Cell culture method for producing tolbuzumab

Abstract

The invention belongs to the field of biology, discloses a cell culture method for producing tolbuzumab, solves the problems of complex production process, low expression quantity, large quality control difference and the like of biosimilar drugs of tolbuzumab at present, and provides powerful support for industrial production of the tolbuzumab.

Description

Technical Field

[0001] The present disclosure belongs to the field of antibody production, and specifically relates to a cell culture method for producing tocilizumab. Background Art

[0002] Rheumatoid arthritis (RA) is a common autoimmune disease, the pathological characteristics of which are inflammation of the synovial membrane and hyperplasia of the synovial tissue, which in turn lead to damage and destruction of the articular cartilage and bone. In the natural course of rheumatoid arthritis, the disability rate is 60% within 5 to 10 years and 90% after 30 years of disease.

[0003] Treatment of RA can improve or cure joint pain and swelling and prevent joint damage. Early treatment leads to better long-term outcomes, and patients who receive early treatment are less likely to develop the type of joint damage that leads to joint replacement. The main treatment goals for rheumatoid arthritis are to control inflammation, relieve pain, and reduce RA-related disability. Treatment usually includes medication, occupational therapy or physical therapy, and regular exercise. Acceptable biologics for the treatment of RA include tumor necrosis factor (TNF) inhibitors and non-TNF inhibitors (such as cytokine inhibitors, T or B cell inhibitors). For example, biologics include tocilizumab (Acemira).

[0004] Tocilizumab ( / Acamira, Roche) is a neutralizing antibody against human interleukin-6 receptor (IL-6R), which can prevent the binding of IL-6 to IL-6R, thus having a good therapeutic effect on rheumatoid arthritis. However, the current biosimilars of tocilizumab have problems such as complex production process, low expression level, and unstable quality control. Summary of the invention

[0005] The purpose of this disclosure is to establish a robust process suitable for the production of recombinant anti-interleukin-6 receptor antibodies, and the quality of the produced recombinant anti-interleukin-6 receptor antibodies is comparable to that of the original drug. quite.

[0006] The present disclosure discloses a cell culture method for producing antibodies, the method comprising a seed culture stage and a production culture stage, the production culture stage comprising the following steps:

[0007] a. Inoculate cells in basal medium;

[0008] b. Feeding:

[0009] i) supplementing a feed medium, wherein the feed medium comprises Maxfeed 401, wherein the concentration of the Maxfeed 401 is 5 g / L to 35 g / L; ii) supplementing a glycoform regulator, wherein the glycoform regulator is supplemented simultaneously with the feed medium;

[0010] The sugar type regulator contains at least galactose or fucose.

[0011] In some embodiments, the concentration of Maxfeed 401 is 5g / L to 25g / L. In some embodiments, the concentration of Maxfeed 401 is, for example, 5 to 8g / L, 8 to 10g / L, 10 to 15g / L, 15 to 20g / L, 20 to 23g / L, 23 to 25g / L; non-limiting examples include about 9g / L, about 10g / L, about 12g / L, about 15g / L, about 20g / L, about 23g / L, about 25g / L, or any value between any two values. In some embodiments, the concentration of Maxfeed 401 is 9g / L to 25g / L; in some embodiments, the concentration of Maxfeed 401 is 9.51g / L. In some embodiments, the concentration of Maxfeed 401 is 23.78g / L.

[0012] In some embodiments, the feed medium further comprises: 0.4 g / L to 1.2 g / L tyrosine salt, 0.5 g / L to 1.1 g / L cysteine ​​salt, and 0.8 g / L to 1.5 g / L asparagine.

[0013] In some embodiments, the concentration of tyrosine salt is, for example, 0.4-0.8 g / L, 0.8-1.0 g / L, 1-1.2 g / L; non-limiting examples include about 0.4 g / L, about 0.8 g / L, about 1 g / L, about 1.2 g / L, or any value between any two values. In some embodiments, the concentration of tyrosine salt in the feed medium is 0.85 g / L.

[0014] In some embodiments, the cysteine ​​salt concentration is, for example, 0.5-0.7 g / L, 0.7-0.9 g / L, 0.9-1.1 g / L; non-limiting examples include about 0.5 g / L, about 0.7 g / L, about 0.9 g / L, about 1.1 g / L, or any value between any two values. In some embodiments, the cysteine ​​salt concentration in the feed medium is 0.75 g / L.

[0015] In some embodiments, the concentration of asparagine is 0.8-1.5 g / L. In some embodiments, the concentration of L-asparagine is, for example, 0.8-1.0 g / L, 1.0-1.2 g / L, 1.2-1.5 g / L; non-limiting examples include about 0.8 g / L, about 1.0 g / L, about 1.2 g / L, about 1.5 g / L, or any value between any two values. In some embodiments, the concentration of asparagine in the feed medium is 1.12 g / L.

[0016] In some embodiments, the feed medium further comprises: 0.8 g / L to 1.0 g / L tyrosine salt, 0.5 g / L to 1.0 g / L cysteine ​​salt, and 1.0 g / L to 1.2 g / L asparagine.

[0017] In some embodiments, the feed medium further comprises: 0.85 g / L tyrosine salts, 0.75 g / L cysteine ​​salts, and 1.12 g / L asparagine.

[0018] In some embodiments, the feed medium further comprises sodium hydroxide, disodium hydrogen phosphate and glucose.

[0019] In some embodiments, the feed medium further comprises 1-3 g / L sodium hydroxide, 3-6 g / L disodium hydrogen phosphate, and 40-70 g / L glucose. In some embodiments, the concentration of sodium hydroxide in the feed medium is 1.75 g / L. In some embodiments, the concentration of disodium hydrogen phosphate in the feed medium is 4.26 g / L. In some embodiments, the concentration of glucose in the feed culture is 55 g / L.

[0020] In some embodiments, the tyrosine salt is tyrosine disodium salt hydrate.

[0021] In some embodiments, the cysteine ​​salt is cysteine ​​hydrochloride monohydrate.

[0022] In some embodiments, the feed medium comprises: 9.51 g / L or 23.78 g / L Maxfeed401, 0.85 g / L tyrosine disodium salt hydrate, 0.75 g / L cysteine ​​hydrochloride monohydrate, 1.12 g / L asparagine, 1-3 g / L sodium hydroxide, 3-6 g / L disodium hydrogen phosphate, 40-70 g / L glucose. In some embodiments, the feed medium consists of the following ingredients: 9.51 g / L or 23.78 g / L Maxfeed401, 0.85 g / L tyrosine disodium salt hydrate, 0.75 g / L cysteine ​​hydrochloride monohydrate, 1.12 g / L asparagine, 1-3 g / L sodium hydroxide, 3-6 g / L disodium hydrogen phosphate and 40-70 g / L glucose.

[0023] In some embodiments, the feed medium consists of 9.51 g / L or 23.78 g / L Maxfeed401, 0.85 g / L tyrosine disodium salt hydrate, 0.75 g / L cysteine ​​hydrochloride monohydrate, 1.12 g / L asparagine, 1.75 g / L sodium hydroxide, 4.62 g / L disodium hydrogen phosphate and 55 g / L glucose.

[0024] In the present disclosure, the concentration of each component in the feed medium is calculated based on the feed volume.

[0025] In some embodiments, the antibody is tocilizumab.

[0026] In some embodiments, the glycoform regulator comprises galactose. In some embodiments, the glycoform regulator further comprises manganese chloride or uridine. In some embodiments, the glycoform regulator is EX-CELL GlycosylationAdjust (GAL+), model 14701C.

[0027] In some embodiments, the amount of the glycoform regulator added is 0.5% to 2% (v / v) of the feed medium. In some embodiments, the amount of the glycoform regulator added is 0.8% to 1.8% (v / v) of the feed medium. In some embodiments, the amount of the glycoform regulator added is 0.8% to 1.5% (v / v) of the feed medium, preferably 1% (v / v).

[0028] In some embodiments, the culture time of the production culture stage is 10 to 14 days. In some embodiments, feeding is performed on the 2nd to 4th day, the 5th to 7th day and / or the 8th to 10th day of the production culture stage; in some embodiments, feeding is performed on the 3rd day, the 6th day and / or the 9th day of the production culture stage;

[0029] In some embodiments, the feed addition amount is 8% to 12% (v / v) of the initial culture volume. In some embodiments, the feed addition amount is 10% (v / v) of the initial culture volume.

[0030] In some embodiments, the cell seeding density in step a is 0.5×10 6 Cells / mL~2×10 6 In some embodiments, the cell seeding density in step a is 1.0×10 6 Cells / mL~1.5×10 6 cells / mL.

[0031] In some embodiments, the cell culture medium can be a chemically defined medium in which all chemical components are known. As will be appreciated by those skilled in the art, a culture medium can be defined without undue experimentation, provided that it is suitable for the specific cells being cultured.

[0032] Commercially available culture media may be used, including but not limited to: Maxpro 301 of Hyclone, Maxpro303 of Hyclone, EX-CELL Advanced CHO basal medium of Millipore, ExpiCHO basal medium of Gibco, CHO CD04 of Zhongshan Kangsheng, CD OptiCHO, Hycell, DN Feed1 of Shanghai Duoning, CHO Feed 02 of Zhongshan Kangsheng, GrowthA, Dynamis, Iscove's Modified Dulbecco's Medium, GMEM with glutamine, or any other culture media prepared for specific cell types known to those skilled in the art. Supplementary components or ingredients, including optional components, may be added to the above exemplary culture media at appropriate concentrations or amounts as needed or as needed and as known and implemented by those skilled in the art using routine skills.

[0033] In some embodiments, the basal medium is Maxpro 301, Maxpro 303, AdvancedCHO, ExpiCHO, CHO CD04, CD OptiCHO, Hycell, DN Feed 1, CHO Feed 02, GrowthA or Dynamis.

[0034] In some embodiments, the basal medium is Maxpro 301.

[0035] In some embodiments, the cell culture methods of the present disclosure are serum-free throughout the entire process.

[0036] In some embodiments, during the production culture stage, the concentration of glucose in the cell culture medium is 3.5 g / L to 6 g / L, and the concentration of glucose is calculated based on the initial culture volume. In some embodiments, during the production culture stage, the concentration of glucose in the cell culture medium is 3.5 g / L to 5 g / L, in some embodiments, during the production culture stage, the concentration of glucose in the cell culture medium is 3.5 g / L to 4.5 g / L, in some embodiments, the concentration of glucose is 4.0 g / L. In some embodiments, the glucose concentration in the cell culture medium is monitored once a day, and when insufficient, it is supplemented to 4.0 g / L.

[0037] In some embodiments, the pH of the production culture stage is 6.8-7.0, and the dissolved oxygen is 20%-60%. In some embodiments, the dissolved oxygen is 40%.

[0038] In some embodiments, the culture temperature in the production culture stage is 34.5-37.5° C. In some embodiments, the culture temperature in the production culture stage is 34.5-35.5° C. In some embodiments, the culture temperature in the production culture stage is 35° C.

[0039] In some embodiments, the cell is a CHO cell; preferably, the CHO cell is selected from CHO-K1, CHO DG44 and CHO-S cells, more preferably CHO-K1 cells.

[0040] The method for constructing the fermentation cell strain in the present disclosure is:

[0041] The host cell is CHO-K1 cell (ATCC CCL-61), and the expression vector pTGS-GS (by modifying the expression vector pTGS-FRT-DHFR, removing the hygromycin and DHFR genes, and adding the GS gene) integrates the light chain gene and heavy chain gene of the antibody to obtain a recombinant plasmid, which can express a recombinant anti-interleukin-6 receptor (IL-6R) humanized monoclonal antibody. The recombinant plasmid is stably transfected into the CHO-K1 host cell, and a monoclonal cell line producing and expressing tocilizumab is screened.

[0042] The amino acid sequences of the heavy chain and light chain of Tocilizumab are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively:

[0043] QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGS LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:1)

[0044] DIQMTQSPSSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPS RFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2).

[0045] In some embodiments, the culture conditions in the seed culture stage include one or more of the following conditions:

[0046] i) the seed culture medium is selected from Maxgrow 201, Advanced CHO, ExpiCHO, CHO CD04, CDOptiCHO, Hycell, DN Feed1, CHO Feed 02, GrowthA or Dynamis;

[0047] ii) The cell seeding density was 0.35×10 6 ~0.75×10 6 cells / mL;

[0048] iii) pH 6.8 to 7.0; and

[0049] iiii) The culture temperature is 36.5°C to 37.5°C.

[0050] In some embodiments, the seed culture medium is Maxgrow 201.

[0051] In some embodiments, the culture temperature in the seed culture stage is 37°C.

[0052] In some embodiments, the cell culture method for producing antibodies further comprises a harvesting stage.

[0053] In some embodiments, antibodies are harvested during the harvest stage.

[0054] In some embodiments, the harvesting stage includes a purification step for the antibody, specifically comprising:

[0055] 1) obtaining a clarified liquid of the fermentation liquid by centrifugation;

[0056] 2) performing affinity chromatography;

[0057] 3) Perform deep filtration;

[0058] 4) performing anion exchange chromatography and cation exchange chromatography in sequence;

[0059] 5) Nanofiltration of the cation exchange chromatography product.

[0060] In some embodiments, the purification step further comprises the following steps:

[0061] The nanofiltration product is subjected to ultrafiltration to obtain an antibody stock solution.

[0062] In some embodiments, affinity chromatography is performed using Mabselect SuRe as the medium.

[0063] In some embodiments, a depth filter is used for depth filtration. Preferably, a depth filter A1HC (purchased from Merck) is used for depth filtration.

[0064] In some embodiments, anion exchange chromatography is performed with Q Sepharose Fast Flow medium and cation exchange chromatography is performed with POROS 50HS medium.

[0065] In some embodiments, nanofiltration is performed using SV4 nanofiltration membrane (PALL, Pall (China) Co., Ltd.

[0066] In some embodiments, ultrafiltration is performed using a tangential flow ultrafiltration membrane P2B050A25 (Merck Millipore) with a molecular weight cutoff of 50 kDa.

[0067] In some embodiments, the purification step specifically comprises in sequence: 1) obtaining a clarified liquid of the fermentation broth. 2) performing affinity chromatography capture with Mabselect SuRe filler and incubating at pH 3.5±0.1 (first virus removal). 3) performing clarification after low pH incubation with a depth filter A1HC (purchased from Merck) to filter out most impurities. 4) performing anion exchange chromatography with Q Sepharose Fast Flow filler and cation exchange chromatography with POROS 50HS filler, this stage is fine purification (second virus removal). 5) performing a third virus removal by filtration using an SV4 nanofiltration membrane (PALL). 6) concentrating the stock solution with a tangential flow ultrafiltration membrane P2B050A25 (Merck Millipore) with a molecular weight cutoff of 50 kDa to obtain a recombinant anti-interleukin-6 receptor (IL-6R) humanized monoclonal antibody.

[0068] In some embodiments, the present disclosure discloses a cell culture method for producing antibodies, the method comprising a seed culture stage and a production culture stage, the production culture stage comprising the following steps:

[0069] a. Inoculate cells in basal medium;

[0070] b. Feeding:

[0071] i) supplementing a feed medium, wherein the feed medium comprises Maxfeed 401, wherein the concentration of the Maxfeed 401 is 5 g / L to 35 g / L; ii) supplementing a glycoform regulator, wherein the glycoform regulator is supplemented simultaneously with the feed medium;

[0072] The sugar type regulator is EX-CELL Glycosylation Adjust (GAL+).

[0073] In some embodiments, the present disclosure discloses a cell culture method for producing antibodies, the method comprising a seed culture stage and a production culture stage, the production culture stage comprising the following steps:

[0074] a. inoculating cells in a basal medium, wherein the basal medium is Maxpro 301;

[0075] b. Feeding:

[0076] i) supplementing a feed medium, wherein the feed medium comprises Maxfeed 401, wherein the concentration of the Maxfeed 401 is 5 g / L to 35 g / L; ii) supplementing a glycoform regulator, wherein the glycoform regulator is supplemented simultaneously with the feed medium;

[0077] The sugar type regulator is EX-CELL Glycosylation Adjust (GAL+); the feed medium also contains: 0.4g / L to 1.2g / L tyrosine salt, 0.5g / L to 1.1g / L cysteine ​​salt and 0.8g / L to 1.5g / L asparagine, and the concentrations of the amino acids and Maxfeed 401 are calculated based on the feed volume;

[0078] The antibody is tocilizumab.

[0079] In some embodiments, in step a, cells (seed solution) are inoculated in a basal medium, wherein the seed solution and the basal medium are inoculated at a volume ratio of 1:1, and the production culture stage is entered. In some embodiments, the culture medium in the production stage comprises a seed culture medium (e.g., Maxgrow 201) and a basal culture medium (e.g., Maxpro 301). In some embodiments, the culture medium in the production stage comprises a seed culture medium (e.g., Maxgrow 201), a feed culture medium (e.g., Maxgrow 401), and a basal culture medium (e.g., Maxpro 301).

[0080] In some embodiments, the present disclosure discloses a cell culture method for producing antibodies, the method comprising a seed culture stage and a production culture stage, the production culture stage comprising the following steps:

[0081] a. inoculating cells in a basal medium, wherein the basal medium is Maxpro 301;

[0082] b. Feeding:

[0083] i) supplementing a feed medium, wherein the feed medium comprises: 9 g / L to 25 g / L Maxfeed401, 0.8 g / L to 1.0 g / L tyrosine salt, 0.5 g / L to 1.0 g / L cysteine ​​salt and 1.0 g / L to 1.2 g / L asparagine; the concentration of each component in the feed medium is calculated based on the feed volume;

[0084] ii) supplementing a glycosylation regulator, wherein the glycosylation regulator is EX-CELL Glycosylation Adjust (GAL+), and the added amount of the glycosylation regulator is 0.5% to 2% (v / v) of the feed medium, and the glycosylation regulator is supplemented simultaneously with the feed medium;

[0085] The culture temperature in the production stage is 34.5-37.5°C, and the antibody is harvested after 10-14 days of culture. The antibody is tocilizumab.

[0086] In some embodiments, the present disclosure discloses a cell culture method for producing antibodies, the method comprising a seed culture stage and a production culture stage, the production culture stage comprising the following steps:

[0087] a. inoculating cells in a basal medium, wherein the basal medium is Maxpro 301;

[0088] b. Feeding:

[0089] i) supplementing a feed medium, the feed medium comprising: 9.51 g / L or 23.78 g / L Maxfeed401, 0.85 g / L tyrosine disodium salt hydrate, 0.75 g / L cysteine ​​hydrochloride monohydrate, 1.12 g / L asparagine, 1-3 g / L sodium hydroxide, 3-6 g / L disodium hydrogen phosphate, and 40-70 g / L glucose; the concentration of each component in the feed medium is calculated based on the feed volume;

[0090] ii) supplementing a glycosylation regulator, wherein the glycosylation regulator is EX-CELL Glycosylation Adjust (GAL+), and the added amount of the glycosylation regulator is 0.5% to 2% (v / v) of the feed medium, and the glycosylation regulator is supplemented simultaneously with the feed medium;

[0091] The culture temperature during the production phase is 34.5-37.5°C, and the antibodies are harvested after 10-14 days of culture;

[0092] The antibody is tocilizumab.

[0093] In some embodiments, the present disclosure discloses a cell culture method for producing antibodies, the method comprising a seed culture stage and a production culture stage, the production culture stage comprising the following steps:

[0094] a. inoculating cells in a basal medium, wherein the basal medium is Maxpro 301;

[0095] b. Feeding:

[0096] i) supplementing a feed medium, the feed medium comprising: 9.51 g / L or 23.78 g / L Maxfeed401, 0.85 g / L tyrosine disodium salt hydrate, 0.75 g / L cysteine ​​hydrochloride monohydrate, 1.12 g / L asparagine, 1.75 g / L sodium hydroxide, 4.26 g / L disodium hydrogen phosphate, and 55 g / L glucose; the concentration of each component in the feed medium is calculated based on the feed volume;

[0097] ii) supplementing a glycosylation regulator, wherein the glycosylation regulator is EX-CELL Glycosylation Adjust (GAL+), and the added amount of the glycosylation regulator is 1% (v / v) of the feed medium, and the glycosylation regulator is supplemented simultaneously with the feed medium;

[0098] The culture temperature during the production phase is 35°C, and the antibodies are harvested after 10 to 14 days of culture;

[0099] The antibody is tocilizumab.

[0100] The present disclosure discloses a cell culture method for producing tocilizumab, which solves the problems of current tocilizumab biosimilars, such as complex production process, low expression level, and large quality control differences, and provides strong support for the industrial production of the antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 Schematic diagram of antibody expression levels under different conditions in Example 1. DETAILED DESCRIPTION

[0102] definition

[0103] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, biochemistry, nucleic acid chemistry, immunology laboratory and other operating procedures used herein are all conventional procedures widely used in the corresponding fields.

[0104] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising", "having", "including", etc. should be understood to have an inclusive sense, rather than an exclusive or exhaustive sense; that is, the sense of "including but not limited to". Unless otherwise specified, "comprising" includes "consisting of".

[0105] As used herein, the term "about" or "approximately" refers to a value that is within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation per practice in the art. Alternatively, "about" or "substantially comprising" can mean a range of up to ±20%, for example, a pH of about 5.5 means pH 5.5±1.1. In addition, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times a value. Unless otherwise indicated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0106] In the present disclosure, the term "antibody" refers to an immunoglobulin that can specifically recognize and bind to an antigen, and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, bispecific antibodies or antibody fragments.

[0107] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the population comprising each antibody is identical, except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. In addition, in contrast to polyclonal antibody preparations comprising different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized without being contaminated by other antibodies. The modifier "monoclonal" should not be interpreted as requiring the production of antibodies by any ad hoc method. For example, the monoclonal antibodies used in accordance with the present invention can be prepared by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods in bacteria, eukaryotic or plant cells (see, e.g., U.S. Pat. No. 4,816,567); phage display technology (see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004) and techniques for producing human or human-like antibodies in animals having partial or complete human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci.USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425 and 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995). .

[0108] The term "cell culture medium" refers to an aqueous solution of nutrients that can be used to grow cells for extended periods of time. Typically, a cell culture medium comprises the following components: an energy source, which will typically be a carbohydrate, preferably glucose, amino acids, preferably amino acids of the basic group, including all essential and non-essential amino acids; vitamins and / or other organic compounds required in low concentrations; free fatty acids; and inorganic compounds, including trace elements, inorganic salts, buffer compounds, and nucleosides and bases.

[0109] The term "basal medium" is a medium used to culture cells, which medium itself is used directly to culture cells and is not used as an additive to other culture media, but various components can be added to the basal medium. For example, if CHO cells are cultured in DMEM (a well-known, commercially available mammalian cell culture medium) and regularly fed with glucose or other nutrients, DMEM would be considered a basal medium.

[0110] "Feed medium" is a medium used as feed in cell culture, which can be a fed batch cell culture. Like the basal medium, the feed medium is designed based on the needs of the specific cells being cultured, and the feed medium can have most of the basal medium at a higher concentration, but not all of the components. For example, some components, such as nutrients including amino acids or carbohydrates, can be about 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 400, 600, 800 times or even about 1000 times their normal concentration in the basal medium. Some components, such as salts, can be maintained at a concentration approximately the same as the basal medium to keep the feed isosmotic with the basal medium. Some components are added to maintain the physiology of the feed, and some components are added because they supplement nutrients in the culture.

[0111] The term "inoculation" refers to the seeding of cells into a culture medium provided for cell culture. Specifically, the culture medium can be provided before the cells are transferred, or can be provided while the cells are transferred to a cell bioreactor. In large-scale animal cell culture, the conventional practice is to provide the culture medium in advance, and after maintaining the temperature and oxygen saturation within a predetermined range, the cells are transferred into the bioreactor.

[0112] The term "cell density" refers to the number of cells present in a given volume of culture medium.

[0113] The term "bioreactor" refers to any container for the growth of mammalian cell cultures. A bioreactor can be of any size, as long as it is used to culture mammalian cells. Typically, such bioreactors will be at least 1 liter and can be 10, 100, 250, 500, 1000, 2500, 5000, 8000, 10,000, 12,000 liters or more, or any volume therebetween. The internal conditions of the bioreactor are usually controlled during the culture period, including but not limited to pH, dissolved oxygen and temperature. A bioreactor can be composed of any material (including glass, plastic or metal) suitable for accommodating a mammalian cell culture suspended in the culture medium under the culture conditions of the present invention.

[0114] The term "fed batch culture" or "fed culture" refers to a method of culturing cells in which additional components are provided to the culture once or more after the start of the culture process. The components so provided generally include nutritional components of the cells that are consumed during the culture process. In the present disclosure, after the seed culture stage is completed, the cells are inoculated into the basal medium, and the fed culture stage is entered, and the day of inoculation of the cells is taken as day 0 of the fed culture.

[0115] The term "initial culture volume" is the culture volume after inoculating cells in the basal medium.

[0116] The term "antibody stock solution" refers to an antibody solution formed by storing the purified antibody in an appropriate buffer system routinely used by those skilled in the art.

[0117] The present disclosure is described below with reference to specific embodiments. It will be appreciated by those skilled in the art that these embodiments are only used to illustrate the present disclosure and do not limit the scope of the present disclosure in any way.

[0118] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur. As used in the specification and claims, the singular forms "a", "an" include the corresponding plural referents unless the content clearly dictates otherwise.

[0119] The present disclosure will be described below in conjunction with specific embodiments, but the content of the present disclosure is not limited thereto.

[0120] Unless otherwise specified, the reagents and instruments used in the following methods are commonly used reagents and instruments in the art and can be obtained commercially; the methods used are conventional methods in the art, and those skilled in the art can undoubtedly perform the methods and obtain corresponding results based on the contents described in the embodiments.

[0121] Determination and detection methods

[0122] (1) Determination of cell viability and density: The cell viability and density were determined by trypan blue staining using a cell viability analyzer.

[0123] (2) Purification of Tocilizumab:

[0124] The purification steps specifically include: 1) obtaining the clarified liquid of the fermentation broth. 2) Affinity chromatography capture with Mabselect SuRe filler and incubation at pH 3.5±0.1 (first virus removal). 3) Clarification after low pH incubation with a deep filter A1HC to filter out most impurities. 4) Anion exchange chromatography with Q Sepharose Fast Flow filler and cation exchange chromatography with POROS 50HS filler. This stage is fine purification (second virus removal). 5) Filtering with SV4 nanofiltration membrane (PALL) for the third virus removal. 6) Concentrating the stock solution through a tangential flow ultrafiltration membrane P2B050A25 with a molecular weight cutoff of 50 kDa to obtain tocilizumab.

[0125] (3) Determination of Tocilizumab Concentration:

[0126] Determine by HPLC, balance the chromatographic column with phosphate buffer, set the standard curve method program in the system program, and then inject the sample. Chromatographic conditions: Chromatographic column: POROS TMA20 (specification: 2.1×30 mm, 20 μm), injection volume: 50 μL, flow rate: 2.00 mL / min, detection wavelength: 280 nm, column temperature: 30°C, running time: gradient running 9 min, injection plate temperature: 6°C. Record relevant data and process them.

[0127] (5) SEC-HPLC determination:

[0128] Determination by HPLC: First, balance the column, that is, use the mobile phase at a flow rate of 0.5 mL / min until the system is stable, and then inject the sample. Chromatographic conditions: Chromatographic column: TSKgel G3000 SW XL (Specifications: 7.8×300mm, 5μm), injection volume: 20μL, flow rate: 0.5mL / min, detection wavelength: 280nm, column temperature: 30℃, running time: isocratic run for 35min.

[0129] (6) CE-SDS assay:

[0130] Capillary electrophoresis was used for detection. The capillary electrophoresis system was cleaned with about 1500 μL of water, about 3000 μL of running buffer, about 1500 μL of 1M NaOH, and about 1500 μL of 1M HCl. Before each injection, the sample was filled with running buffer at 4.0 bar for 10 minutes. The capillary was 33 cm long, with an effective length of 8.5 cm, a detection wavelength of 214 nm, a column temperature of 20 °C, and a sample tray of 15 °C.

[0131] (7) IEC-HPLC determination:

[0132] Determination by HPLC: First, balance the chromatographic column, and then inject the sample. Chromatographic conditions: chromatographic column: PropacWCX-10 (specification: 4×250mm, 10μm), injection volume of 10μL, flow rate: 0.8mL / min, detection wavelength of 214nm, column temperature: 30℃, running time: gradient running 60min.

[0133] Table 1 Gradient operation table

[0134] Time / min Mobile phase A% Mobile phase B% 0 85 15 3 85 15 6 70 30 36 30 70 38 0 100 43 0 100 45 85 15 60 85 15

[0135] (8) N-Glycan Profiling Method:

[0136] Determination by HPLC: The sample was processed by Gly-X InstantPC N-sugar rapid analysis kit 96Kit. Before detection, the chromatographic column was balanced with water and acetonitrile, and the sample was injected after equilibration. Chromatographic conditions: Chromatographic column: XBridge GlycanBEH Amide XP Column (specification: 2.1×150mm, 2.5μm, 130A), injection volume: 20μL, detection wavelength: λex=285nm, λem=345nm, flow rate was 0.3mL / min, 0.1mL / min, 0.15mL / min, column temperature 60℃, sample chamber temperature: 8℃; running time: gradient run 65min (see the table below), post-run 10min (75min in total).

[0137] Table 2 Gradient run table for N-sugar spectrum determination method

[0138] Time (min) Mobile phase A (%) Mobile phase B (%) Flow rate (ml / min) 0.00 25.0 75.0 0.3 14.00 28.5 71.5 0.3 46.00 36.5 63.5 0.3 50.00 80.0 20.0 0.1 52.00 80.0 20.0 0.15 58.00 25.0 75.0 0.3 65.00 25.0 75.0 0.3

[0139] Example 1.2 L bioreactor feed medium optimization

[0140] In our original process (seeding density 1×10 6 cells / mL~1.5×10 6 cells / mL, temperature 37°C, 200rpm, 40% DO, pH=6.90±0.10, glucose supplemented to 4.0g / L every day, 10% F solution supplemented on the 3rd, 6th and 9th days), the feed medium was optimized and analyzed in a 2L reactor, and the effects of different feed media on cell growth, etc. were systematically investigated.

[0141] All parameter settings start from the inoculation culture, that is, they remain unchanged during the culture process. Three 2L bioreactors from Electrolab, UK, were used for parallel optimization. The experimental design is shown in Table 3-1.

[0142] Table 3-1 Experimental plan for optimizing feed medium

[0143]

[0144] The cells are inoculated into the seed culture medium for cultivation. After the seeds are expanded, the seed solution and the basal culture medium are mixed at a ratio of 1:1 and transferred to the bioreactor for cultivation, entering the production culture stage. The seed culture medium is liquid C, which is Maxgrow201 culture medium (purchased from Cytiva), and the basal culture medium is liquid A, which is Maxpro 301 culture medium (purchased from Cytiva). The ingredients of the feed are shown in Table 3-2 below.

[0145] Table 3-2 Composition of feed

[0146]

[0147] The fermentation broth was harvested and centrifuged to obtain the supernatant, which was measured by affinity high performance liquid chromatography. The analysis results are shown in Tables 4, 5 and Figure 1 The control group directly purchased the finished product of Actemra (Roche) for chromatographic testing.

[0148] Table 4 Optimized expression data of 2L tank feed medium

[0149]

[0150] Table 5 Optimization analysis results of 2L tank feed medium

[0151]

[0152] From Table 4 and Figure 1 It can be seen that under the feeding conditions of F1 and F2, the expression of the antibody is significantly better than that of F, among which F1 is the best. As can be seen from Table 5, the main peak content of TOC161002-2# (feeding condition is F1) and TOC161002-6# (feeding condition is F2) is equivalent to the main peak content of the original drug Actelion, and the main peak content of TOC161101-2# (feeding condition is F) is significantly lower than the main peak content of the original drug Actelion.

[0153] Example 2. Optimization of 2L bioreactor temperature

[0154] Based on the optimization process of Example 1 (inoculation density 1.00E+06 cells / mL~1.50E+06 cells / mL, temperature 37°C, 200rpm, 40%DO, pH=6.90±0.10, glucose supplemented to 4.0g / L every day, 10%F1 liquid supplemented on the 3rd, 6th and 9th days), the culture temperature was optimized and analyzed on a 2L bioreactor, and the influence of different culture temperatures on cell growth, protein expression and quality was systematically investigated to determine the key process parameters of cell culture. The pH setting value was 6.90±0.10, and the dissolved oxygen setting value was 40%. All parameter settings were fixed from the start of inoculation culture, i.e., during the culture process. Three 2L bioreactors of Electrolab, UK, were used for parallel optimization, and the experimental design was shown in Table 6.

[0155] Table 6 Experimental plan for optimizing the culture temperature in 2L tanks

[0156]

[0157] The fermentation broth was harvested and centrifuged to obtain the supernatant, which was measured by affinity high performance liquid chromatography, and the analysis was shown in Table 7. The results showed that the temperature had little effect on the expression amount and antibody quality, but cooling to 35°C could slightly increase the percentage of the CE main peak. Therefore, the culture temperature of 35°C was optimal.

[0158] Table 7

[0159]

[0160] Example 3.2 Optimization of sugar type regulators in L bioreactor

[0161] Based on the optimized processes of Examples 1 and 2, the process of TOC161201#2 was selected, namely: inoculation density 1.00E+06 cells / mL to 1.50E+06 cells / mL, temperature 35°C, 200rpm, 40% DO, pH = 6.90±0.10, glucose supplemented to 4.0 g / L every day, 10% F1 solution supplemented on days 3, 6 and 9, and a glycoform regulator was supplemented at the same time during feeding. The concentration of the glycoform regulator EX-CELL Glycosylation Adjust (GAL+) (purchased from Sigma, model 14701C) was optimized in a 2L reactor, and the effects of different concentrations of the glycoform regulator EX-CELL Glycosylation Adjust (GAL+) on cell growth, antibody expression and quality were systematically investigated to determine the key process parameters for cell culture.

[0162] Three 2L bioreactors were used for parallel optimization. The experimental design is shown in Table 8.

[0163] Table 8 Optimization of culture process with different concentrations of sugar type regulators

[0164]

[0165] Note: The amount of sugar type regulator is calculated based on the feed volume.

[0166] The fermentation broth was harvested and centrifuged to obtain a supernatant, and the supernatant was measured by affinity high performance liquid chromatography, and the analysis was shown in Table 9 below, wherein. From the data analysis in Table 9, it can be seen that when the concentration of the glycoform regulator is 2% (v / v) of the feed volume, the G0F% value decreases significantly, so the concentration of the glycoform regulator is preferably 1% (v / v) or 1.5% (v / v) of the feed volume.

[0167] Table 9

[0168]

[0169] Example 4.300 L bioreactor pilot scale production process parameter control

[0170] The process TOC170101-1# optimized in the above embodiment was used as the process in the production culture stage in this embodiment, that is, the culture temperature was 35°C, the dissolved oxygen was 40%, the pH value was 6.9, the reactor speed was 200rpm, 10% F1 solution was fed on the 3rd, 6th and 9th days, the concentration of the sugar type regulator EX-CELL Glycosylation Adjust (GAL+) was 1% (calculated relative to the feed volume), and glucose was fed to 4.0g / L every day. In the seed culture stage, a step-by-step amplification culture was adopted, and the amplification culture was carried out through 20mL, 80mL, 320mL, 5L, and 30L. The culture temperature was 36.5-37.5°C, the pH was 6.9-7.1, and the seed culture medium was Maxgrow 201. When the seed cell density in the 30L bioreactor was 1.5×10 6 ~3.0×10 6 When the concentration of 100 cells / mL was 0.1, the cells were inoculated into a 300 L bioreactor for the production culture stage, and the antibodies were harvested after 10 to 14 days of culture.

[0171] This example was produced in a 300 L cell culture scale (batch number 0162817002). Product testing and analysis showed that the average antibody expression level was about 2.05±0.01 g / L, and the SEC-HPLC purity was over 98%.

[0172] The fermentation broth was harvested and centrifuged to obtain a supernatant, which was measured by affinity high performance liquid chromatography. The analysis results are shown in Tables 10 and 11. The control group directly purchased the finished product of Actemra (Roche) for chromatographic detection.

[0173] The results of IEC-HPLC showed that the acidic peak, main peak and alkaline peak of the pilot sample were consistent with those of the original drug Acteluo. The results of CE-SDS showed that the main peak and HHL peak of the non-reduced CE-SDS of the pilot sample were consistent with those of the original drug Acteluo. The results of N-sugar spectrum analysis showed that the glycoform of the pilot sample was consistent with that of the original drug Acteluo.

[0174] Table 10 300L scale pilot production analysis data

[0175]

[0176] Table 11 Sugar type analysis data of 300L pilot production

[0177]

[0178] In summary, the 300L (harvest volume 210L) pilot production data showed that the process was stable and reliable, and the product quality met the quality standards. The quality comparison study results showed that the product prepared by the disclosed process was The quality is highly similar, which proves the feasibility of the product process disclosed herein.

Claims

1. A cell culture method for producing antibodies, the method comprising a seed culture stage and a production culture stage, the production culture stage comprising the following steps: a. Inoculate cells in basal medium; b. Feeding: i) supplementing a feed medium, wherein the feed medium comprises Maxfeed 401, wherein the concentration of the Maxfeed 401 is 5 g / L to 35 g / L; ii) supplementing a glycoform regulator, wherein the glycoform regulator is supplemented simultaneously with the feed medium; The sugar type regulator contains at least galactose or fucose.

2. The method for producing antibodies according to claim 1, wherein: The concentration of Maxfeed 401 is 9 g / L to 25 g / L; preferably, The feed medium further comprises: 0.4 g / L to 1.2 g / L tyrosine salt, 0.5 g / L to 1.1 g / L cysteine ​​salt and 0.8 g / L to 1.5 g / L asparagine; More preferably, the feed medium further comprises: 0.8 g / L to 1.0 g / L tyrosine salt, 0.5 g / L to 1.0 g / L cysteine ​​salt and 1.0 g / L to 1.2 g / L asparagine.

3. The cell culture method for producing antibodies according to claim 1 or 2, wherein the glycosylation regulator comprises galactose; more preferably, the glycosylation regulator is EX-CELL Glycosylation Adjust (GAL+).

4. The cell culture method for producing antibodies according to any one of claims 1 to 3, wherein the amount of the glycoform regulator added is 0.5% to 2% (v / v) of the feed medium; preferably, the amount of the glycoform regulator added is 0.8% to 1.8% (v / v) of the feed medium; more preferably, the amount of the glycoform regulator added is 0.8% to 1.5% (v / v) of the feed medium.

5. The cell culture method for producing antibodies according to any one of claims 1 to 4, wherein the feed addition amount is 8% to 12% (v / v) of the initial culture volume; preferably 10% (v / v); more preferably, the feed is added on the 2nd to 4th day, the 5th to 7th day and / or the 8th to 10th day of the production culture stage; most preferably, the feed is added on the 3rd day, the 6th day and / or the 9th day of the production culture stage.

6. The cell culture method for producing antibodies according to any one of claims 1 to 5, wherein in the production culture stage, the culture temperature is 34.5-37.5°C; preferably 34.5-35.5°C; more preferably 35°C.

7. The cell culture method for producing antibodies according to any one of claims 1 to 6, wherein the culture conditions in the production culture stage include one or more of the following: i) The cell seeding density in step a is 0.5×10 6 Cells / mL~2×10 6 cells / mL; preferably, the cell seeding density in step a is 1.0×10 6 cells / mL~1.5×10 6 cells / mL; ii) pH 6.8 to 7.0; iii) dissolved oxygen is 20% to 60%; preferably, the dissolved oxygen is 40%; and iiii) The concentration of glucose in the cell culture medium is 3.5 g / L to 5 g / L, preferably 4.0 g / L, and the glucose concentration is calculated based on the initial culture volume.

8. The cell culture method for producing an antibody according to any one of claims 1 to 7, wherein the basal medium is Maxpro 301, Maxpro 303, Advanced CHO, ExpiCHO, CHO CD04, CD OptiCHO, Hycell, DNFeed1, CHO Feed 02, GrowthA or Dynamis.

9. The cell culture method for producing antibodies according to any one of claims 1 to 8, wherein the cells are CHO cells; preferably, the CHO cells are selected from CHO-K1, CHO DG44 and CHO-S cells, more preferably CHO-K1 cells; most preferably, the antibody is tocilizumab.

10. The method for producing antibodies according to any one of claims 1 to 9, wherein The culture conditions in the seed culture stage include one or more of the following conditions: i) a seed culture medium selected from the group consisting of Maxgrow 201, Advanced CHO, ExpiCHO, CHO CD04, CD OptiCHO, Hycell, DN Feed1, CHO Feed 02, GrowthA and Dynamis; ii) The cell seeding density was 0.35×10 6 ~0.75×10 6 cells / mL; and iii) pH 6.8 to 7.0; iiii) The culture temperature is 36.5°C to 37.5°C.

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