAR1)单克隆抗体包含重链可变区和轻链可变区，其中，
    所述重链可变区的氨基酸序列如SEQ ID NO：7所示；且，
    所述轻链可变区的氨基酸序列如SEQ ID NO：8所示。
  3. 根据权利要求2所述的亲和纯化方法，其中，所述抗人干扰素α受体1(IFNAR1)单克隆抗体的重链的氨基酸序列如SEQ ID NO：10所示；轻链的氨基酸序列如SEQ ID NO：11所示。
  4. 根据权利要求1-3中任一项所述的亲和纯化方法，其中，所述亲和层析介质为配基交联到琼脂糖、聚乙烯醚、羟基化聚醚树脂、聚丙烯酸树脂、聚苯乙烯二乙烯苯基树脂、聚甲基丙烯酸树脂、聚苯乙烯树脂、羟基磷灰石或玻璃基质上的层析介质，优选的，所述亲和层析介质为配基交联到聚乙烯醚的层析介质；
    优选的，所述配基为Protein A、Protein G或Protein L，优选为Protein A。
  5. 根据权利要求1-4中任一项所述的亲和纯化方法，其中，所述第一平衡缓冲液为磷酸盐缓冲液、Tris-HCl缓冲液或硼酸-硼砂缓冲液，所述第一平衡缓冲液中的盐浓度为5mM-0.25M，pH为5.5-8.0。
  6. 根据权利要求1-5中任一项所述的亲和纯化方法，其中，中间预洗脱缓冲液为中性缓冲液和/酸性缓冲液；优选的，所述中性缓冲液为磷酸盐缓冲液、三羟甲基氨基甲烷缓冲液或甘氨酸缓冲液；所述酸性缓冲液为柠檬酸-磷酸氢二钠缓冲液、醋酸-醋酸钠缓冲液或柠檬酸-柠檬酸三钠缓冲液；
    优选的，所述中间预洗脱缓冲液的pH为5.0-7.5。
  7. 根据权利要求6所述的亲和纯化方法，其中，在所述中间预洗脱缓冲液中加入预洗脱活性剂，优选的，所述预洗脱活性剂为盐酸胍、聚山梨酯80或氯化钠，进一步优选的，所述预洗脱活性剂为盐酸胍。
  8. 根据权利要求7所述的亲和纯化方法，其中，所述盐酸胍的浓度为0.01-1M。
  9. 根据权利要求1-8中任一项所述的亲和纯化方法，其中，在中间预洗脱淋洗后，在进行终洗脱之前还包括下述步骤：
    使用第二平衡缓冲液进行平衡，优选的，所述第二平衡缓冲液为磷酸盐缓冲液、Tris-HCl缓冲液或硼酸-硼砂缓冲液。
  10. 根据权利要求1-9中任一项所述的亲和纯化方法，其中，终洗脱缓冲液选自柠檬酸-磷酸氢二钠缓冲液、醋酸缓冲液、甘氨酸-HCl缓冲液和柠檬酸-柠檬酸钠缓冲液中的一种或两种以上，优选为柠檬酸-磷酸氢二钠缓冲液；
    优选的，所述柠檬酸-磷酸氢二钠缓冲液的pH为2.0-7.0。
  11. 一种抗人干扰素α受体1(IFNAR1)单克隆抗体浓缩溶液的制备方法，其包括下述步骤：
    第一超滤浓缩：在流速为120-300L/m 2·h和跨膜压差(TMP)为0.6-1.5bar的条件下将含有抗人干扰素α受体1(IFNAR1)单克隆抗体的溶液浓缩至蛋白质浓度为20-60mg/ml，得到浓缩样品；
    超滤置换：采用置换缓冲液置换浓缩样品，当置换缓冲液的使用量为浓缩样品重量的6-10倍时得到超滤置换溶液；
    第二超滤浓缩：将超滤置换溶液和碱性氨基酸母液混匀使得氨基酸的浓 度为100-150mM，进行超滤浓缩后得到蛋白质浓度为100-200mg/ml的浓缩溶液；
    所述抗IFNAR1单克隆抗体包含三个重链互补决定区(CDR-H1、CDR-H2和CDR-H3)和三个轻链互补决定区(CDR-L1、CDR-L2和CDR-L3)，其中：
    (a)CDR-H1的氨基酸序列如SEQ ID NO：1所示；
    (b)CDR-H2的氨基酸序列如SEQ ID NO：2所示；
    (c)CDR-H3的氨基酸序列如SEQ ID NO：3所示；
    (d)CDR-L1的氨基酸序列如SEQ ID NO：4所示；
    (e)CDR-L2的氨基酸序列如SEQ ID NO：5所示；且
    (f)CDR-L3的氨基酸序列如SEQ ID NO：6所示。
  12. 根据权利要求11所述的制备方法，其中，所述抗人干扰素α受体1(IFNAR1)单克隆抗体包含重链可变区和轻链可变区，其中，
    所述重链可变区的氨基酸序列如SEQ ID NO：7所示；且，
    所述轻链可变区的氨基酸序列如SEQ ID NO：8所示。
  13. 根据权利要求12所述的制备方法，其中，所述抗人干扰素α受体1(IFNAR1)单克隆抗体的重链的氨基酸序列如SEQ ID NO：10所示；轻链的氨基酸序列如SEQ ID NO：11所示。
  14. 根据权利要求11-13中任一项所述的制备方法，其中，在第一超滤浓缩中，将含有抗人干扰素α受体1(IFNAR1)单克隆抗体的溶液浓缩至蛋白质浓度为30-50mg/ml。
  15. 根据权利要求11-14中任一项所述的制备方法，其中，在超滤置换中，置换缓冲液的浓度为5-50mM，优选为20mM；优选的，置换缓冲液的pH为6.0-7.0，优选为6.2-6.8。
  16. 根据权利要求15所述的制备方法，其中，所述置换缓冲液为组氨酸-盐酸缓冲液、柠檬酸缓冲液、磷酸盐缓冲液或醋酸钠-醋酸缓冲液，优选为组氨酸-盐酸缓冲液。
  17. 根据权利要求111-6中任一项所述的制备方法，其中，在第二超滤浓缩中，所述碱性氨基酸为盐酸精氨酸、赖氨酸和/或脯氨酸，优选为盐酸精氨酸。
  18. 根据权利要求11-17中任一项所述的制备方法，其中，所述碱性氨 基酸母液的浓度为0.5-2.0mol/L，优选为1mol/L～1.5mol/L。
  19. 根据权利要求11-18中任一项所述的制备方法，其中，所述浓缩溶液的粘度≤20cP。
  20. 根据权利要求11-19中任一项所述的制备方法，其中，所述含有抗人干扰素α受体1(IFNAR1)单克隆抗体的溶液通过对表达抗人干扰素α受体1(IFNAR1)单克隆抗体的细胞发酵液进行亲和层析、低pH灭活、阴离子层析、阳离子层析和纳滤得到。
  21. 一种包含抗人干扰素α受体1(IFNAR1)单克隆抗体的液体制剂，所述液体制剂包含蛋白质浓度为100-200mg/ml的抗人干扰素α受体1(IFNAR1)单克隆抗体以及氨基酸浓度为5-300mM的碱性氨基酸；
    所述抗IFNAR1单克隆抗体包含三个重链互补决定区(CDR-H1、CDR-H2和CDR-H3)和三个轻链互补决定区(CDR-L1、CDR-L2和CDR-L3)，其中：
    (a)CDR-H1的氨基酸序列如SEQ ID NO：1所示；
    (b)CDR-H2的氨基酸序列如SEQ ID NO：2所示；
    (c)CDR-H3的氨基酸序列如SEQ ID NO：3所示；
    (d)CDR-L1的氨基酸序列如SEQ ID NO：4所示；
    (e)CDR-L2的氨基酸序列如SEQ ID NO：5所示；且
    (f)CDR-L3的氨基酸序列如SEQ ID NO：6所示。
  22. 根据权利要求21所述的液体制剂，其中，所述抗人干扰素α受体1(IFNAR1)单克隆抗体包含重链可变区和轻链可变区，其中，
    所述重链可变区的氨基酸序列如SEQ ID NO：7所示；且，
    所述轻链可变区的氨基酸序列如SEQ ID NO：8所示。
  23. 根据权利要求22所述的液体制剂，其中，所述抗人干扰素α受体1(IFNAR1)单克隆抗体的重链的氨基酸序列如SEQ ID NO：10所示；轻链的氨基酸序列如SEQ ID NO：11所示。
  24. 根据权利要求21-23中任一项所述的液体制剂，其中，所述碱性氨基酸选自精氨酸、赖氨酸、组氨酸和脯氨酸中的一种或两种以上。
  25. 根据权利要求24所述的液体制剂，其中，所述碱性氨基酸包含100-200mM的精氨酸和5-50mM的组氨酸。
  26. 根据权利要求24所述的液体制剂，其中，所述碱性氨基酸包含 100-200mM的赖氨酸和5-50mM的组氨酸。
  27. 根据权利要求24所述的液体制剂，其中，所述碱性氨基酸包含100-200mM的脯氨酸和5-50mM的组氨酸。
  28. 根据权利要求21-27中任一项所述的液体制剂，其中，所述液体制剂还包含20-150mg/ml的蔗糖，优选为50-100mg/ml的蔗糖。
  29. 根据权利要求21-24中任一项所述的液体制剂，其中，所述液体制剂还包含0.5-50mg/ml的山梨醇，优选为1-20mg/ml的山梨醇。
  30. 根据权利要求21-24中任一项所述的液体制剂，其中，所述液体制剂还包含20-300mg/ml的氯化钠，优选为100-200mg/ml的氯化钠；
    优选的，所述液体制剂的pH为5.5～6.5。
PCT/CN2021/114952 2021-07-13 2021-08-27 降低单克隆抗体生产中宿主细胞蛋白含量的亲和纯化方法、抗人ifnar1单克隆抗体浓缩溶液的制备方法以及液体制剂 WO2023284073A1 (zh)

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CN202110790531.5A CN113521276B (zh) 2021-07-13 2021-07-13 包含抗人干扰素α受体1(IFNAR1)单克隆抗体的液体制剂
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