WO2022041390A1 - Formulation liquide à faible viscosité comprenant un anticorps monoclonal anti-interleukine 23 humaine à concentration élevée, et son procédé de préparation - Google Patents

Formulation liquide à faible viscosité comprenant un anticorps monoclonal anti-interleukine 23 humaine à concentration élevée, et son procédé de préparation Download PDF

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WO2022041390A1
WO2022041390A1 PCT/CN2020/118635 CN2020118635W WO2022041390A1 WO 2022041390 A1 WO2022041390 A1 WO 2022041390A1 CN 2020118635 W CN2020118635 W CN 2020118635W WO 2022041390 A1 WO2022041390 A1 WO 2022041390A1
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cdr
amino acid
seq
acid sequence
monoclonal antibody
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PCT/CN2020/118635
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Chinese (zh)
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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 CN202010888836.5A external-priority patent/CN111944046B/zh
Priority claimed from CN202010898618.XA external-priority patent/CN111956606B/zh
Priority claimed from CN202010904172.7A external-priority patent/CN112159473B/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
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • 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
    • 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
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons

Definitions

  • the present invention relates to the field of antibody pharmaceutical preparations. Specifically, the present invention relates to a monoclonal antibody against human interleukin 23 (hIL-23), a low-viscosity liquid preparation comprising a high concentration of the monoclonal antibody, and a preparation method thereof, and the liquid preparation can be used as an injection, especially a subcutaneous injection .
  • hIL-23 human interleukin 23
  • a low-viscosity liquid preparation comprising a high concentration of the monoclonal antibody
  • a preparation method thereof a preparation method thereof
  • IL-23 is mainly produced by activated dendritic cells, macrophages and monocytes, and is a member of the IL-12 heterodimeric cytokine family, mainly composed of IL-23p19 and IL-12/IL-23p40. composed of subunits.
  • IL-23 receptor includes IL-12 receptor ⁇ 1 and IL-23 receptor 2 subunits.
  • IL-23 expresses receptor IL-23 on the surface of T cells, NK cells, monocyte macrophages/dendritic cells. 23R and IL-12R ⁇ 1 act to activate downstream signaling pathways to exert biological functions.
  • IL-23 mainly acts on Th17 cells, inducing them to produce IL-17A, IL-17F, IL-21, IL-22 and other pro-inflammatory cytokines. It plays an important role in autoimmune and inflammatory diseases such as encephalopathy and inflammatory bowel disease.
  • IL-23-mediated signaling and biological effects are associated with many types of diseases, including rheumatoid arthritis, juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, psoriatic arthritis, ankylosis Spondylitis, osteoarthritis, gastric ulcer, inflammatory bowel disease, ulcerative colitis, acute pancreatitis, primary biliary sclerosis, Hashimoto's thyroiditis, systemic lupus erythematosus, iridocyclitis, grapevine Meningitis, optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/Wegener's granulomatosis, allergic/atopic disease, asthma, allergic rhinitis, eczema, adult respiratory distress syndrome, allergic exposure Atopic dermatitis, vitiligo, psoriasis, alopecia areata, pemphigus, scleroderma, allergic/a
  • the preferred dosage form of anti-hIL-23 monoclonal antibody drugs is subcutaneous injection. Since the dose of subcutaneous injection is usually in the range of 100 mg to 600 mg, and the maximum subcutaneous injection volume is generally limited to less than 2 ml, the concentration of hIL-23 monoclonal antibody in hIL-23 monoclonal antibody injection administered by subcutaneous injection is usually It needs to reach more than 100 mg/mL, for example, 100-150 mg/mL.
  • a high concentration of a monoclonal antibody usually causes an increase in the viscosity of the solution containing the monoclonal antibody, and too high a viscosity will make it impossible to manually push the needle plunger to inject the drug subcutaneously. Therefore, usually for different monoclonal antibody molecules, it is necessary to specially adjust the components of the solution, pH and other conditions to obtain a low-viscosity liquid preparation containing a high concentration of the monoclonal antibody molecule, which can be used as injections, Especially subcutaneous injections.
  • HCP host cell protein
  • Quantitative determination of residual HCP in genetically engineered drugs is for quality control. An important means to help maintain the efficiency and consistency of the purification process. In antibody affinity purification, while ensuring high-efficiency antibody recovery, it is necessary to effectively remove HCP produced by engineered cells during the fermentation process.
  • the HCP content of the precipitated antibody samples was significantly reduced, but this process was not suitable for process scale-up, and the precipitation may have a certain impact on the activity of the antibody. Therefore, there are many process methods for removing HCP. Due to the differences in the fermentation process of the antibody samples and the properties of the antibodies, the selection process needs to be comprehensively considered.
  • the object of the present invention is to provide a monoclonal antibody against human interleukin 23 (hIL-23), a low-viscosity liquid preparation comprising a high concentration of the monoclonal antibody and a preparation method thereof, the liquid
  • hIL-23 human interleukin 23
  • a low-viscosity liquid preparation comprising a high concentration of the monoclonal antibody and a preparation method thereof, the liquid
  • the formulations can be used as injections, especially subcutaneous injections.
  • the first aspect of the present invention includes:
  • a liquid formulation comprising:
  • the anti-human interleukin-23 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-L3 represents light chain CDR3
  • SEQ ID NO: 6 QSGYVFAGLT
  • the basic amino acid refers to one or two selected from arginine (Arg, R), lysine (Lys, L), histidine (His, H) and proline (Pro, P). kind or three or four.
  • the above-mentioned liquid preparation is substantially free of impurity proteins (other proteins other than the anti-human interleukin-23 monoclonal antibody, the impurity protein content is less than 1%)
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and its amino acid sequence is EVQLVESGGGLVQPGGSLRLSCAASGFSLSNHEMSWVRQAPGKGLEWIGIITTSDTTYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVDIVLLSVTSRIWGQGTLVTVSS; and,
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8, and its amino acid sequence is DVVMTQSPSSLSASVGDRVTITCQASQSVSTYLSWYQQKPGKAPKLLIYGASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSGYVFAGLTFGGGTKVEIK.
  • the aforementioned liquid formulation further comprising 20-150 mg/mL (eg 50-100 mg/mL) of sucrose.
  • the aforementioned liquid preparation comprising 130 mg/mL or more of anti-human interleukin-23 monoclonal antibody, and having a viscosity of 15 cP or less.
  • the aforementioned liquid preparation comprising 150 mg/mL or more of anti-human interleukin-23 monoclonal antibody, and having a viscosity of 30 cP or less.
  • the aforementioned liquid preparation comprising 100 mg/mL or more of an anti-human interleukin-23 monoclonal antibody and having a viscosity of 10 cP or less.
  • the aforementioned liquid preparation which has a pH value of 5.5 to 6.5, preferably 6.0.
  • a first aspect of the present invention provides a liquid preparation comprising a novel anti-human interleukin 23 (hIL-23) monoclonal antibody, which comprises a high concentration (above 100 mg/mL) of the anti-human interleukin 23 monoclonal antibody and has a low Viscosity (less than 30cP), it can be easily injected with a syringe, so it is suitable for injection, especially subcutaneous injection.
  • hIL-23 novel anti-human interleukin 23
  • the novel anti-human interleukin 23 (hIL-23) monoclonal antibody compared with the existing anti-human interleukin 23 monoclonal antibodies (Guselkumab and Risankizumab), binds hIL-23 with comparable affinity, but at the cellular level
  • the antagonistic activity was superior to that of Guselkumab and comparable to that of Risankizumab.
  • Risankizumab It has been approved for marketing in Japan, the United States and the European Union, and its dosage form is subcutaneous injection.
  • liquid preparation of the present invention is also a subcutaneous injection, and the monoclonal antibody contained therein shows an antagonistic activity comparable to that of Risankizumab at the cellular level, the liquid preparation of the present invention is expected to show good clinical effects in the prevention and treatment of related diseases.
  • a second aspect of the present invention includes:
  • a preparation method of anti-human IL-23 monoclonal antibody concentrated solution it comprises the following steps:
  • Step A The first ultrafiltration concentration step: for the solution containing the anti-human IL-23 monoclonal antibody, the flow rate is 110-320 L/m 2 ⁇ h, and the transmembrane pressure difference (TMP) is maintained at 0.7-1.4 bar. Concentrating the solution containing the anti-human IL-23 monoclonal antibody to a protein concentration of 40-50 mg/mL to obtain a first ultrafiltration concentrate;
  • Step B Equal volume buffer replacement step: use a peristaltic pump to pump the buffer to be replaced into the first ultrafiltration concentrate, continue ultrafiltration, and adjust the pumping speed and permeability of the buffer to be replaced. The flow rate is consistent, and when the pumping amount of the buffer to be replaced reaches 6 to 10 times the weight of the first ultrafiltration concentrate, the equal volume buffer replacement is completed, and the ultrafiltration after the equal volume buffer replacement is obtained.
  • Concentrate wherein, the buffer to be replaced is selected as 10-30 mM histidine-hydrochloric acid buffer, and the pH range is between 5.5 and 6.0;
  • Step C The second ultrafiltration concentration step: adjust the arginine concentration in the ultrafiltration concentrated solution after the equal volume of buffer replacement, so that the arginine concentration is 100mM to 200mM, and then perform ultrafiltration concentration, so that the protein concentration 100 ⁇ 200mg/mL, obtain the second ultrafiltration concentrated solution (that is, the concentrated solution of anti-human IL-23 monoclonal antibody); wherein, add the concentration of 1M to the ultrafiltration concentrated solution after the equal volume buffer replacement ⁇ 2M arginine stock solution, so that the final concentration of arginine is 100mM ⁇ 200mM;
  • the anti-human interleukin-23 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-L3 represents light chain CDR3
  • SEQ ID NO: 6 QSGYVFAGLT
  • the anti-human interleukin-23 monoclonal antibody comprises a heavy chain variable region and a light chain variable region
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and its amino acid sequence is EVQLVESGGGLVQPGGSLRLSCAASGFSLSNHEMSWVRQAPGKGLEWIGIITTSDTTYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVDIVLLSVTSRIWGQGTLVTVSS; and,
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8, and its amino acid sequence is DVVMTQSPSSLSASVGDRVTITCQASQSVSTYLSWYQQKPGKAPKLLIYGASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSGYVFAGLTFGGGTKVEIK.
  • the protein concentration of the solution comprising the anti-human IL-23 monoclonal antibody in the step A is less than 15 mg/mL.
  • the protein concentration of the solution comprising the anti-human IL-23 monoclonal antibody in the step A is less than 10 mg/mL.
  • the buffer to be replaced in the step B is a 20 mM histidine-hydrochloric acid buffer with a pH value of 6.0.
  • the arginine concentration in the ultrafiltration concentrate after the equal volume of buffer replacement in the step C is 130 mM to 160 mM.
  • the protein concentration of the second ultrafiltration concentrate in the step C is 150 mg/mL or more, and the viscosity thereof is 30 cP or less.
  • the preparation method is suitable for ultrafiltration concentration of 200L pilot scale.
  • the solution comprising the anti-human IL-23 monoclonal antibody in the step A is carried out by affinity chromatography, low pH inactivation, anion chromatography, cation chromatography and nanofiltration.
  • the method of reducing the viscosity of the high-concentration antibody liquid and improving the stability in the ultrafiltration liquid exchange process is simple and feasible, can be scaled up, can ensure the high purity of the sample and obtain a high recovery rate.
  • a concentrated solution of high-concentration and low-viscosity anti-human IL-23 monoclonal antibody with a protein concentration of 130 mg/mL or more and a viscosity of 10 cP or less can be prepared on a 200L pilot scale.
  • Antibody concentrate solution can be used to prepare anti-human IL-23 monoclonal antibody subcutaneous injection.
  • a third aspect of the present invention includes:
  • a purification method of a recombinant humanized anti-human interleukin-23 monoclonal antibody comprising carrying out affinity chromatography on a cell fermentation broth supernatant comprising a recombinant humanized anti-human interleukin-23 monoclonal antibody, the And chromatography includes the following steps:
  • Step A Before sample loading, use equilibration buffer A to equilibrate the affinity chromatography column;
  • Step B Load the cell fermentation broth supernatant containing the recombinant humanized anti-human interleukin-23 monoclonal antibody onto the affinity chromatography column; (Is the cell fermentation broth supernatant directly loaded?)
  • Step C after loading, use the equilibration buffer A to equilibrate the affinity chromatography column again;
  • Step D pre-eluting the affinity column with a pre-elution buffer
  • Step E Equilibrate the affinity chromatography column with Equilibration Buffer B.
  • Step F final elution of the affinity column with elution buffer, and sample collection
  • the anti-human interleukin-23 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-L3 represents light chain CDR3
  • SEQ ID NO: 6 QSGYVFAGLT
  • the anti-human interleukin-23 monoclonal antibody comprises a heavy chain variable region and a light chain variable region
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and its amino acid sequence is EVQLVESGGGLVQPGGSLRLSCAASGFSLSNHEMSWVRQAPGKGLEWIGIITTSDTTYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVDIVLLSVTSRIWGQGTLVTVSS; and,
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8, and its amino acid sequence is DVVMTQSPSSLSASVGDRVTITCQASQSVSTYLSWYQQKPGKAPKLLIYGASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSGYVFAGLTFGGGTKVEIK.
  • the affinity chromatography column is a Protein A column.
  • the equilibration buffer A is phosphate buffer, Tris-HCl buffer or boric acid-borax buffer, the salt concentration is 5mM-0.25M, and the pH is 5.5-8.0.
  • the equilibration buffer A may contain NaCl or Na 2 SO 4 to reduce the non-specific adsorption between non-antibody proteins and Protein A filler, and the concentration of NaCl and/or Na 2 SO 4 is preferably below 250mM.
  • the equilibration buffer A is preferably a phosphate buffer.
  • the salt concentration of the equilibration buffer A is preferably 5mM-0.15M, more preferably 10mM-50mM; its pH is preferably pH6.5-7.5.
  • the pre-eluting buffer is phosphate buffer, citrate buffer, Tris-HCl buffer or sodium acetate-acetate buffer, and its salt concentration is 0.001M-0.5 M, its pH is 5.0-7.5.
  • the salt concentration of the pre-elution buffer is preferably 0.05M to 0.2M.
  • the pH of the pre-elution buffer is preferably 5.5-6.5.
  • the pre-elution buffer comprises 0.01M-1.0M guanidine hydrochloride.
  • concentration of guanidine hydrochloride is preferably between 0.05M and 0.3M.
  • the elution buffer is a citric acid-disodium hydrogen phosphate buffer, an acetate buffer, a glycine-HCl buffer or a citric acid-sodium citrate buffer, and its salt concentration is 5 ⁇ 100mM, its pH is 2.5-4.0.
  • the salt concentration of the elution buffer is preferably 20-50 mM, and its pH is preferably 2.9-3.8.
  • the equilibration buffer B is a phosphate buffer Tris-HCl buffer or a boric acid-borax buffer, and its salt concentration is 5mM-0.15M, and its pH is 5.5-8.0.
  • the equilibration buffer B may contain NaCl or Na 2 SO 4 to maintain conductivity while maintaining partial buffering capacity, and the concentration of NaCl and/or Na 2 SO 4 is preferably below 250 mM, for example, may be 10-100 mM.
  • a method for reducing host cell protein content in a recombinant humanized anti-human interleukin-23 monoclonal antibody preparation comprising using the purification method described in any one of the preceding paragraphs to quantify a recombinant humanized anti-human interleukin-23 monoclonal antibody.
  • the antibody is purified from the cell fermentation supernatant.
  • the antibody Protein A affinity purification process in the present invention is simple and feasible, and can be amplified and purified for production, the cell fermentation supernatant does not need to be pre-treated, the elution sample yield is high, and the HCP residue is also kept at a low level. (residual control amount is not higher than 0.5%), thereby reducing the pressure of removing HCP in subsequent purification steps, thereby ensuring that the residual amount of HCP in the final antibody sample is at a very low level.
  • the verification of the affinity purification process for different batches of fermentation supernatant in the invention proves that the affinity purification process in the invention has good stability.
  • the possible mechanism that the present invention can achieve the above technical effect is to use the pre-elution of affinity chromatography to reduce the interaction between HCP and the antibody and the filler matrix, that is, the active reagent guanidine hydrochloride can weaken the HCP with the antibody protein and the filler to varying degrees.
  • the force between the media plays a role in removing HCP.
  • FIG. 1 is a graph showing the results of nucleic acid electrophoresis for the construction of the QX004N (HZD90-32) transient expression plasmid.
  • M Marker
  • Band 1 PCR product 90VH-Hu18
  • Band 2 pHZDCH, HindIII/NheI
  • Band 3 PCR product 90VK-Hu9
  • Band 4 pHZDCK, HindIII/BsiWI.
  • Figure 2 is a flow chart of transient expression.
  • Figure 3 is the electrophoresis detection chart of QX004N (HZD90-32).
  • human interleukin 23 refers to a human-derived protein, which is a heterodimer composed of two subunits, p19 and p40.
  • the amino acid sequence of p19 is shown in SEQ ID NO: 9
  • the amino acid sequence of p40 is shown in SEQ ID NO: 10, wherein the underlined part represents the signal peptide.
  • anti-human interleukin-23 monoclonal antibody refers to a monoclonal antibody capable of binding human interleukin-23 with sufficient affinity such that the monoclonal antibody can be used as a diagnostic agent targeting human interleukin-23 and /or therapeutic agent.
  • the experimental results show that the novel anti-human interleukin 23 (IL-23) monoclonal antibody can specifically bind to the p19 subunit of human interleukin 23 (IL-23).
  • the new anti-human interleukin 23 (IL-23) monoclonal antibody is comparable to or superior to the same type of monoclonal antibody products on the market in many biological activities.
  • the biological activities include, for example, inhibition of IL-23-induced STAT3 phosphorylation in cells, inhibition of IL23-induced IL-17A release from mouse splenocytes, and IL-23-induced IL-23-induced IFN- ⁇ release from human NK cells.
  • amino acid sequence of the heavy chain of the novel anti-human interleukin 23 (IL-23) monoclonal antibody is shown in SEQ ID NO: 11; the amino acid sequence of the light chain is shown in SEQ ID NO: 12 .
  • SEQ ID Nos: 11 and 12 are both humanized sequences.
  • anti-human interleukin-23 monoclonal antibody QX004N in the following examples is the new anti-human interleukin-23 monoclonal antibody.
  • Procured human interleukin 23 (IL-23) from Shanghai Nearshore Technology Co., Ltd., used to immunize New Zealand rabbits, used B cell cloning technology to obtain antigen-binding specific antibody clones, and then screened for binding to IL-23 and having IL-23 inhibitory activity of monoclonal antibodies.
  • the cell supernatant was detected by Binding ELISA to select clones that bind to IL-23; then, Blocking ELISA was used to detect the clones with IL-23 inhibitory activity.
  • the above immunization and screening processes are entrusted to commercial companies.
  • the 90# clone was humanized.
  • the homology alignment of human IgG germline sequence (Germline) was performed using NCBI IgBlast, IGHV3-66*01 was selected as the heavy chain CDR transplantation template, and the CDR region of the 90# cloned heavy chain (ie CDR-H1 (SEQ ID No: 1), CDR-H2 (SEQ ID No: 2) and CDR-H3 (SEQ ID No: 3)) were transplanted into the framework region of IGHV3-66*01; IGKV1-39*01 was selected as the light chain CDR transplant template, and the 90# CDR regions of cloned light chain (i.e.
  • CDR-L1 (SEQ ID No: 4), CDR-L2 (SEQ ID No: 5) and CDR-L3 (SEQ ID No: 6)) were grafted into IGKV1-39*01
  • QX004N variable region QX004N variable region.
  • 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 gene of the above-mentioned heavy chain variable region (SEQ ID NO:7) is obtained by PCR amplification; the gene of the light chain variable region (SEQ ID NO:8) is obtained by PCR amplification.
  • the heavy chain expression plasmid pHZDCH was digested with HindIII and NheI; the light chain expression plasmid pHZDCK was digested with HindIII and BsiWI; the PCR amplified genes were inserted into the corresponding expression plasmids with Infusion recombinase to construct the heavy chain expression plasmid pHZDCH- 90VH-Hu18 and light chain expression plasmid pHZDCK-90VK-Hu9.
  • Figure 1 shows the results of double-enzyme digestion of plasmids 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 double-enzyme digestion of the heavy chain and light chain expression plasmids, wherein the plasmid sizes of the heavy chain and light chain are about 10000bp, the light chain variable region is about 438bp, and the heavy chain variable region is about 459bp.
  • ExpiCHO-S cells were co-transfected with the correct heavy chain expression plasmid and light chain expression plasmid. One day before transfection, ExpiCHO-S cells were diluted to 3 ⁇ 10 6 cells/mL for pre-transfection passage. On the day of transfection, the cell density was diluted to 6 ⁇ 10 6 cells/mL, and 25 mL of cells were placed in 125 mL shake flasks, waiting for transfection. The transfection and expression process is shown in Figure 2.
  • the culture supernatant was harvested for one-step purification with ProteinA.
  • the purified antibody was detected by SDS-PAGE electrophoresis and named as QX004N (HZD90-32).
  • the results of detection of the antibody by protein electrophoresis are shown in FIG. 3 .
  • the protein electrophoresis was detected by denaturing reducing gel, and the results in Figure 3 showed that there were two bands, the sizes of the two bands were about 50kDa and 25kDa respectively, which were consistent with the theoretical molecular weights of the heavy chain (49.1kDa) and the light chain (23.1kDa).
  • the affinity of QX004N (HZD90-32) to IL-23 was detected by BiacoreT200, and all procedures were performed at 25°C.
  • a commercial Protein A chip was used, and an appropriate amount of antibody was immobilized by the capture method, so that the Rmax was around 50RU, and the capture flow rate was 10 ⁇ l/min.
  • the antigen was serially diluted, the flow rate of the instrument was switched to 30 ⁇ l/min, and the concentration flowed through the reference channel and the immobilized antibody channel in order of concentration from low to high, and the buffer was used as a negative control.
  • the chip was regenerated with pH 1.5 glycine after each binding and dissociation was completed.
  • the 1:1 binding model in the Kinetics option was selected by the instrument's own analysis software for fitting, and the on-rate constant ka , the dissociation rate constant k d and the dissociation equilibrium constant K D value of the antibody were calculated.
  • the data in the table are: each sample was tested twice and the average value was calculated.
  • QX004N can inhibit IL-23-induced STAT3 phosphorylation activity in HEK Blue TM IL-23 cells with IC 50 of 3.21ng/mL; Guselkumab and Risankizumab can also inhibit IL-23-induced HEK Blue TM IL-23 cells
  • the phosphorylation activity of STAT3 in IL-23 and its IC 50 were 6.18ng/mL and 3.51ng/mL, respectively, indicating that QX004N inhibited the signal transduction activity induced by IL-23 and the currently commercialized monoclonal antibody against IL-23, Risankizumab. Activity was comparable and superior to Guselkumab.
  • QX004N can inhibit IL-23-induced IL-17A release from mouse spleen cells with an IC 50 of 11.7ng/mL; Guselkumab and Risankizumab can also inhibit IL-23-induced IL-17A release from mouse spleen cells, Its IC 50s were 13.5ng/mL and 8.43ng/mL, respectively, indicating that QX004N has a strong inhibitory activity against IL-23-induced IL-17A release from mouse splenocytes, and its ability to compete with existing commercial products (Guselkumab and Risankizumab) quite.
  • QX004N can inhibit IL-23-induced release of IFN- ⁇ from human NK cells with an IC 50 of 10.4ng/mL; Guselkumab and Risankizumab can also inhibit IL-23-induced release of IFN- ⁇ from human NK cells with an IC 50 of 10.4 ng/mL; 50 were 16.8ng/mL and 11.1ng/mL, respectively, indicating that the activity of QX004N in inhibiting the release of IFN- ⁇ from human NK cells induced by IL-23 was stronger than that of the currently commercialized product Guselkumab, and was comparable to that of Risankizumab.
  • QX004N is superior to Guselkumab in the three biological activities measured at the cellular level, but it is incomparable with Risankizumab. Given Risankizumab It has been confirmed in clinical trials that it has a significant therapeutic effect on moderate to severe plaque psoriasis, and QX004N is also expected to show good clinical effects in the prevention and treatment of related diseases.
  • an ultrafiltration concentrate (containing 20 mM) was obtained. His-HCl, pH 6.0).
  • the SEC-HPLC method was used to analyze, and it was determined that the QX004N monomer in the ultrafiltration concentrate was more than 99%, the polymer was less than 1%, and basically did not contain impurity proteins.
  • the concentration of QX004N in the ultrafiltration concentrate was determined by UV spectrophotometry to be 170 mg/mL.
  • the viscosity of the ultrafiltration concentrate was determined to be 63.4 cP using a Riosen's ⁇ VISC viscometer.
  • liquid formulations with a viscosity of less than 30 cP are suitable for use as subcutaneous injections. It can be seen from Table 3 that the subcutaneous administration concentration of QX004N can reach at least 100-150 mg/mL through the above-mentioned liquid preparation.
  • an ultrafiltration concentrate (containing 20 mM) was obtained. His-HCl, pH 6.0).
  • the SEC-HPLC method was used to analyze, and it was determined that the QX004N monomer in the ultrafiltration concentrate was more than 99%, the polymer was less than 1%, and basically did not contain impurity proteins.
  • the concentration of QX004N in the ultrafiltration concentrate was determined by UV spectrophotometry to be 130 mg/mL.
  • the viscosity of the ultrafiltration concentrate was determined to be 63.4 cP using a Riosen's ⁇ VISC viscometer.
  • CHO cells are used as host cells to ferment QX004N in a 2L-scale bioreactor.
  • the fermentation broth is obtained by centrifuge or deep membrane filtration, and Protein A chromatography is used to capture the target protein, and then undergo low pH virus inactivation. Clarification, anion and cation chromatography remove impurities and obtain intermediate samples to be ultrafiltered.
  • the above-mentioned intermediate was used to investigate the ultrafiltration process, and the ultrafiltration equipment was selected from the Labscale small ultrafiltration apparatus of Merck Millipore (holding two 50cm 2 Pellicon XL membrane packs, the interception amount was 30KD).
  • the above intermediate sample was concentrated to about 40 to 50 mg/mL, and then replaced with an equal volume of buffer solution.
  • the intermediate sample after liquid exchange is concentrated to 170 mg/mL using a 30kDa ultrafiltration centrifuge tube of Merck Millipore, and the sample is filtered with a 0.2 ⁇ m filter membrane.
  • the viscosity value was measured after the buffer solution or additive mother solution was mixed, and the specific preparation scheme and viscosity value results are shown in Table 2 below.
  • the viscosity of QX004N solution increases exponentially. If no additives are added, the viscosity of the monoclonal antibody will reach 63cP when the concentration is 170mg/mL. Such a high viscosity value cannot be used.
  • the concentration process is achieved using conventional ultrafiltration membranes. Taking 150mg/mL protein concentration as an example, by adding 100mM-200mM basic amino acid or its combination, the antibody viscosity value under the same protein concentration condition can be reduced to less than 50% of the original, less than 20cP. Based on the above experimental results, the administration concentration of QX004N solution can be realized to be 100-150 mg/mL.
  • the nanofiltered sample (UF0) was subjected to the first step concentration (UF1) at a flow rate of 120 to 300 L/m 2 ⁇ h, and the TMP was controlled at 0.6 to 1.5 bar.
  • the sample was concentrated to 40 to 50 mg/mL; then UA was used.
  • Fermentation of antibody QX004N CHO cells were used as host cells, Dynamis was used as fermentation basal medium, and cells were cultured by conventional cell culture process.
  • the harvest was started, and the harvested liquid was subjected to deep filtration using the primary filter MDOHC10FS1 and the secondary filter MX0HC10FS1, and the clarified cell culture supernatant was collected and recorded as the fermentation broth intermediate.
  • the fermentation broth intermediate was subjected to affinity chromatography and loaded on a Protein A column (Merck 0.15L), and the loading capacity was set to 45mg/ml.
  • equilibrate with equilibration buffer A 12 mmol/L Na 2 HPO 4 , 8 mmol/L NaH 2 PO 4 , 0.15 mol/L NaCl; after loading, equilibrate with equilibration buffer A again until the fermentation broth flows completely Then use different pre-elution buffers to rinse, the composition of pre-elution buffers is shown in Table 1 below; then use equilibration buffer B: 6mmol/L Na 2 HPO 4 , 4mmol/L NaH 2 PO 4 , equilibrated at pH 7.2; then eluted with elution buffer PB: 7mmol/L Na 2 HPO 4 , 15mmol/L citric acid, pH 3.1 and collected the elution samples, and the collected elution samples were subjected to antibody concentration
  • Antibody concentration determination method :
  • 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 sample dilution in one step should not exceed 10 times, and the minimum sampling amount should not be less than 5 ⁇ l.
  • Loading Add standard, sample, and spiked samples in a certain arrangement (two duplicate wells for each, standard products do not need duplicate wells), 50 ⁇ l/well, and seal the plate. Place in a horizontal shaker at room temperature, 180 rpm, for 2 hours, protected from light.
  • Wash the plate discard the liquid in the well, add 300 ⁇ l/well of washing solution with a multi-channel pipette, let it stand for 30 seconds, shake off the liquid, pat dry on absorbent paper, and wash the plate 4 times. After the last plate wash, try to pat dry the residual wash solution in the wells.
  • Color development and termination reading add 100 ⁇ l/well of TMB reagent (TMB Substrate), let stand for color development for 30 minutes, and protect from light. After 30 minutes, 100 ⁇ l/well of Stop Solution was added, and the microplate reader was read at 450 nm, with 650 nm as the reference.
  • TMB reagent TMB Substrate
  • CHO cell protein residue (%) average measured value of sample (ng/ml) ⁇ dilution multiple/protein content of undiluted sample (mg/ml) ⁇ 10 -4 (%).
  • 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 experimental group containing guanidine hydrochloride was used.
  • the HCP residue of the sample is less than 0.05%, the load of removing HCP 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; in the experimental group using polysorbate 80 or NaCl, the HCP residue in the sample after affinity chromatography is still greater than 0.2%, and the removal effect of HCP residue is worse than that of guanidine hydrochloride.
  • Example 8 Comparison of HCP scavenging effects of different batches of recombinant humanized anti-IL-23 monoclonal antibody (QX004N) fermentation broth
  • Example 1 The preparation of the fermentation broth intermediate and the method of affinity chromatography are the same as those in Example 1.
  • a total of three batches of fermentation broth intermediates were prepared, and the pre-eluting buffer solution containing guanidine hydrochloride was used for pre-eluting and washing: 0.1 mol/L Sodium citrate, 0.1 mol/L guanidine hydrochloride, 11 mmol/L citric acid, pH 5.8 for washing.
  • the test methods for the yield of the affinity process and the residual HCP content are as described in Example 1.
  • the HCP residues of the intermediate QX004N 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 and chromatography were all lower than 0.05%, and the removal of HCP by guanidine hydrochloride and elution eluting was stable.
  • a monoclonal antibody against human interleukin 23 hIL-23
  • a low-viscosity liquid preparation comprising a high concentration of the monoclonal antibody, and a preparation method thereof, and the liquid preparation can be used as an injection, especially subcutaneously injection.

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Abstract

L'invention concerne une formulation liquide de faible viscosité comprenant un anticorps monoclonal anti-interleukine 23 humaine de haute concentration, et son procédé de préparation. La formulation liquide peut être utilisée comme une injection, en particulier une injection sous-cutanée. La formulation liquide comprend 100 mg/ml ou plus d'un anticorps monoclonal anti-interleukine 23 humaine et de 10 à 500 mM d'un acide aminé basique. La concentration d'administration par injection sous-cutanée de l'anticorps monoclonal anti-interleukine 23 humaine peut atteindre au moins de 100 à 150 mg/ml. Le procédé de préparation de la formulation liquide comprend un procédé de concentration et de purification et un procédé d'élimination des protéines de la cellule hôte.
PCT/CN2020/118635 2020-08-28 2020-09-29 Formulation liquide à faible viscosité comprenant un anticorps monoclonal anti-interleukine 23 humaine à concentration élevée, et son procédé de préparation WO2022041390A1 (fr)

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CN202010888836.5A CN111944046B (zh) 2020-08-28 2020-08-28 高浓度、低粘度抗人il-23单克隆抗体溶液的制备方法
CN202010888836.5 2020-08-28
CN202010898618.X 2020-08-31
CN202010898618.XA CN111956606B (zh) 2020-08-31 2020-08-31 包含高浓度抗人白介素23单克隆抗体的低粘度液体制剂
CN202010904172.7 2020-09-01
CN202010904172.7A CN112159473B (zh) 2020-09-01 2020-09-01 重组人源化抗人白介素23单克隆抗体的纯化方法

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