WO2016127954A1 - 获得高产稳定表达细胞克隆的方法及由此获得的抗体分子 - Google Patents

获得高产稳定表达细胞克隆的方法及由此获得的抗体分子 Download PDF

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WO2016127954A1
WO2016127954A1 PCT/CN2016/076135 CN2016076135W WO2016127954A1 WO 2016127954 A1 WO2016127954 A1 WO 2016127954A1 CN 2016076135 W CN2016076135 W CN 2016076135W WO 2016127954 A1 WO2016127954 A1 WO 2016127954A1
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cell
antibody
cells
protein
clones
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French (fr)
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美林
胡里奥
米盖尔
路明
塔摩拉
罗兰多
白帜
刘月茂
肖凯珩
陈晓
和振花
蔡杨柳
杨振华
白先宏
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百泰生物药业有限公司
分子免疫中心
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Priority to JP2017560865A priority Critical patent/JP2018506306A/ja
Priority to KR1020177022618A priority patent/KR20180029948A/ko
Priority to MYPI2017702967A priority patent/MY192147A/en
Priority to NZ735485A priority patent/NZ735485A/en
Priority to EA201791828A priority patent/EA201791828A1/ru
Priority to AU2016218641A priority patent/AU2016218641C1/en
Priority to EP16748755.2A priority patent/EP3257935A4/en
Priority to CUP2017000106A priority patent/CU20170106A7/xx
Application filed by 百泰生物药业有限公司, 分子免疫中心 filed Critical 百泰生物药业有限公司
Priority to CA2974027A priority patent/CA2974027C/en
Priority to SG11201706595TA priority patent/SG11201706595TA/en
Priority to TNP/2017/000302A priority patent/TN2017000302A1/en
Priority to US15/549,087 priority patent/US10457747B2/en
Priority to MX2017010421A priority patent/MX2017010421A/es
Priority to BR112017017303A priority patent/BR112017017303B8/pt
Publication of WO2016127954A1 publication Critical patent/WO2016127954A1/zh
Priority to IL253932A priority patent/IL253932A0/en
Priority to CONC2017/0008087A priority patent/CO2017008087A2/es

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    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3084—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated gangliosides
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
    • 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
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention relates to the field of biotechnology, and in particular to an efficient method for obtaining a highly productive stable expression cell clone for producing a therapeutic antibody in a perfusion fermentation process.
  • Therapeutic antibodies constitute a major class in the biotech drug market (Walsh G, 2014, Nature Biotechnology 2014, 32: 992–1000). Some therapeutic antibodies have been approved for the treatment of cancer, autoimmune diseases and other chronic diseases, and dozens of recombinant antibodies are also at different clinical stages (Biologic Medicines in Development, Phrma Report 2013, www.phrma.org). Usually, patients receive therapeutic antibodies, which are several hundred milligrams per dose. Therefore, the current demand for antibody production worldwide is enormous.
  • the perfusion fermentation-based production process allows for high density of cell culture and potentially high antibody concentrations in the fermented harvest.
  • long-term high-density cell culture requires high-yield, stably expressed cell clones to truly optimize antibody production.
  • Protein-free media has been developed for the production of biopharmaceuticals, for example, PFHMII cell culture media (purchased from Hyclone, USA).
  • the NSH cell line produced by recombinant antibodies has been successfully adapted to protein-free medium (WO 2004/038010 A1), however, long-term fermentation processes adapted to serum-free medium and the next serum-free medium are usually accompanied by cells. Loss of productivity (Barnes et al., 2003, Biotechnol Bioeng 81: 631-639; Barnes et al., 2004, Biotechnol Bioeng 85: 115-121). High-yield, stable-expressing cell clones adapted to protein-free media can be re-obtained from unstable recombinant myeloma cell lines (CN104152415A).
  • Biological drugs are very complex glycoprotein molecules. Any change in the production process can result in changes in product characteristics. The concept of comparability has therefore emerged to evaluate these variants of human biological products, including therapeutic biotechnology-derived Products,Center for Biologics Evaluation and Research (CBER), Center for DrugEvaluation and Research (CDER)April 1996.www.fda.gov/Drugs/GuidanceComplianceRegulatory Information/Guidances/ucm122879.htm;EU Guideline on Comparability of Medicinal Products containing Biotechnology- Derived Proteins as Active Substances: Quality issues (CPMP December 2003).
  • CBER Center for Biologics Evaluation and Research
  • CDER Center for DrugEvaluation and Research
  • ICH Q5E Comparability of Biotechnological/Biological Products Subject to Changes in their Manufacturing Process.EU: Adopted by CMPM, December 1, 2004, CPMP/ICH/5721/03, date for coming into operation: June 2005; MHLW: Adopted 26April 2005, PFSB/ELD Notification No. 0426001; FDA: Published in the Federal Register, Vol. , No. 125, June 30, 2005; 37861-2www.ich.org/fileadmin/Public Web Site/ICH Products/Guidelines/Quality/Q5E/Step4/Q5E Guideline.pdf).
  • 14F7 is a monoclonal antibody that specifically binds to the tumor associated antigen N-hydroxyacetyl-GM3 ganglioside [GM3(Neu5Gc)] (ZL 99800261.5; Carr et al., 2000, Hybridoma 19, 241-247).
  • GM3(Neu5Gc) antigen has been used in breast cancer (Marquina et al., 1996, Cancer Res 56, 5165-5171; Oliva et al., 2006, Breast Cancer Res Treat 96, 115-121), melanoma (Osorio et al., 2008).
  • 14F7 mAb is capable of killing ganglioside-expressing tumor cells in a complement-dependent manner (Carr et al., 2002, Hybrid Hybridomics 21, 463-468; Roque-Navarro et al., 2008, Mol Cancer Ther 7, 2033-2041).
  • Humanized antibodies obtained by modification of potential human T cell epitopes (CN1809592A; Mateo et al., 2000, Hybridoma 19, 463-471), designated 14F7h, retain the properties of murine and chimeric antibodies (Fernandez-Marrero et Al., 2011, Immunobiology 216, 1239-1247).
  • the humanized 14F7h mAb has the potential to treat tumors expressing GM3 (NeuGc).
  • NSO myeloma cell lines expressing 14F7h antibodies lost their ability to survive in high-density cell culture media. Force, because these cells express the antigen GM3 (Neu5Gc) and are then killed by secreted cytotoxic antibodies.
  • the recombinant humanized 14F7h antibody was expressed by the N-glycolylation-sugar complex-deficient murine NSO myeloma cell line due to the action of CMP-N-acetylneuraminidase (Fernandez-Marrero et al., 2011, Immunobiology 216, 1239-1247).
  • Such cell lines have shown difficulty in adapting to growth in serum-free medium.
  • the method comprises the adaptation of a recombinant myeloma cell line from a serum-free medium to a protein-free medium, which is unlikely to occur or takes a long time due to the emergence of a non-producing cell population, and is accompanied by antibody production. loss.
  • a key innovative step of the invention resides in the adaptation process of growth at high cell densities (8–10) x 10 6 cells/ml in protein-free medium, which increases the frequency of high-yield, stable expression of cell clones.
  • the method of the invention consists of three steps:
  • Adapt to protein-free medium use low-density stationary cell culture to gradually reduce lipid-rich supplements into chemical medium;
  • a cell agonist rich in cholesterol, lipids and other nutrients
  • PHMII protein-free medium
  • the maximum cell concentration (Xv) varies from (0.9–1.2) x 10 6 cells/ml. Pass through successively until Xv reaches a constant value. The cell concentration starting after one passage was adjusted to (0.4–0.5) x 10 6 cells/ml. After 7 passages, the cytostatic supplement was reduced to (1-2) g/L, and after 4 passages, the cytostatic supplement was completely removed. The cells were then allowed to grow for more than 60 days in protein-free medium without any supplements. The Xv value reached (1.5–1.8) x 10 6 cells/ml, and the cell viability value was around 95%.
  • cells grown in PFHMII cell culture medium for more than 60 days were inoculated into a 5 L bioreactor.
  • the perfusion external device was used to grow the cell concentration from 0.5x10 6 cells/ml to 10x10 6 cells/ml, and the perfusion process lasted for 24 days. From day 0 to day 16, cell viability remained above 90%, but after day 16, cell viability dropped below 80%.
  • the average antibody concentration in the cell harvest was at (30–40) mg/ml.
  • the cells grown for more than 500 hours in protein-free medium
  • a cell clone having high yield and stable expression after fermentation is collected, and cells thawed from EPCB are subjected to cell dilution by limiting dilution method (Freshney R., 2010, Culture of animal cells: a manual) Of basic technique and specialized applications (6th ed.). Hoboken, N.J.: Wiley-Blackwell.pp. 208-211).
  • the secreted antibody in the cell culture supernatant was detected by an ELISA method using an anti-14F7h anti-idiotype antibody 4G9 as a capture antigen.
  • the cell culture supernatant was diluted to 1/500. Clones with absorbance values above the median were selected for structural confirmation for the next flow cytometry method.
  • Intracellular immunostaining was performed by coupling FITC with an anti-human IgG antibody and flow cytometry (Pluschke et al., 2011, BMC Proceedings 5, Suppl 8: P97).
  • the mean fluorescence intensity (MFI) and the percentage of positive cells were detected for each of the isolated clones. Surprisingly, more than 80% of the clones evaluated showed the dominant antibody-production subpopulation.
  • Antibodies with more than 95% cell viability - clones producing subpopulations and higher MFI were selected for kinetic studies in roller bottles and 5 L bioreactors to evaluate cell concentration (Xv), cells in cell culture supernatants Viability (%), specific growth rate ( ⁇ ), and IgG concentration.
  • the cell concentration was (4–5) x 10 6 cells/ml, and the cell viability in the logarithmic growth phase was approximately 85%.
  • the specific growth rate variation range is (0.015–0.025) h -1 .
  • the IgG concentration is (70–120) mg/L.
  • long-term stability studies of highly productive and stably expressing cell clones were performed by kinetic studies at 30, 60, and 90 days of continuous cell culture. Specific growth rate ( ⁇ ) and specific production rate (qp) were detected.
  • the characteristic properties of immunoglobulin secreted by high-yield, stably expressed cell clones are identified.
  • the disclosure of this characteristic property defines the molecular phenotype of this therapeutic antibody 14F7h from an operational level.
  • the antitumor activity of the recombinant antibody 14F7h in vitro and in vivo was evaluated.
  • Figure 1 Cell growth in PFHM-II medium adapted to the addition of different concentrations of Cell Boost 5.
  • Figure 3 Identification of ELISA absorbance values. The black line is the intermediate value of the result. Purified 1 ug/mL T1h and hR3 were used as negative controls. Screening clones 35D8, 35D6, 31E7, 31C7, 23E9, 23C2, 73F4, 73E5, 72G5, 72F6, 72D3 (grey), discarding clones 35F7, 33E8, 23E4, 22D8, 21E6, 21C5, 73C4, 72D9, 71F2, 62E7, 61E3 (black).
  • Figure 4 Cells screened by flow cytometry analysis to determine the percentage of high expressing cell subpopulations.
  • the NS0 myeloma cell line served as a negative control, and the recombinant NSO myeloma cell line expressing the hR3 antibody served as a positive control.
  • Clones 31E7, 23E9, 33E8, 35D6, 23C2, 72F6, 73F4, 72G5 (grey) were screened and clones 72D3, 73E5 (black) were discarded.
  • Figure 5 Determination of MFI values for high expression cell subpopulations.
  • Myeloma cell lines expressing hR3 and T1h antibodies served as positive controls (light grey).
  • Clones 31E7, 35D6, 72G5 (moderate gray) were screened and clones 23E9, 33E8, 72D3, 73E5, 23C2, 72F6, 73F4 (black) were discarded.
  • Figure 6 Kinetic study (y-axis) of clonal growth curves, cell viability, overall viable cell count over time (x-axis) in roller bottles and 5 L bioreactors.
  • Figure 7 Comparability analysis of maximum specific growth rate and maximum IgG concentration of screened clones in roller bottles and 5L fermenters.
  • Figure 8 14F7h antibody produced by different screening clones recognizes NeuGcGM3 gangliosides. The supernatant was taken from a 5 L bioreactor. The anti-EGFR antibody was used as a negative control, and the 14F7h antibody produced by the parental cell was used as a positive control.
  • Figure 9 Intracellular concentration of IgG measured by flow cytometry at different screening stages.
  • Figure 10 Comparison of kinetic studies of parental cells and clone 31E7 in a 5L bioreactor.
  • XV viable cell concentration
  • SXV overall viable cell number
  • IgG maximum antibody concentration.
  • Figure 11 Cellular thawing for 30 days, 60 days and 90 days post kinetic studies evaluated the stability of clone 31E7.
  • Cell concentration (Xv), cell activity (%), overall viable cell number (SX V ), IgG concentration, maximum growth rate ( ⁇ ), specific production rate (QP) were measured.
  • Figure 12 Determination of intracellular IgG content from clone 31E7 stability study samples by flow cytometry.
  • Figure 13 Deconvoluted mass spectrum of the 14F7h light chain in the native state (top panel) and deglycosylation (bottom panel, using PNGase F), the sample is reduced/alkylated. Conventional LC-MS conditions, sample separation/desalting on a C8 column, and acetonitrile/formic acid elution buffer system operation. The inserted graph is an enlarged view of the main peak region.
  • Figure 14 Deconvoluted mass spectrum of the 14F7h heavy chain in the native state (top panel) and deglycosylation (bottom panel, using PNGase F), the sample is reduced/alkylated. Conventional LC-MS conditions, sample separation/desalting on a C8 column, and acetonitrile/formic acid elution buffer system operation. The inserted graph is an enlarged view of the main peak region.
  • Figure 15 Deconvoluted mass spectrum of the 14F7h state of the entire molecule in its native state (top panel) and deglycosylation (bottom panel, using PNGase F). Conventional LC-MS conditions, sample separation/desalting on a C8 column, and acetonitrile/formic acid elution buffer system operation. The inserted graph is an enlarged view of the main peak region.
  • Figure 16 Peptide map of 14F7h obtained after trypsinization, separated by reverse phase HPLC using a C4 column and a conventional acetonitrile/TFA buffer system.
  • Figure 17 Circular dichroism spectroscopy obtained at 25 ° C using a 2 mm path length cuvette, 14F7 in the far ultraviolet (205-260 nm) region. The spectrum was obtained at a sample concentration of 0.6 mg/mL.
  • Figure 18 Fluorescence emission spectra of tryptophan were read between 310 and 390 nm after stimulation of 14F7h molecules at 280 nm using a Varioskan Flash device. A 96-well plate was used, 200 uL per well, and the sample concentration was 0.2 mg/mL.
  • Figure 19 Analysis of glycosylation properties of 2-AB-labeled polysaccharide isolated from 14F7h, separated by normal phase HPLC fluorescence detection (Ex: 330 nm/Em: 420 nm). The dotted line in the box indicates an enlarged view of the secondary peak.
  • Figure 21 Weak cation exchange map of 14F7h obtained at 280 nm with a Propac-WCX10 column. A 30 ug sample was injected.
  • Figure 22 SEC-HPLC plot of 14F7 obtained at 280 nm using a TSK-G3000sxl column.
  • Figure 23 Dose-response curve of flow cytometry obtained by different antibody samples on different days using L1210 target cells (log value of positive % versus 14F7h concentration (ug/mL)). .
  • Figure 24 Cytotoxic effect induced by 14F7h mAb in X63 mouse myeloma cells.
  • A Binding properties of the 14F7h monoclonal antibody. X63 cells were stained with 10 ⁇ g/mL antibody and then stained with FITC-conjugated rabbit anti-human IgG antibody. Herceptin mAb (anti-human Her-2) was used as a negative control.
  • B X63 cells were treated with 100 ⁇ g/mL of monoclonal antibody. After incubation at 37 ° C for 6 hours, cell viability was assessed by uptake of propidium iodide (PI) and flow cytometry analysis. Cytotoxicity is expressed as the percentage of cells stained with PI. Herceptin monoclonal antibody was used as a negative control.
  • PI propidium iodide
  • Figure 25 In vivo anti-tumor effect of 14F7h monoclonal antibody in a mouse myeloma model.
  • A Monoclonal administration schedule. On day 0, BALB/c mice were subcutaneously inoculated with X63 mouse myeloma cells (0.2 x 10 6 ), and antibodies (300 ⁇ g) were injected intravenously at 2-5 days.
  • B Kaplan-Meier curve without tumor survival until 106 days.
  • the humanized monoclonal antibody T1h (anti-human CD6) was used as a negative control. Statistical analysis was performed using a log-rank test.
  • Example 1 Adaptation to protein-free medium, low-density resting cell culture, and stepwise reduction of lipid-rich supplements into chemical medium;
  • the 14F7htb58 cell line is suitable for growth in PFHMII (Protein-free Hybridoma Medium) cell culture medium that does not supplement Cell Boost 5.
  • PFHMII Protein-free Hybridoma Medium
  • the process of reducing the supplemental Cell Boost 5 was carried out in a 75 cm 2 flask, shaker stirring (80 rpm), 36.5 ° C, 5% CO 2 .
  • the concentration of viable cells and the percentage of cell viability were determined every 48-72 hours. In each subculture, the cell concentration was adjusted to (0.4-0.5) x 10 6 cells/ml.
  • 14F7htb58 cells were thawed in PFHMII supplemented with 3.5 g/L cell boost 5. Thawed to a cell viability of 75% and then increased to over 90% (Figure 1). In the adaptation process, subculture was carried out to maintain the maximum concentration of living cells in the range of 0.9-1.8 ⁇ 10 6 cells/ml, and the percentage of cell viability was higher than 90%. After 11 subcultures, the concentration of Cell Boost 5 was reduced to zero, the cell viability was 95%, and the concentration was (1.2-1.8) x 10 6 cells/ml. The adaptation process was approximately 60 days, and there was no lipid or cholesterol in the PFHMII cell culture medium.
  • Example 2 Adaptation to protein-free medium: high-density cell culture, perfusion fermentation system on a laboratory scale; 14F7htb58 cells adapted to grow in protein-free PFHMII cell culture medium were seeded in a 5 L fermentor at a cell concentration of (0.4 -0.5) x10 6 cell/ml.
  • the operating parameters of the fermenter are: pH 6.9-7.0, 105 RPM, 40% dissolved oxygen, (36.5-37.0) ° C, working volume 3.5L. Cell viability and cell concentration were routinely monitored by using the Neubauer chamber trypan blue exclusion (Sigma). When the cell concentration reaches 2.5 x 10 6 cells/ml, the fermentor is operated in perfusion mode.
  • the perfusion mode uses a hollow fiber cartridge with a dilution rate of (0.3-0.7) VVD.
  • the cell viability remained above 90% during the fermentation process for 384 hours and then reduced to 80%.
  • the antibody concentration is in the range of (30-40) mg/L.
  • the maximum cell concentration reached 9x10 6 cell / ml (FIG. 2).
  • cells were seeded in roller bottles at the end of the fermentation process, at a concentration of 0.8x10 6 cell / ml, after 72 hours of incubation, cells were 12x10 6 cells / freezing vial (final production cell banks, End Production Cell Bank, EPC) .
  • the cell viability was 90% after thawing, and the cell viability was 97% after 96 hours of culture.
  • Example 3 Screening for high-yield, stable expression of cell clones from cells at the end of fermentation
  • Intracellular IgG levels were determined by flow cytometry (FACS) in screened clones.
  • Anti-human IgG antibody was coupled to a 1:200 dilution of FITC (fluorescein isothiocyanate) (Sigma) for use as a probe. 4x10 5 cells/sample were analyzed to determine the percentage of markers. More than 95% of clones of high expressing cell subsets were screened (Fig. 4).
  • Another screening criterion is the standardized median fluorescence intensity (FMI), and the screened clones are 35D6, 72G5, and 31E7 ( Figure 5).
  • the samples were subjected to kinetic studies in a 5 L bioreactor for evaluation of the biological activity of the secreted antibodies. Detection by sandwich ELISA method.
  • the Polysorp plate was coated with a NeuGcGM3 ganglioside solution (10 ⁇ g/ml) dissolved in methanol ( ⁇ g/ml).
  • An anti-human heavy chain goat antibody conjugated to alkaline phosphatase was used as the second antibody. All samples were adjusted to an antibody concentration of 1 ⁇ g/ml and diluted 1/10 in the ELISA test. All tested samples showed recognition of ganglioside antigens, clones 31E7 and 35D6 relative to positive controls, with values ranging from 70% to 80% ( Figure 8).
  • the intracellular IgG content is a function of phenotypic adaptation and cloning.
  • the parental cell 14F7htB58 was grown in PFHMII with Cell Boost5 and exhibited a bimodal distribution due to the presence of a non-producing cell subpopulation.
  • 14F7htB58 cells were enriched to produce a subpopulation of cells (monomodal distribution), whereas for clone 31E7, a narrow single peak was obtained suggesting a more uniform subpopulation of cells ( Figure 9).
  • clone 31E7 has a higher maximum cell concentration, overall viable cell count, and antibody production rate than the parental cells (Fig. 10).
  • the production stability of the clone 31E7 antibody was evaluated during 90 days of cell culture. Kinetic studies were performed in roller bottles at 30, 60 and 90 days after cell thawing. The extracted samples were subjected to flow cytometry studies. There were no significant differences at different stages of cell culture. The maximum cell concentration from 3.5x10 6 cell / ml to 4.5x10 6 cell / ml variation, cell viability greater than 90%, at an antibody concentration of 50mg / L to 80mg / L (FIG. 10) change. The specific growth rate ( ⁇ ) was maintained above 0.025 h -1 and the specific production rate (QP) was higher than 0.18 pg/cell * h (Fig. 11). After 90 days of cell culture, clone 31E7 showed a narrow single peak, which is representative of the same fluorescence intensity values of the same high expression cell population (Fig. 12).
  • Example 5 Secreted immunoglobulin characteristic properties of screened stable high production cell clone 31E7
  • the high-level structure was analyzed at two levels, and the secondary structure (Fig. 17) and intrinsic fluorescence characteristics were analyzed by using far ultraviolet CD spectra, which were able to detect conformational differences and protein stability (Weichel et al, 2008, BioProcess). International, June).
  • Figure 18 shows the test results. In this case, the maximum emission wavelength was obtained at 333 nm, and the absorbance ratio at 330 nm/350 nm was 1.23.
  • G0F%, G1F%, G2F% fucosylated galactose-free, fucosylated monogalactose, fucosylated digalactose, fucosyl glycan%;
  • WCX weak cation exchange
  • WCX weak cation exchange
  • WCX weak cation exchange
  • WCX weak cation exchange
  • the result of this method is primarily to monitor C-terminal lysine truncation, a conventional change found on hIgG1 molecules (Dionex Application Note 127, http://www.dionex-france.com/ Library/literature/application_notes_updates/AN127_LPN1047.pdf)).
  • Figure 21 and Table 3 show the results of the integration.
  • SEC Size Exclusion Chromatography
  • Figure 22 shows the spectra obtained for the different samples
  • Table 4 shows the results of the analysis.
  • Example 6 Evaluation of anti-tumor effects in vivo and in vitro
  • Antigen expression of X63 mouse myeloma cells was determined by flow cytometry. 100% of the X63 mouse myeloma cells were stained with 14F7h mAb (Fig. 24A). X63 cells were incubated with 10 ⁇ g/mL antibody and then incubated with FITC-conjugated rabbit anti-human IgG antibody. Herceptin mAb (anti-human Her-2) was used as a negative control.
  • the in vitro cytotoxic effect induced by 14F7h monoclonal antibody was evaluated in X63 mouse myeloma cells.
  • X63 cells were treated with 100 ⁇ g/mL of 14F7h mAb. After incubation at 37 ° C for 6 hours, use ingested propidium iodide (PI) and flow cytometry Valence cell activity. Cytotoxicity is expressed as the percentage of cells stained with PI. More than 50% of tumor cells died after treatment (Fig. 24B).
  • Herceptin monoclonal antibody was used as a negative control.

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Abstract

提供了一种在无蛋白培养基中从骨髓瘤细胞系获得高产稳定表达细胞克隆的方法，该方法用于工业上生产重组抗体，包括三个阶段：（1）适应无蛋白培养基，低密度静止细胞培养，逐步减少富含脂质的补充物到化学培养基中；（2）适应无蛋白培养基：以高密度细胞培养，在实验室规模采用灌流发酵系统；和（3）发酵结束时从细胞中筛选高产稳定表达的细胞克隆。该细胞克隆可用于生产人源化的抗NeuGcGM3 14F7h重组抗体。

Description

获得高产稳定表达细胞克隆的方法及由此获得的抗体分子 技术领域
本发明涉及生物技术领域，尤其是涉及一种有效的获得高产稳定表达细胞克隆的方法，该方法用于在灌流发酵工艺中生产治疗性抗体。
背景技术
治疗性抗体构成了生物技术药物市场的主要类别(Walsh G,2014,Nature Biotechnology2014,32:992–1000)。一些治疗性抗体已经获得注册批准用于治疗癌症、自身免疫性疾病和其他慢性病，几十种重组抗体也正处于不同的临床阶段(Biologic Medicines in Development,Phrma Report 2013,www.phrma.org)。通常，病人接受治疗性抗体，每一个剂量下要几百毫克，因此，目前全世界范围内对抗体产能的需求是巨大的。
几种方法已经被用于提高工业细胞系的产能，例如，基因扩增系统，细胞培养基的优化和选择高产稳定表达的细胞克隆。基因修饰和重组骨髓瘤细胞系的表观遗传适应来生产治疗性抗体是目前非常有竞争性的研究领域(Barnes et al.,2000,Cytotechnology 32:109-123；Barnes et al.,2007,Biotechnol Bioeng 96:337-349)。
基于灌流发酵的生产工艺允许在发酵收获液中有高密度的细胞培养和潜在的高抗体浓度，然而，长期的高密度细胞培养需要高产稳定表达的细胞克隆来真正优化抗体的生产。无蛋白培养基已经被开发出来用于生物药的生产，例如，PFHMII细胞培养基(购自美国Hyclone公司)。
重组抗体生产的NS0细胞系已经被成功适应于无蛋白培养基(WO 2004/038010 A1)，然而，适应于无血清培养基和下一步的无血清培养基中的长时间发酵工艺通常伴随着细胞系产能的损失(Barnes et al.,2003,Biotechnol Bioeng 81:631-639；Barnes et al.,2004,Biotechnol Bioeng 85:115-121)。适应于无蛋白培养基的高产稳定表达细胞克隆能从不稳定的重组骨髓瘤细胞系中重新获得(CN104152415A)。
但是实践中，一些重组骨髓瘤细胞系从无血清培养基到无蛋白培养基的适应过程是不可能的或者需要很长的时间，同时由于非生产细胞群的出现而导致抗体产能的损失。具有工业潜能的细胞系的选择工艺必须不断改进。
生物药品，尤其治疗性抗体是很复杂的糖蛋白分子。生产工艺中的任何变化都可能会导致产品特性的变化。可比较性的概念因此出现用于评估产品特性上的这些变(Demonstration of comparability of human biological products,including therapeutic biotechnology-derived  products,Center for Biologics Evaluation and Research(CBER),Center for DrugEvaluation and Research(CDER)April 1996.www.fda.gov/Drugs/GuidanceComplianceRegulatory Information/Guidances/ucm122879.htm；EU Guideline on Comparability of Medicinal Products containing Biotechnology-derived Proteins as Active Substances:Quality issues(CPMP December2003).www.emea.europa.eu/pdfs/human/bwp/320700en.pdf；ICH Q5E:Comparability of Biotechnological/Biological Products Subject to Changes in their Manufacturing Process.EU:Adopted by CMPM,December 1,2004,CPMP/ICH/5721/03,date for coming into operation:June2005；MHLW:Adopted 26April 2005,PFSB/ELD Notification No.0426001；FDA:Published in the Federal Register,Vol.70,No.125,June 30,2005；37861-2www.ich.org/fileadmin/Public Web Site/ICH Products/Guidelines/Quality/Q5E/Step4 /Q5E Guideline.pdf)。细胞系的改变被认为是生产工艺的重要修饰，因此，尽管具有工业潜能的细胞系的选择在生产工艺的开发中是绝对必要的，但是任何选择的具有稳定性和高表达量特性的细胞系是否适合是不确定的，因为分泌的免疫球蛋白的一些变化可能会影响它的生物学性质，对于生产工艺改造而言，如生产规模或生产场所，每一个特异性抗体的属性特征的确认都是下一步可比性研究的先决条件。
14F7是一种特异性结合肿瘤相关抗原N-羟乙酰基-GM3神经节苷脂[GM3(Neu5Gc)](ZL 99800261.5；Carr et al.,2000,Hybridoma 19,241-247)的一种单克隆抗体。GM3(Neu5Gc)抗原已经在乳腺癌(Marquina et al.,1996,Cancer Res 56,5165-5171；Oliva et al.,2006,Breast Cancer Res Treat 96,115-121)、黑色素瘤(Osorio et al.,2008,Cancer BiolTher 7,488-495)、非小细胞肺癌(van Cruijsen et al.,2009,BMC Cancer 9,180；Hayashi et al.,2013,Cancer Sci 104,43-47)、Wilms瘤(Scursoni et al.,2010,Pediatr Dev Pathol 13,18-23)、神经外胚瘤(Scursoni et al.,2011,Clin Dev Immunol 245181)、肉瘤和甲状腺癌(Blanco et al.,2013,Journal of Biomarkers,602417)和消化系统(Blanco et al.,2011,ISRN Gastroenterol 645641)以及泌尿生殖系统(Blanco et al.,2011,ISRN Pathology,953803)的肿瘤中被检测出来。
14F7单抗能够杀死补体依赖方式表达神经节苷脂的肿瘤细胞(Carr et al.,2002,Hybrid Hybridomics 21,463-468；Roque-Navarro et al.,2008,Mol Cancer Ther 7,2033-2041)。通过潜在的人T细胞表位的修饰获得的人源化抗体(CN1809592A；Mateo et al.,2000,Hybridoma 19,463-471)，命名为14F7h，保留了鼠和嵌合抗体的性质(Fernandez-Marrero et al.,2011,Immunobiology 216,1239-1247)。人源化的14F7h单抗具有潜在的治疗GM3(NeuGc)表达的肿瘤的价值。
然而，能表达14F7h抗体的重组NS0骨髓瘤细胞系在高密度细胞培养基中丧失生存能 力，因为这些细胞表达抗原GM3(Neu5Gc)，然后被分泌的细胞毒抗体所杀死。因此，重组人源化的14F7h抗体被N-羟乙酰基化-糖复合物缺陷的鼠NS0骨髓瘤细胞系表达，因为CMP-N-乙酰神经氨酸酶的作用(Fernandez-Marrero et al.,2011,Immunobiology 216,1239-1247)。这样的细胞系显示出很难适应在无血清培养基中生长。
在本发明中我们建立了一套新的方法用于开发高产稳定表达的细胞克隆用于灌流发酵生产工艺。采用这种方法，细胞克隆从生产抗-GM3(NeuGc)的单抗的重组NS0骨髓瘤细胞系中重新获得。这种治疗性抗体的属性特征也在本发明中被公开，这种属性特征能够确定生物药的分子表型。
发明内容
本方法包含重组骨髓瘤细胞系从无血清培养基到无蛋白培养基的适应过程，因为非生产细胞群的出现，该过程是不可能发生的或需要很长的时间，并且伴随着抗体产量的损失。本发明关键的创新步骤在于无蛋白培养基中以高细胞密度(8–10)x106cells/ml生长的适应过程，这个过程能够增加高产稳定表达细胞克隆的频率。
本发明的方法由三个步骤组成：
1、适应无蛋白培养基：以低密度静止细胞培养，逐步减少富含脂质的补充物到化学培养基中；
2、适应无蛋白培养基：以高密度细胞培养，在实验室规模采用灌流发酵系统；
3、发酵结束后，从收获液细胞中挑选高产稳定表达的细胞克隆。
第一步，在无蛋白培养基(PFHMII)中补充(3-4)g/L的细胞促剂(富含胆固醇、脂质和其他营养物)，解冻细胞。最大细胞浓度(Xv)的变动范围是(0.9–1.2)x106cell/ml。连续传代，直到Xv达到一个恒定值。一次传代后起始的细胞浓度被调整到(0.4–0.5)x106cell/ml。7次传代后，细胞促剂补充物减少至(1-2)g/L，再经4次传代后，细胞促剂补充物被完全转移走。然后，细胞被允许在无任何补充物的无蛋白培养基中生长超过60天。Xv值达到(1.5–1.8)x106cell/ml，细胞活力值在95％左右。
第二步，在PFHMII细胞培养基中生长超过60天的细胞被接种到5L的生物反应器中。应用灌流外部装置，细胞浓度从0.5x106cells/ml生长到10x106cells/ml，灌流过程持续24天。从第0天到第16天，细胞活力维持在90％以上，但是第16天之后，细胞活力降到80％以下。细胞收获液中平均的抗体浓度在(30–40)mg/ml。发酵结束后，细胞(在无蛋白培养基中生长超过500小时)被冻存到液氮中(最终生产细胞库,EPCB)。
在本发明的又一个实施例中，发酵结束后高产稳定表达的细胞克隆被收集，从EPCB中解冻的细胞经有限稀释法进行细胞克隆(Freshney R.,2010,Culture of animal cells:a manual  of basic technique and specialized applications(6th ed.).Hoboken,N.J.:Wiley-Blackwell.pp.208–211)。细胞培养上清中的分泌抗体通过ELISA方法来检测，采用抗-14F7h抗独特型抗体4G9作为捕获抗原。细胞培养上清稀释到1/500。吸光值超过中值的克隆被选择出来用于下一步的流式细胞仪方法进行的结构确证。细胞内免疫印染用抗人IgG抗体偶联FITC进行，流式细胞仪检测(Pluschke et al.,2011,BMC Proceedings 5,Suppl 8:P97)。每一个分离的克隆都检测了平均荧光强度(MFI)和阳性细胞百分比，令人吃惊的是，超过80％的被评价的克隆都显示出其为占主导的抗体——生产亚群。
细胞活力超过95％的抗体——生产亚群和更高MFI的克隆被挑选出来在转瓶和5L的生物反应器中进行动力学研究来评价细胞培养上清中的细胞浓度(Xv)、细胞活力(％)、比生长速率(μ)和IgG浓度。细胞浓度为(4–5)x106cells/ml，对数生长期的细胞活力为大约85％。比生长速率变动范围是(0.015–0.025)h-1。IgG浓度是(70–120)mg/L。
本发明的又一个实施例中，进行了高产稳定表达细胞克隆的长期稳定性研究，通过在持续细胞培养的30、60、90天的动力学研究来进行。比生长速率(μ)和比生产速率(qp)被检测。
仍旧在本发明的又一个实施例中，高产稳定表达的细胞克隆分泌的免疫球蛋白的特征属性被鉴别。这种特征属性的公开从操作层面上定义了这种治疗性抗体14F7h的分子表型。
Figure PCTCN2016076135-appb-000001
仍旧在本发明的又一个实施例中，评价了重组抗体14F7h的体内体外的抗肿瘤活性。
附图说明
图1：适应添加不同浓度Cell Boost 5的PFHM-II培养基中的细胞生长。
图2、5L生物反应器发酵运转中，适应无cell boost 5的PFHM-II培养基中的细胞生长。
图3：识别ELISA吸光度值。黑线是结果的中间值。纯化的1ug/mL T1h和hR3作为阴性对照。筛选克隆35D8、35D6、31E7、31C7、23E9、23C2、73F4、73E5、72G5、72F6、72D3(灰色)，丢弃克隆35F7、33E8、23E4、22D8、21E6、21C5、73C4、72D9、71F2、62E7、 61E3(黑色)。
图4：通过流式细胞仪分析筛选的细胞，测定高表达细胞亚群的百分比。NS0骨髓瘤细胞系作为阴性对照，表达hR3抗体的重组NS0骨髓瘤细胞系作为阳性对照。筛选克隆31E7、23E9、33E8、35D6、23C2、72F6、73F4、72G5(灰色)，丢弃克隆72D3、73E5(黑色)。
图5：测定高表达细胞亚群的MFI值。表达hR3和T1h抗体的骨髓瘤细胞系作为阳性对照(着浅灰色)。筛选克隆31E7、35D6、72G5(中度灰色)，丢弃克隆23E9、33E8、72D3、73E5、23C2、72F6、73F4(黑色)。
图6：在滚瓶和5L生物反应器中评估随时间(x轴)的克隆生长曲线、细胞活力、整体活细胞数的动力学研究(y轴)。
图7：滚瓶和5L发酵罐中筛选克隆的最大比生长速率和最大IgG浓度的可比性分析。
图8：不同筛选克隆生产的14F7h抗体识别NeuGcGM3神经节苷脂。上清液取自5L生物反应器。抗EGFR抗体作为阴性对照，亲本细胞生产的14F7h抗体作为阳性对照。
图9：在不同筛选阶段用流式细胞仪测定的IgG细胞内浓度。
图10：5L生物反应器中亲本细胞和克隆31E7的动力学研究比较。XV：活细胞浓度；SXV：整体活细胞数；IgG：最大抗体浓度。
图11：细胞解冻30天，60天和90天后动力学研究评价克隆31E7稳定性。测定细胞浓度(Xv)，细胞活性(％)，整体活细胞数(SXV)，IgG浓度，最大生长速率(μ)，比生产速率(QP)。
图12：用流式细胞仪测定来自克隆31E7稳定性研究样品的IgG细胞内含量。
图13：14F7h轻链在天然状态(上图)和去糖基化(下图，使用PNGase F)状态的解卷积质谱，样品是还原/烷基化的。常规LC-MS条件，C8柱进行样品分离/脱盐，乙腈/甲酸洗脱缓冲系统运行。插入的图是主峰区的放大图。
图14：14F7h重链在天然状态(上图)和去糖基化(下图，使用PNGase F)状态的解卷积质谱，样品是还原/烷基化的。常规LC-MS条件，C8柱进行样品分离/脱盐，乙腈/甲酸洗脱缓冲系统运行。插入的图是主峰区的放大图。
图15：14F7h整个分子在天然状态(上图)和去糖基化(下图，使用PNGase F)状态的解卷积质谱。常规LC-MS条件，C8柱进行样品分离/脱盐，乙腈/甲酸洗脱缓冲系统运行。插入的图是主峰区的放大图。
图16：胰蛋白酶消化后，用C4柱和常规乙腈/TFA缓冲系统反相HPLC分离后获得的14F7h的肽图。
图17：25℃，使用2mm路径长度比色皿，14F7在远紫外线(205-260nm)区得到的圆二色光谱分析。光谱是样品浓度为0.6mg/mL时获得的。
图18：使用Varioskan Flash设备，280nm处刺激14F7h分子后，在310和390nm中间读取色氨酸的荧光发射光谱。使用96孔板，每孔200uL，样品浓度为0.2mg/mL。
图19：从14F7h分离的2-AB标记多糖的糖基化性能分析，用正相HPLC荧光检测分离(Ex:330nm/Em:420nm)。方框内的虚线表示次峰的放大图。
图20：14F7h不同样品(N＝3)的糖基化参数，用于研究糖基化性能分析的偏差范围。
图21：用Propac-WCX10柱，在280nm处获得的14F7h的弱阳离子交换图。注射30ug样品。
图22：用TSK-G3000sxl柱，在280nm处获得的14F7的SEC-HPLC图。
图23：使用L1210靶细胞对不同抗体样品在不同日期检测获得的流式细胞仪的剂量-反应曲线(阳性％对14F7h浓度(ug/mL)的log值)。。
图24：X63小鼠骨髓瘤细胞内由14F7h单抗诱导的细胞毒性作用。A：14F7h单克隆抗体的结合特性。X63细胞用10μg/mL抗体染色，然后用FITC-偶联的兔抗人IgG抗体染色。赫赛汀单抗(抗人Her-2)作为阴性对照。B：用100μg/mL单抗处理X63细胞。37℃、6小时孵育后，通过碘化丙啶(PI)的摄取和流式细胞术分析来评价细胞活性。细胞毒性用PI染色的细胞百分比表示。赫赛汀单克隆抗体作为阴性对照。
图25：小鼠骨髓瘤模型中14F7h单抗的体内抗肿瘤作用。A：单抗给药时间表。第0天对BALB/c小鼠皮下接种X63小鼠骨髓瘤细胞(0.2×106)，在2-5天静脉注射抗体(300μg)。B：直到106天没有肿瘤生存的Kaplan-Meier曲线。人源化单克隆抗体T1h(抗人CD6)作为阴性对照。用对数秩检验进行统计分析。
具体实施方式
下面的实施例用于说明本发明但并不限于它的范围。现有技术方法的详细说明未提供。
实施例1：适应无蛋白培养基，低密度静止细胞培养，逐步减少富含脂质的补充物到化学培养基中；
14F7htb58细胞系适于生长在不补充Cell Boost 5的PFHMII(Protein-free Hybridoma Medium)细胞培养基中。在75cm2的烧瓶中进行减少补充Cell Boost 5的过程，摇床搅拌(每分钟80转)，36.5℃，5％CO2。每48-72小时测定活细胞的浓度和细胞活力的百分比。在每个传代培养中，细胞浓度调节到(0.4-0.5)x106cell/ml。
在补充3.5g/L cell boost 5的PFHMII中解冻14F7htb58细胞。解冻到细胞活力为75％， 然后提高到90％以上(图1)。在适应过程中，进行传代培养，维持活细胞的最大浓度在0.9-1.8×106cell/ml的范围内，细胞活力百分比高于90％。11次传代培养后Cell Boost 5的浓度降至零，细胞活力为95％，浓度为(1.2-1.8)x106cell/ml。适应过程大约60天，PFHMII细胞培养液中没有脂质和胆固醇。
实施例2：适应无蛋白培养基：以高密度细胞培养，在实验室规模采用灌流发酵系统；适应生长在无蛋白质PFHMII细胞培养基中的14F7htb58细胞接种在5L发酵罐中，细胞浓度为(0.4-0.5)x106cell/ml。发酵罐的运行参数为：pH值为6.9-7.0，105RPM，40％溶氧，(36.5-37.0)℃，工作容积3.5L。通过使用纽鲍尔室(Neubauer chamber)台盼蓝排除法(Sigma)日常监控细胞活力和细胞浓度。细胞浓度达到2.5x106cell/ml时，在灌流模式下操作发酵罐，。灌流模式使用中空纤维筒，稀释率为(0.3-0.7)VVD。
在发酵过程中细胞活力保持90％以上，达384个小时，然后将其降低到80％。抗体浓度在(30-40)mg/L的范围内。稀释率为0.7VVD时，最大细胞浓度达到9x106cell/ml(图2)。在发酵过程结束时取细胞接种在滚瓶中，浓度为0.8x106cell/ml，培养72小时后，细胞以12x106细胞/小瓶冷冻(最终生产细胞库，End Production Cell Bank,EPC)。解冻后细胞活力为90％，96小时培养后细胞活力为97％。
实施例3：发酵结束时从细胞中筛选高产稳定表达的细胞克隆
采用补充(5-10)％胎牛血清的DMEM-F12 1:1细胞培养基，有限稀释法将从EPC中解冻的细胞克隆到96孔板中，每孔一个细胞。36.5℃、5％CO2培养板孵育。克隆效率低于2％。细胞克隆20天后，取培养基上清液，通过夹层式ELISA评价IgG浓度。抗独特型抗体4G9(3ug/ml)用作捕获抗原，偶联碱性磷酸酶的抗人重链羊抗体作为探针。所有样品1/500稀释，检测上清液吸光度的中值。选择高于中值的克隆(23C2,23E9,31C7,31E7,35D6,35D8,72D3,72F6,72G5,73E5,73F4)(图3)。选择的克隆被扩增到24孔板中，补充(5-10)％胎牛血清的DMEM-F12 1：1细胞培养基。进一步将细胞扩增到T培养瓶中，使用PFHMI细胞培养基，摇床搅拌(每分钟80转)，36.5℃，5％CO2。
在筛选的克隆中用流式细胞仪(FACS)测定细胞内IgG含量。抗人IgG抗体偶联1:200稀释的FITC(荧光素异硫氰酸酯)(Sigma)用作探针。对4x105细胞/样品进行分析，确定标记的百分比。筛选95％以上的高表达细胞亚群的克隆(图4)。另一筛选标准是标准化的荧光强度中值(FMI)，筛选的克隆为35D6、72G5和31E7(图5)。
这些克隆在滚瓶和5L生物反应器(36.5-37)℃，(100-105)RPM，pH值<7，溶解氧高于40％)中扩增，进行动力学研究。克隆31E7在滚瓶和5L生物反应器中表现出最高细胞 浓度，分别为5x106cell/ml和4x106cell/ml。整体活细胞数也显示类似的结果，在滚瓶中浓度值为4.5x108cell/ml*h，5L生物反应器中浓度值为2.5x108cell/ml*h，这些浓度值比亲本细胞获得的浓度值(图6)高1.3-1.4倍。对所有评估的克隆来说，细胞活力在对数生长期高于85％(图6)。克隆31E7在滚瓶和5L生物反应器中都表现出最高比生长速率(>0.025h-1)。所有克隆在5L生物反应器中抗体浓度为(50–70)mg/L，而31E7在滚瓶中表现出最高抗体浓度(图7A和7B)。
样品在5L生物反应器中进行动力学研究，用于评价所分泌抗体的生物活性。夹层式ELISA方法检测。用溶于甲醇(μg/ml)的NeuGcGM3神经节苷脂溶液(10μg/ml)包被Polysorp板。使用偶联碱性磷酸酶的抗人重链的羊抗体作为第二抗体。所有的样品都调节至1μg/ml的抗体浓度，ELISA测试中1/10稀释。所有测试的样品都表现出识别神经节苷脂抗原，克隆31E7和35D6相对于阳性对照，数值为70％-80％(图8)。
细胞内的IgG含量是随着表型适应和克隆的过程而变化的。亲代细胞14F7htB58在加Cell Boost5的PFHMII中生长，由于非生产细胞亚群的存在而呈现出双峰分布。适应于在PFHMII培养基中高密度生长后，14F7htB58细胞富集生产细胞亚群(单峰分布)，然而对于克隆31E7，获得的一个窄单峰暗示了更均匀的细胞亚群(图9)。实际上，克隆31E7比亲本细胞具有更高的最大细胞浓度、整体活细胞数和抗体产生率(图10)。
实施例4：高产细胞克隆的长期稳定性研究
细胞培养90天期间，对克隆31E7抗体的生产稳定性进行评价。细胞解冻后的30、60和90天在滚瓶中进行动力学研究。被抽取的样品进行流式细胞术研究。在细胞培养的不同时期没有显著差异。最大细胞浓度从3.5x106cell/ml到4.5x106cell/ml变化，细胞活力高于90％，抗体浓度在50mg/L到80mg/L(图10)变化。比生长速率(μ)保持高于0.025h-1，比生产速率(QP)高于0.18pg/cell*h(图11)。细胞培养90天后，克隆31E7出现一个窄单峰，是同类高表达细胞群的相同荧光强度值的代表(图12)。
实施例5：筛选的稳定高生产细胞克隆31E7的分泌免疫球蛋白特征属性
以覆盖基本分子属性定义几个特征属性，来评估分子质量，监测产品一致性以及细胞系的稳定性。一级结构通过测定整个分子及其单链质量，通过LC-ESI-MS分析亲本和二硫键减少/烷基化的样品(糖基化和去糖基化)来研究。结果总结见表1和图13、14、15。此外，该分子的肽图用于监测一级结构，并进一步排除任何因翻译后修饰截断或者序列改变的可能(图16)。
表1：完整分子的质量，及其有糖基化和无糖基化的链的质量
Figure PCTCN2016076135-appb-000002
a：考虑在两条重链C末端区域缺少K
b：减少二硫苏糖，碘乙酸烷基化
c：考虑在C末端区域缺少K和碘乙酸修饰
d：考虑碘乙酸修饰
在两个层次上分析高级结构，通过使用远紫外CD光谱分析二级结构(图17所示的图)和固有荧光特性，其能够检测构象的不同和蛋白质稳定性(Weichel et al,2008,BioProcess International,June)。图18显示了试验结果。在这种情况下，在333nm处获得了最大发射波长，在330nm/350nm处吸光度比值为1.23。
糖基化，发生在人IgG1抗体分子上的主要翻译后修饰，是通过正相HPLC谱图来研究的。图19显示了三种不同样品的结果，在表2中显示了它们的糖基化参数(Montesino et al,2012,Biologicals 40:288-298)。图20显示了这些糖基化参数的特性和分散。
表2：14F7h不同样品(N＝3)的糖基化参数，用于研究糖基化性能分析的偏差范围
Figure PCTCN2016076135-appb-000003
G0F％，G1F％，G2F％：岩藻糖化无半乳糖，岩藻糖化单半乳糖，岩藻糖化二半乳糖，岩藻糖基聚糖％；
Fuc％：岩藻聚糖％；
SIAL％：唾液酸化聚糖％
分子的异质性是由两个正交的方式来定义的。首位弱阳离子交换(WCX)用于检测不同电荷的物质，用于检测产品截断、脱酰胺基作用和一些糖基化变化等。对于抗体而言，这种方法的结果主要是监测C-末端赖氨酸截断，是一种在hIgG1分子上发现的常规改变(Dionex Application Note 127,http://www.dionex-france.com/library/literature/application_notes_updates/AN127_LPN1047.pdf))。对于不同样品获得的图谱， 图21和表3显示了整合的结果。此外，尺寸排阻色谱法(SEC)用于监测该分子的聚集状态。图22显示了不同样品获得的图谱，表4显示了其分析结果。
表3：同14F7h样品的WCX-HPLC整合结果(主峰百分比)
样品 主峰％
1 96.2
2 95.0
3 95.7
4 96.5
5 96.6
平均值 96.0±1.8
表4：同14F7h样品的WCX-HPLC整合结果(主峰百分比)
Figure PCTCN2016076135-appb-000004
最后，通过流式细胞仪研究分子识别靶细胞上抗原的能力，由此来评价分子的功能。图23显示了不同样品获得的剂量-反应曲线的结果，表5显示了从中计算的EC50值。
表5.使用L1210靶细胞对不同抗体样品在不同日期检测获得的流式细胞仪的剂量-反应曲线(阳性％对14F7h浓度(ug/mL)的log值)的EC50结果
Figure PCTCN2016076135-appb-000005
实施例6：体内和体外抗肿瘤效应的评价
通过流式细胞仪测定X63小鼠骨髓瘤细胞的抗原表达。100％的X63小鼠骨髓瘤细胞被14F7h单抗染色(图24A)。用10μg/mL抗体孵育X63细胞，然后用FITC-偶联的兔抗人IgG抗体孵化。赫赛汀单抗(抗-人Her-2)作为阴性对照。
X63小鼠骨髓瘤细胞内评价14F7h单抗诱导的体外细胞毒性效应。X63细胞用100μg/mL的14F7h mAb处理。37℃，6小时孵化后，用摄取的碘化丙啶(PI)和流式细胞仪评 价细胞活性。细胞毒性用PI染色的细胞百分比表示。处理后50％以上的肿瘤细胞死亡(图24B)。赫赛汀单克隆抗体作为阴性对照。
为了评价X63小鼠骨髓瘤模型中14F7h单抗的体内抗肿瘤作用，在第0天对BALB/c小鼠皮下接种0.2×106的X63肿瘤细胞，在2-5天静脉注射抗体(300ug)(图25A)。图25B显示直到106天无瘤生存率的Kaplan-Meier曲线。14F7h治疗的60％小鼠，随着时间，无瘤生存率获得统计学上显著增长。人源化单克隆抗体T1h测定(抗人CD6)作为阴性对照。用对数秩检验进行统计分析。

Claims (10)

  1. 一种在无蛋白培养基中从骨髓瘤细胞系获得高产稳定表达细胞克隆的方法，所述方法用于工业上生产重组抗体，其特征在于，所述的方法包含三个阶段：
    I.适应无蛋白培养基，低密度静止细胞培养，逐步减少富含脂质的补充物到化学培养基中；
    II.适应无蛋白培养基：以高密度细胞培养，在实验室规模采用灌流发酵系统；
    III.发酵结束时从细胞中筛选高产稳定表达的细胞克隆。
  2. 根据权利要求1所述的方法，其特征在于，所述阶段I包含以下步骤：
    I.适应生长在补充3.5g/L Cell Boost 5的PFHMII细胞培养基,传代培养，直到活细胞的最大浓度(Xv)达到一个恒定值；
    II.适应生长在补充1g/L Cell Boost 5的PFHMII细胞培养基，传代培养，直到活细胞的最大浓度(Xv)达到一个恒定值；
    III.适应生长在PFHMII细胞培养基中,细胞允许在无任何补充剂的无蛋白培养基中生长60天。
  3. 根据权利要求1所述的方法，其特征在于，所述阶段II包含以下步骤：
    I.适应生长在无蛋白质PFHMII细胞培养基中，以高细胞密度，在5L生物反应器中使用灌流发酵系统生长超过21天，所述高细胞密度为(5-10)×106cell/ml；
    II.发酵结束后，细胞被冻存在液氮中，即最终生产细胞库EPBC。
  4. 根据权利要求1所述的方法，其特征在于，所述阶段III包含以下步骤：
    I.从最终生产细胞库中解冻的细胞采用有限稀释法进行细胞克隆；
    II.分泌抗体的克隆采用高灵敏度和特异性ELISA方法检测，使用抗独特型抗体作为捕获抗原；
    III.在选择的克隆中使用细胞内免疫印染FACS进行抗体生产细胞亚群的定量检测；
    IV.选择比生长速率(μ)和比生产速率(qp)高的克隆进行动力学研究。
  5. 根据权利要求1-4所述的方法，其特征在于，所述的骨髓瘤细胞系是NS0细胞系。
  6. 根据权利要求5所述的方法，其特征在于，所述的骨髓瘤细胞系包含编码人源化重组抗-NeuGcGM3的抗体14F7h的序列。
  7. 根据权利要求6的方法获得的细胞系分泌的人源化重组抗-NeuGcGM3的抗体14F7h。
  8. 根据权利要求7所述的细胞分泌的人源化重组抗-NeuGcGM3的抗体14F7h，其特征在于，所述的细胞系是克隆B58-31E7。
  9. 根据权利要求8所述的人源化重组抗-NeuGcGM3的抗体14F7h，其特征在于，所述的抗体具有如下的分子表型：
    Figure PCTCN2016076135-appb-100001
  10. 根据权利要求9所述的人源化重组抗-NeuGcGM3的抗体14F7h具有体内和体外抗肿瘤活性。
PCT/CN2016/076135 2015-02-14 2016-03-11 获得高产稳定表达细胞克隆的方法及由此获得的抗体分子 WO2016127954A1 (zh)

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