A cell culture method for producing tocilizumab

Abstract

The present disclosure belongs to the field of biology, and discloses a cell culture method for producing tocilizumab, solves the problems of complex production process, low expression, and large quality control difference of the current tocilizumab biosimilar, and provides strong support for the industrialized production of the antibody.

Description

Technical Field

[0001] This disclosure pertains to the field of antibody production, specifically relating to a cell culture method for producing tocilizumab. Background Technology

[0002] Rheumatoid arthritis (RA) is a common autoimmune disease characterized by inflammation and proliferation of the synovial membrane, leading to damage and destruction of articular cartilage and bone. In the natural course of RA, the disability rate is 60% after 5-10 years and 90% after 30 years.

[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 goals of rheumatoid arthritis treatment are to control inflammation, reduce pain, and reduce RA-related disability. Treatment typically includes medication, occupational or physical therapy, and regular exercise. Recognized biologics for treating RA include tumor necrosis factor (TNF) inhibitors and non-TNF inhibitors (such as cytokine inhibitors, T or B cell inhibitors), for example, biologics such as tocilizumab (Acmetab).

[0004] Tocilizumab ( Tocilizumab (Roche) is a neutralizing antibody against human interleukin-6 receptor (IL-6R), which prevents IL-6 from binding to IL-6R, thus having a good therapeutic effect on rheumatoid arthritis. However, current biosimilars of tocilizumab suffer from problems such as complex manufacturing processes, low expression levels, 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 to ensure that the quality of the produced recombinant anti-interleukin-6 receptor antibodies is comparable to that of the original drug. quite.

[0006] This 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 culture medium;

[0008] b. Replenishment:

[0009] i) Supplementing with feed medium containing Maxfeed 401 at a concentration of 5 g / L to 35 g / L; ii) Supplementing with a saccharide form regulator, which is added simultaneously with the feed medium;

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

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

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

[0013] In some embodiments, the concentration of the tyrosine salt is, for example, 0.4–0.8 g / L, 0.8–1.0 g / L, or 1–1.2 g / L; non-limiting embodiments 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 of these. In some embodiments, the concentration of the tyrosine salt in the feed medium is 0.85 g / L.

[0014] In some embodiments, the cysteine ​​concentration is, for example, 0.5–0.7 g / L, 0.7–0.9 g / L, or 0.9–1.1 g / L; non-limiting embodiments 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 of these. In some embodiments, the cysteine ​​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 other embodiments, the concentration of L-asparagine is, for example, 0.8–1.0 g / L, 1.0–1.2 g / L, or 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 of these. In some embodiments, the concentration of asparagine in the fed culture medium is 1.12 g / L.

[0016] In some embodiments, the feed culture medium further comprises: 0.8 g / L to 1.0 g / L tyrosine, 0.5 g / L to 1.0 g / L cysteine, 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, 0.75 g / L cysteine, and 1.12 g / L asparagine.

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

[0019] In some embodiments, the fed culture 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 fed culture medium is 1.75 g / L. In some embodiments, the concentration of disodium hydrogen phosphate in the fed culture medium is 4.26 g / L. In some embodiments, the concentration of glucose in the fed culture is 55 g / L.

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

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

[0022] In some embodiments, the fed culture medium comprises: 9.51 g / L or 23.78 g / L Maxfeed401, 0.85 g / L disodium tyrosine 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. In some embodiments, the fed culture medium consists of: 9.51 g / L or 23.78 g / L Maxfeed401, 0.85 g / L disodium tyrosine 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 the following components: 9.51 g / L or 23.78 g / L Maxfeed 401, 0.85 g / L tyrosine disodium 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 this disclosure, the concentrations of each component in the fed culture medium are calculated based on the fed volume.

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

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

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

[0028] In some embodiments, the culture time for the production culture phase is 10 to 14 days. In some embodiments, feed is added on days 2 to 4, 5 to 7, and / or 8 to 10 of the production culture phase; in some embodiments, feed is added on days 3, 6, and / or 9 of the production culture phase.

[0029] In some embodiments, the feed rate is 8% to 12% (v / v) of the initial culture volume. In some embodiments, the feed rate 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. 6 cells / mL ~ 2 × 10 6 Cells / mL. In some embodiments, the cell seeding density in step a is 1.0 × 10⁻⁶ cells / mL. 6 Cells / mL ~ 1.5 × 10⁻⁶ 6 Cells / mL.

[0031] In some specific embodiments, the cell culture medium may have a defined chemical composition, wherein all chemical components are known. As those skilled in the art will understand, a culture medium can be determined by those skilled in the art without excessive experimentation, provided it is suitable for the specific cells being cultured.

[0032] Commercially available culture media can be used, including but not limited to: Hyclone's Maxpro 301, Hyclone's Maxpro 303, Millipore's EX-CELL Advanced CHO basal medium, Gibco's ExpiCHO basal medium, Zhongshan Kangcheng CHO CD04, CD OptiCHO, Hycell, Shanghai Duoning DN Feed1, Zhongshan Kangcheng CHO Feed 02, GrowthA, Dynamis, Iscove's Modified Dulbecco's Medium, GMEM containing glutamine, or any other culture media known to those skilled in the art for specific cell types. Supplementary components or ingredients, including optional components, may be added to the above exemplary culture media in appropriate concentrations or amounts, as needed or as required and as known and practiced by those skilled in the art using conventional skills.

[0033] In some embodiments, the basal culture medium is Maxpro 301, Maxpro 303, AdvancedCHO, ExpiCHO, CHO CD04, CD OptiCHO, Hycell, DN Feed1, CHO Feed 02, GrowthA, or Dynamis.

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

[0035] In some implementations, the cell culture method disclosed herein is a serum-free culture process.

[0036] In some embodiments, during the production culture phase, the glucose concentration in the cell culture medium is 3.5 g / L to 6 g / L, calculated based on the initial culture volume. In some embodiments, the glucose concentration in the cell culture medium is 3.5 g / L to 5 g / L during the production culture phase; in some embodiments, the glucose concentration is 3.5 g / L to 4.5 g / L during the production culture phase; and in some embodiments, the glucose concentration is 4.0 g / L. In some embodiments, the glucose concentration in the cell culture medium is monitored daily, and if insufficient, it is replenished to 4.0 g / L.

[0037] In some embodiments, the pH of the production culture phase 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 during the production culture stage is 34.5–37.5°C. In some embodiments, the culture temperature during the production culture stage is 34.5–35.5°C. In some embodiments, the culture temperature during the production culture stage is 35°C.

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

[0040] The method for constructing the fermentation cell line disclosed herein is as follows:

[0041] The host cell was CHO-K1 cells (ATCC CCL-61). A recombinant plasmid was obtained by integrating the light and heavy chain genes of the antibody into the expression vector pTGS-GS (which was modified by removing the hygromycin and DHFR genes and adding the GS gene from the expression vector pTGS-FRT-DHFR). This recombinant plasmid could express a recombinant humanized monoclonal antibody against interleukin-6 receptor (IL-6R). The recombinant plasmid was stably transfected into CHO-K1 host cells, and monoclonal cell lines producing tocilizumab were obtained through screening.

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

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

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

[0045] In some implementations, the culture conditions for 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 is 0.35 × 10⁻⁶. 6 ~0.75×10 6 cells / mL;

[0048] iii) pH is 6.8–7.0; and

[0049] iiii) The culture temperature is 36.5℃~37.5℃.

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

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

[0052] In some implementations, the cell culture method for producing antibodies also includes a harvesting phase.

[0053] In some implementations, antibodies are harvested during the harvest phase.

[0054] In some implementations, the harvest phase includes a purification step for the antibody, specifically including:

[0055] 1) Obtain the clarified fermentation broth by centrifugation;

[0056] 2) Perform affinity chromatography;

[0057] 3) Perform deep filtration;

[0058] 4) Perform anion exchange chromatography and cation exchange chromatography sequentially;

[0059] 5) Perform nanofiltration on the cation exchange chromatography products.

[0060] In some implementations, the purification step further includes the following steps:

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

[0062] In some implementations, Mabselect SuRe is used as the packing material for affinity chromatography.

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

[0064] In some implementations, anion exchange chromatography is performed using Q Sepharose Fast Flow packing material and cation exchange chromatography is performed using POROS50HS packing material.

[0065] In some implementations, SV4 nanofiltration membranes (PALL, Pall (China) Co., Ltd.) are used for nanofiltration.

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

[0067] In some implementations, the purification steps specifically include the following steps in sequence: 1) obtaining a clear solution of the fermentation broth. 2) affinity chromatography capture using Mabselect SuRe packing material, followed by incubation at pH 3.5 ± 0.1 (first virus removal). 3) clarification after low-pH incubation using a depth filter A1HC (purchased from Merck), filtering out most impurities. 4) fine purification using anion exchange chromatography with Q Sepharose Fast Flow packing material and cation exchange chromatography with POROS 50HS packing material (second virus removal). 5) third virus removal using SV4 nanofiltration membrane (PALL). 6) concentration of the stock solution using 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, this disclosure provides 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 culture medium;

[0070] b. Replenishment:

[0071] i) Supplementing with feed medium containing Maxfeed 401 at a concentration of 5 g / L to 35 g / L; ii) Supplementing with a saccharide form regulator, which is added simultaneously with the feed medium;

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

[0073] In some embodiments, this disclosure provides 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. Inoculate cells in a basal medium, wherein the basal medium is Maxpro 301;

[0075] b. Replenishment:

[0076] i) Supplementing with feed medium containing Maxfeed 401 at a concentration of 5 g / L to 35 g / L; ii) Supplementing with a saccharide form regulator, which is added simultaneously with the feed medium;

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

[0078] The antibody is tocilizumab.

[0079] In some embodiments, in step a, cells are inoculated into a basal medium (seed culture) at a 1:1 volume ratio with the basal medium, thus entering the production culture stage. In some embodiments, the culture medium for the production stage comprises a seed culture medium (e.g., Maxgrow 201) and a basal medium (e.g., Maxpro 301). In some embodiments, the culture medium for the production stage comprises a seed culture medium (e.g., Maxgrow 201), a fed culture medium (e.g., Maxgrow 401), and a basal medium (e.g., Maxpro 301).

[0080] In some embodiments, this disclosure provides 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. Inoculate cells in a basal culture medium, wherein the basal culture medium is Maxpro 301;

[0082] b. Replenishment:

[0083] i) Supplemented feeding medium, wherein the supplemented feeding medium comprises: 9 g / L to 25 g / L Maxfeed401, 0.8 g / L to 1.0 g / L tyrosine, 0.5 g / L to 1.0 g / L cysteine, and 1.0 g / L to 1.2 g / L asparagine; the concentrations of each component in the supplemented feeding medium are calculated based on the feeding volume;

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

[0085] The culture temperature during the production stage is 34.5–37.5℃, and the antibody is harvested after 10–14 days of culture. The antibody is tocilizumab.

[0086] In some embodiments, this disclosure provides 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. Inoculate cells in a basal culture medium, wherein the basal culture medium is Maxpro 301;

[0088] b. Replenishment:

[0089] i) Supplemental feeding medium, wherein the supplemental feeding medium comprises: 9.51 g / L or 23.78 g / L Maxfeed 401, 0.85 g / L tyrosine disodium 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 concentrations of each component in the supplemental feeding medium are calculated based on the feeding volume;

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

[0091] The culture temperature during the production stage is 34.5–37.5℃, and antibodies are harvested after 10–14 days of culture.

[0092] The antibody is tocilizumab.

[0093] In some embodiments, this disclosure provides 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. Inoculate cells in a basal culture medium, wherein the basal culture medium is Maxpro 301;

[0095] b. Replenishment:

[0096] i) Supplemented feeding medium, wherein the supplemented feeding medium comprises: 9.51 g / L or 23.78 g / L Maxfeed 401, 0.85 g / L tyrosine disodium 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 concentrations of each component in the supplemented feeding medium are calculated based on the feeding volume;

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

[0098] The culture temperature during the production stage is 35℃, and antibodies are harvested after 10–14 days of culture.

[0099] The antibody is tocilizumab.

[0100] This disclosure presents a cell culture method for producing tocilizumab, which solves the problems of complex production processes, low expression levels, and large quality control differences in current tocilizumab biosimilars, providing strong support for the industrial production of this antibody. Attached Figure Description

[0101] Figure 1 This is a schematic diagram of antibody expression levels under different conditions in Example 1. Detailed Implementation

[0102] definition

[0103] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are all conventional procedures widely used in their respective fields.

[0104] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood as encompassing rather than exclusive or exhaustive; that is, meaning “including but not limited to.” Unless otherwise stated, “comprising” includes “consisting of.”

[0105] As used herein, the terms “about” or “approximately” mean a numerical value within an acceptable margin of error for a specific value determined by a person skilled in the art, the numerical value depending in part on how it is measured or determined (i.e., the limits of the measurement system). For example, in every practice in the art, “about” may mean within or above a standard deviation of 1. Alternatively, “about” or “substantially comprises” may mean a range of up to ±20%, for example, about 5.5 pH means pH 5.5 ± 1.1. Furthermore, particularly for biological systems or processes, the term may mean up to an order of magnitude or up to five times the numerical value. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of “about” or “substantially comprises” should be assumed to be within an acceptable margin of error for that specific value.

[0106] In this disclosure, the term "antibody" refers to an immunoglobulin that specifically recognizes and binds to an antigen, encompassing a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, bispecific antibodies, or antibody fragments.

[0107] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning the group comprising the antibodies is identical, except for the possibility of a small number of naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike polyclonal antibody formulations which contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, a key advantage of monoclonal antibodies is that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" should not be interpreted as requiring the production of the antibody through any particular method. For example, the monoclonal antibodies used according to the present invention can be prepared by a variety of techniques, including, for example, hybridoma methods (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, (ColdSpring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., In Monoclonal Antibodies and T-Cell Hybridoma). Methods for recombinant DNA in bacterial, eukaryotic, or plant cells (see, for example, U.S. Patent No. 4,816,567); phage display techniques (see, for example, 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 generating human or human-like antibodies in animals having some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, for example, WO1998 / 24893; WO1996 / 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); US Patent 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. Typically, a cell culture medium contains the following components: an energy source, usually carbohydrates, preferably glucose; amino acids, preferably the basic group of amino acids, 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" refers to a culture medium used to culture cells. This medium is used directly for culturing cells and is not used as an additive to other culture media, but various components can be added to a basal medium. For example, if CHO cells are cultured in DMEM (a well-known, commercially available mammalian cell culture medium) and periodically fed with glucose or other nutrients, DMEM would be considered a basal medium.

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

[0111] The term "seeding" refers to seeding cells into a culture medium provided for cell culture. Specifically, the culture medium can be provided before cell transfer or simultaneously with cell transfer to a cell bioreactor. In large-scale animal cell culture, the conventional practice is to pre-provide the culture medium, maintain the temperature and oxygen saturation within predetermined ranges, and then transfer the cells 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 used 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 in between. Internal conditions of the bioreactor, including but not limited to pH, dissolved oxygen, and temperature, are typically controlled during culture. The bioreactor can be composed of any material (including glass, plastic, or metal) suitable for containing mammalian cell cultures suspended in a culture medium under the culture conditions of the present invention.

[0114] The terms "feed-batch culture" or "feed culture" refer to a method of culturing cells in which additional components are provided to the culture one or more times after the culture process has begun. The components thus provided typically include the nutrient components of the cells that are consumed during the culture process. In this disclosure, the fed-batch culture phase begins after the cells are seeded into basal medium following the completion of the seed culture phase, with the day of cell seeding designated as day 0 of the fed-batch culture.

[0115] The term "initial culture volume" refers to the culture volume after cells have been inoculated in basal medium.

[0116] The term "antibody stock solution" refers to an antibody solution formed by preserving purified antibodies using a suitable buffer system that is routinely used by those skilled in the art.

[0117] The present disclosure is illustrated below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the disclosure in any way.

[0118] "Optional" or "optionally" means that the events or circumstances described below may, but do not necessarily, occur. The singular forms "a" and "an" as used in the specification and claims include their corresponding plural references, unless otherwise clearly stated in the text.

[0119] The following will describe this disclosure with reference to specific embodiments, but the content of this disclosure is not limited thereto.

[0120] Unless otherwise specified, the reagents and instruments used in the following methods are commonly used in the art and can be obtained commercially; the methods used are conventional methods in the art, and those skilled in the art can perform the methods and obtain the corresponding results without any doubt based on the description of the embodiments.

[0121] Measurement and detection methods

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

[0123] (2) Purification of tocilizumab:

[0124] The purification steps specifically include the following steps in sequence: 1) Obtaining a clear solution of the fermentation broth. 2) Affinity chromatography capture using Mabselect SuRe packing material, followed by incubation at pH 3.5 ± 0.1 (first virus removal). 3) Clarification after low-pH incubation using a depth filter A1HC to filter out most impurities. 4) Anion exchange chromatography using Q Sepharose Fast Flow packing material and cation exchange chromatography using POROS 50HS packing material; this stage is for fine purification (second virus removal). 5) Third virus removal using SV4 nanofiltration membrane (PALL). 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] The analysis was performed by HPLC. The column was equilibrated with phosphate buffer, and a standard curve program was set in the system program before injection. Chromatographic conditions: Column: POROS TMA20 (specifications: 2.1×30mm, 20μm), injection volume: 50μL, flow rate: 2.00mL / min, detection wavelength: 280nm, column temperature: 30℃, runtime: gradient run 9min, injection tray temperature: 6℃. Record relevant data and process them.

[0127] (5) SEC-HPLC determination:

[0128] HPLC determination: Before detection, the column was first equilibrated by using mobile phase at a flow rate of 0.5 mL / min until stable, then the sample was injected. Chromatographic conditions: Column: TSKgel G3000 SW XL (Specifications: 7.8×300mm, 5μm), injection volume: 20μL, flow rate: 0.5mL / min, detection wavelength: 280nm, column temperature: 30℃, runtime: isocratic run for 35min.

[0129] (6) CE-SDS determination:

[0130] Detection was performed using capillary electrophoresis. The capillary electrophoresis system was washed with approximately 1500 μL of water, approximately 3000 μL of running buffer, approximately 1500 μL of 1M NaOH, and approximately 1500 μL of 1M HCl, respectively. Before each injection, the system was filled with running buffer at 4.0 bar for 10 min before injection. The capillary was 33 cm long with an effective length of 8.5 cm. The detection wavelength was 214 nm, the column temperature was 20 °C, and the sample tray temperature was 15 °C.

[0131] (7) IEC-HPLC determination:

[0132] Determination by HPLC: The column was first equilibrated before injection. Chromatographic conditions: Column: Propac WCX-10 (size: 4×250mm, 10μm), injection volume: 10μL, flow rate: 0.8mL / min, detection wavelength: 214nm, column temperature: 30℃, run time: gradient run for 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-glucose spectrum determination method:

[0136] The analysis was performed by HPLC: The sample was treated with the Gly-X InstantPC N Sugar Rapid Analysis Kit 96 Kit. Before detection, the column was equilibrated with water and acetonitrile respectively, and then injected. Chromatographic conditions: Column: XBridge GlycanBEH Amide XP Column (size: 2.1×150mm, 2.5μm, 130A); Injection volume: 20μL; Detection wavelength: λex=285nm, λem=345nm; Flow rates: 0.3mL / min, 0.1mL / min, 0.15mL / min sequentially; Column temperature: 60℃; Sample chamber temperature: 8℃; Run time: 65min gradient run (see table below), followed by 10min (total 75min).

[0137] Table 2 Gradient Operation Table for N-Glycosyl Profile Determination

[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: Optimization of Feeding Culture Medium in a L Bioreactor

[0140] In our company's original process (inoculation density 1×10) 6 cells / mL ~1.5×10 6 Based on the following conditions (cells / mL, temperature 37℃, 200rpm, 40% DO, pH=6.90±0.10, glucose supplemented to 4.0g / L daily, 10% F solution supplemented on days 3, 6 and 9), the feeding medium was optimized and analyzed in a 2L reactor to systematically investigate the effects of different feeding media on cell growth.

[0141] All parameters were set to remain constant from the start of inoculation and culture. Parallel optimization was performed using three 2L bioreactors from Electrolab, UK. The experimental design is shown in Table 3-1.

[0142] Table 3-1 Optimization Scheme for Feeded Culture Medium

[0143]

[0144] Cells were seeded into seed culture medium and cultured. After seed amplification, the seed culture and basal culture medium were mixed 1:1 and transferred to a bioreactor for further cultivation, entering the production culture stage. The seed culture medium was solution C, i.e., Maxgrow 201 medium (purchased from Cytiva), and the basal culture medium was solution A, i.e., Maxpro 301 medium (purchased from Cytiva). The composition of the feed is shown in Table 3-2 below.

[0145] Table 3-2 Composition of Feed

[0146]

[0147] After harvesting the fermentation broth, the supernatant was obtained by centrifugation. The supernatant was then analyzed by affinity high-performance liquid chromatography (HPLC). The results are shown in Tables 4 and 5 below. Figure 1 As shown in the figure. The control group consisted of Roche's (Amerex) finished product, which was purchased directly and analyzed by chromatography.

[0148] Table 4. Optimized expression levels in 2L fed culture medium

[0149]

[0150] Table 5 Results of Optimization Analysis of Feeded Culture Medium in 2L Tanks

[0151]

[0152] From Table 4 and Figure 1 It can be seen that the antibody expression level is significantly better under feeding conditions F1 and F2 than under condition F, with F1 being the optimal condition. Table 5 shows that the peak content of TOC161002-2# (feeding condition F1) and TOC161002-6# (feeding condition F2) is comparable to that of the original drug, Actemra, while the peak content of TOC161101-2# (feeding condition F) is significantly lower than that of the original drug, Actemra.

[0153] Example 2.2 Optimization of temperature in a L bioreactor

[0154] Based on the optimized process in Example 1 (seeding density 1.00E+06 cells / mL~1.50E+06 cells / mL, temperature 37℃, 200 rpm, 40% DO, pH=6.90±0.10, glucose supplemented daily to 4.0 g / L, 10% F1 solution supplemented on days 3, 6 and 9), the culture temperature was optimized and analyzed in a 2L bioreactor. The effects of different culture temperatures on cell growth, protein expression and quality were systematically investigated, and key process parameters for cell culture were determined. The pH was set at 6.90±0.10, and the dissolved oxygen was set at 40%. All parameters were kept constant from the start of the inoculation culture. Parallel optimization was performed using three 2L bioreactors from Electrolab, UK. The experimental design is shown in Table 6.

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

[0156]

[0157] After harvesting the fermentation broth, the supernatant was obtained by centrifugation. The supernatant was then analyzed by affinity high-performance liquid chromatography (HPLC), and the results are shown in Table 7 below. The results showed that temperature had little effect on expression levels and antibody quality, but cooling to 35℃ slightly increased the percentage of the CE main peak. Therefore, a culture temperature of 35℃ is optimal.

[0158] Table 7

[0159]

[0160] Example 3.2 Optimization of glycoform regulators for L-type bioreactors

[0161] Based on the optimized processes of Examples 1 and 2, the TOC161201#2 process was selected, namely: seeding density of 1.00E+06 cells / mL to 1.50E+06 cells / mL, temperature of 35℃, 200 rpm, 40% DO, pH = 6.90±0.10, glucose supplemented to 4.0 g / L daily, and 10% F1 solution supplemented on days 3, 6 and 9. Glycosylation adjuster was added simultaneously with the feed. The concentration of the glycosylation adjuster EX-CELL Glycosylation Adjust (GAL+) (purchased from Sigma, model 14701C) was optimized in a 2L reactor. The effects of different concentrations of the glycosylation adjuster 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] Parallel optimization was performed using three 2L bioreactors, and the experimental design is shown in Table 8.

[0163] Table 8 Optimized culture process for different concentrations of sugar form regulators

[0164]

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

[0166] After harvesting the fermentation broth, centrifugation was performed to obtain the supernatant. The supernatant was then analyzed by affinity high-performance liquid chromatography (HPLC), and the results are shown in Table 9 below. Analysis of the data in Table 9 shows that when the concentration of the sugar form regulator is 2% (v / v) of the feed volume, the G0F% value decreases significantly. Therefore, a sugar form regulator concentration of 1% (v / v) or 1.5% (v / v) of the feed volume is optimal.

[0167] Table 9

[0168]

[0169] Example 4. Control of process parameters for pilot-scale production in a 300L bioreactor

[0170] The optimized process TOC170101-1# obtained in the above embodiments was used as the process for the production and cultivation stage in this embodiment. Specifically, the cultivation temperature was 35℃, dissolved oxygen was 40%, pH was 6.9, reactor rotation speed was 200 rpm, and 10% F1 solution was added on days 3, 6, and 9. The concentration of the glycosylation adjuster EX-CELL Glycosylation Adjust (GAL+) was 1% (calculated relative to the feed volume), and glucose was added daily to 4.0 g / L. During the seed culture stage, a step-by-step expansion culture was adopted, progressing through 20 mL, 80 mL, 320 mL, 5 L, and 30 L scale-up cultures. The cultivation temperature was 36.5–37.5℃, pH was 6.9–7.1, and the seed culture medium was Maxgrow 201. The seed cell density in the 30 L bioreactor was 1.5 × 10⁶ cells / mL. 6 ~3.0×10 6 When the cell count is 100 / mL, the antibody is inoculated into a 300L bioreactor for the production culture stage. The antibody is harvested after 10-14 days of culture.

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

[0172] After harvesting the fermentation broth, the supernatant was obtained by centrifugation. The supernatant was then analyzed by affinity high-performance liquid chromatography (HPLC), and the results are shown in Tables 10 and 11 below. The control group consisted of Roche's commercially available Armero (Acetate) product, which was purchased directly and analyzed by chromatography.

[0173] IEC-HPLC analysis showed that the acidic peak, main peak, and basic peak of the pilot-scale sample were consistent with those of the original drug, Actemere. CE-SDS analysis showed that the main peak and HHL peak of the non-reducing CE-SDS in the pilot-scale sample were consistent with those of the original drug, Actemere. N-glucose spectroscopy analysis showed that the glycoform of the pilot-scale sample was consistent with that of the original drug, Actemere.

[0174] Table 10 Analysis Data of 300L-scale Pilot Production

[0175]

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

[0177]

[0178] In summary, the pilot-scale production data of 300L (harvest volume 210L) demonstrates that the process is stable and reliable, and the product quality meets the standards. The quality comparison study results show that the product prepared by this disclosed process is comparable to... The high degree of similarity in quality demonstrates the feasibility of the product process disclosed herein.

Claims

1. A cell culture method for producing an antibody, the method comprising a seed culture stage and a production culture stage, the production culture stage comprising the following steps: a. inoculating cells in a basal medium; the basal medium is Maxpro 301; the cells are CHO cells; b. feeding: i) supplementing a feed medium, the feed medium comprising Maxfeed 401, wherein the concentration of the Maxfeed 401 is 9 g / L to 25 g / L; 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; ii) supplementing a glycosylation adjuster, the glycosylation adjuster being supplemented simultaneously with the feed medium; the glycosylation adjuster is EX-CELL Glycosylation Adjust (GAL+), the glycosylation adjuster is added at an amount of 0.8% to 1.8% (v / v) of the feed medium; the feed is fed at an amount of 8% to 12% (v / v) of the initial culture volume, the feed is fed at the 2nd to 4th day, the 5th to 7th day, and / or the 8th to 10th day of the production culture stage; the temperature of the production culture stage is 34.5 to 37.5 °C; the culture conditions of the production culture stage comprise one or more of the following: i) the cell seeding density in step a is 0.5 x 10 6 cells / mL to 2 x 10 6 cells / mL; ii) pH is 6.8 to 7.0; iii) dissolved oxygen is 20% to 60%; and iiii) the concentration of glucose in the cell culture medium is 3.5 g / L to 5 g / L, the concentration of glucose is calculated based on the initial culture volume; the antibody is tocilizumab.

2. The cell culture method for producing an antibody according to claim 1, wherein: 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 antibody-producing cell culture method according to claim 1, wherein, the glycosylation adjuster is added at an amount of 0.8% to 1.5% (v / v) of the feed medium.

4. The cell culture method for producing an antibody according to claim 1, the feed is fed at an amount of 10% (v / v) of the initial culture volume.

5. The antibody-producing cell culture process according to claim 1, wherein, the feed is fed at the 3rd day, the 6th day, and / or the 9th day of the production culture stage.

6. The cell culture method for producing an antibody according to any one of claims 1 to 5, the temperature of the production culture stage is 34.5 to 35.5 °C.

7. The cell culture method for producing an antibody according to claim 6, the temperature of the production culture stage is 35 °C.

8. The cell culture method for producing an antibody according to any one of claims 1 to 5, in the culture conditions of the production culture stage, The cell seeding density in step a is 1.0 x 10 6 cells / mL ~ 1.5 x 10 6 cells / mL.

9. The antibody-producing cell culture process according to any one of claims 1 to 5, wherein, in the culture conditions of the production culture stage, the dissolved oxygen is 40%.

10. The antibody-producing cell culture process according to any one of claims 1 to 5, wherein, in the culture conditions of the production culture stage, the concentration of glucose in the cell culture medium is 4.0 g / L.

11. The antibody-producing cell culture method according to any one of claims 1 to 5, wherein, the CHO cells are selected from CHO-K1, CHO DG44, and CHO-S cells.

12. The antibody-producing cell culture process according to any one of claims 1 to 5, wherein, The culture conditions of the seed culture stage include one or more of the following conditions: i) the seed culture medium is selected from Maxgrow 201, Advanced CHO, ExpiCHO, CHO CD04, CD OptiCHO, Hycell, DN Feed1, CHO Feed 02, GrowthA and Dynamis; ii) a cell seeding density of 0.35 x 10 6 ~0.75 x 10 6 cells / mL; and iii) the pH is 6.8-7.0; iiii) the culture temperature is 36.5-37.5°C.

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