CN119241710A

CN119241710A - A cell culture method for producing tocilizumab

Info

Publication number: CN119241710A
Application number: CN202411639703.9A
Authority: CN
Inventors: 焦静雨; 孙龑; 杨亚平; 苟金霞; 高栋; 王海彬
Original assignee: Zhejiang Borui Biopharmaceutical Co ltd; Hangzhou Bozhirui Biopharmaceutical Co ltd
Current assignee: Zhejiang Borui Biopharmaceutical Co ltd; Hangzhou Bozhirui Biopharmaceutical Co ltd
Priority date: 2024-11-15
Filing date: 2024-11-15
Publication date: 2025-01-03

Abstract

本披露属于生物学领域，其公开了一种用于生产托珠单抗的细胞培养方法，解决了目前托珠单抗的生物类似药存在的生产工艺复杂，表达量低，质控差异大等问题，为该抗体的工业化生产提供了有力支持。The present disclosure belongs to the field of biology, and discloses a cell culture method for producing tocilizumab, which solves the problems of complex production process, low expression level, large quality control differences, etc. of current biosimilars of tocilizumab, and provides strong support for the industrial production of the antibody.

Description

Cell culture method for producing tolizumab

Technical Field

The present disclosure is in the field of antibody production, and in particular relates to a cell culture method for producing tolizumab.

Background

Rheumatoid arthritis (Rheumatoid arthritis, RA) is a common autoimmune disease, whose pathological features are joint synovial inflammation and synovial tissue proliferation, which in turn leads to injury and destruction of articular cartilage and bone. In the natural course of rheumatoid arthritis, the disability rate is 60% for 5-10 years, and the disability rate is 90% for 30 years.

Pain and swelling of the joint can be ameliorated or cured by treatment of RA and joint damage prevented. Early treatment may lead to better long term results and the type of joint damage that may lead to joint replacement is less likely to occur in patients receiving early treatment. The main therapeutic goals of rheumatoid arthritis are to control inflammation, reduce pain and reduce RA-related disability. Treatment typically includes medication, physical therapy, or physical therapy, and regular exercise. Well-known biological agents for treating RA include Tumor Necrosis Factor (TNF) inhibitors and non-TNF inhibitors (e.g., cytokine inhibitors, T or B cell inhibitors), for example, biological agents include tolizumab (yamero) and the like.

Tuozhu monoclonal antibodyYamilo, roche) is a neutralizing antibody against human interleukin-6 receptor (IL-6R) which prevents the binding of IL-6 to IL-6R and thus has a good therapeutic effect on rheumatoid arthritis. However, the existing biological similar medicine of tolizumab has the problems of complex production process, low expression quantity, unstable quality control and the like.

Disclosure of Invention

The aim of the present disclosure is to establish a robust process suitable for the production of recombinant anti-interleukin-6 receptor antibodies, and the quality and pro-drug of the produced recombinant anti-interleukin-6 receptor antibodiesEquivalent.

The present disclosure discloses 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 steps of:

a. inoculating cells in a basal medium;

b. And (3) material supplementing:

i) Supplementing a feed medium, wherein the feed medium comprises Maxfeed g/L-35 g/L Maxfeed, ii) supplementing a sugar-type regulator, and supplementing the sugar-type regulator and the feed medium simultaneously;

the glycoform modifier comprises at least galactose or fucose.

In some embodiments, the Maxfeed g/L to 25g/L concentration of Maxfeed. In some embodiments, the Maxfeed.about.8 g/L, 8.about.10 g/L, 10.about.15 g/L, 15.about.20 g/L, 20.about.23 g/L, 23.about.25 g/L, non-limiting examples include about 9g/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 therebetween. In some embodiments, the Maxfeed401,401 concentration is 9g/L to 25g/L, and in some embodiments, the Maxfeed401,401 concentration is 9.51g/L. In some embodiments, the Maxfeed401 concentration is 23.78g/L.

In some embodiments, the feed medium further comprises 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.

In some embodiments, the concentration of the tyrosine salt is, for example, 0.4 to 0.8g/L, 0.8 to 1.0g/L, 1 to 1.2g/L, with non-limiting examples including about 0.4g/L, about 0.8g/L, about 1g/L, about 1.2g/L, or any value therebetween. In some embodiments, the concentration of tyrosine salt in the feed medium is 0.85g/L.

In some embodiments, the cysteine salt concentration is, for example, 0.5 to 0.7g/L, 0.7 to 0.9g/L, 0.9 to 1.1g/L, non-limiting examples include about 0.5g/L, about 0.7g/L, about 0.9g/L, about 1.1g/L, or any value therebetween. In some embodiments, the concentration of cysteine salts in the feed medium is 0.75g/L.

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 0.8g/L, about 1.0g/L, about 1.2g/L, about 1.5g/L, or any value in between any two values. In some embodiments, the concentration of asparagine in the feed medium is 1.12g/L.

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

In some embodiments, the feed medium further comprises 0.85g/L tyrosine salt, 0.75g/L cysteine salt, and 1.12g/L asparagine.

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

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

In some embodiments, the tyrosine salt is tyrosine disodium salt hydrate.

In some embodiments, the cysteine hydrochloride is cysteine hydrochloride monohydrate.

In some embodiments, the feed medium comprises 9.51g/L or 23.78g/L Maxfeed g/L tyrosine disodium salt hydrate, 0.85g/L cysteine hydrochloride monohydrate, 1.12g/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 9.51g/L or 23.78g/L Maxfeed g/L of 401, 0.85g/L of tyrosine disodium salt hydrate, 0.75g/L of cysteine hydrochloride monohydrate, 1.12g/L of asparagine, 1-3 g/L of sodium hydroxide, 3-6 g/L of disodium hydrogen phosphate, and 40-70 g/L of glucose.

In some embodiments, the feed medium consists of 9.51g/L or 23.78g/L Maxfeed g/L of 401, 0.85g/L of tyrosine disodium salt hydrate, 0.75g/L of cysteine hydrochloride monohydrate, 1.12g/L of asparagine, 1.75g/L of sodium hydroxide, 4.62g/L of disodium hydrogen phosphate, and 55g/L of glucose.

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

In some embodiments, the antibody is tobrazumab.

In some embodiments, the glycoform modulator comprises galactose. In some embodiments, the glycoform modulator further comprises manganese chloride or uridine. In some embodiments, the glycoform modifier is EX-CELL Glycosylation Adjust (GAL+), model 14701C.

In some embodiments, the sugar-type modifier is added in an amount of 0.5% -2% (v/v) of the feed medium. In some embodiments, the sugar-type modifier is added in an amount of 0.8% -1.8% (v/v) of the feed medium. In some embodiments, the sugar-type modifier is added in an amount of 0.8% -1.5% (v/v), preferably 1% (v/v), of the feed medium.

In some embodiments, the culturing time of the production culturing stage is 10 days to 14 days. In some embodiments, the feed is fed on days 2-4, 5-7, and/or 8-10 of the production culture stage, in some embodiments, the feed is fed on days 3, 6, and/or 9 of the production culture stage;

in some embodiments, the feed is fed in an amount of 8% -12% (v/v) of the initial culture volume. In some embodiments, the feed is fed in an amount of 10% (v/v) of the initial culture volume.

In some embodiments, the cell seeding density in step a is 0.5X10 ⁶ cells/mL to 2X 10 ⁶ cells/mL. In some embodiments, the cell seeding density in step a is 1.0X10 ⁶ cells/mL to 1.5X10 ⁶ cells/mL.

In some embodiments, the cell culture medium may be chemically defined, wherein all chemical components are known. As will be appreciated by those skilled in the art, the medium may be determined by those skilled in the art without undue experimentation, as appropriate to the particular cells being cultured.

Commercially available media may be utilized, including but not limited to Maxpro for Hyclone, maxpro for Hyclone, EX-CELL ADVANCED CHO basal medium for Millipore, expiCHO basal medium for Gibco, zhongshan Kang CHO CD04, CD OptiCHO, hycell, shanghai dorning DN Feed1, CHO Feed 02 for Zhongshan Kang, growthA, dynamis, dulbecco's modified by Iscove (Iscove's Modified Dulbecco ' sMedium), GMEM with glutamine or any other medium formulated for specific cell types known to those skilled in the art. Additional components or ingredients, including optional components, may be added to the above-described exemplary media in appropriate concentrations or amounts as needed or desired and as known and practiced by those of ordinary skill in the art.

In some embodiments, the basal medium is Maxpro, maxpro, advanced CHO, expiCHO, CHO CD04, CD OptiCHO, hycell, DN Feed1, CHO Feed 02, growthA, or Dynamis.

In some embodiments, the basal medium is Maxpro a 301.

In some embodiments, the cell culture methods of the present disclosure are serum-free throughout.

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

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%.

In some embodiments, the culture temperature of the production culture stage is 34.5-37.5 ℃. In some embodiments, the incubation temperature in the production incubation stage is 34.5-35.5 ℃. In some embodiments, the culture temperature of the production culture stage is 35 ℃.

In some embodiments, wherein the cell is a CHO cell, preferably the CHO cell is selected from the group consisting of CHO-K1, CHO DG44 and CHO-S cells, more preferably a CHO-K1 cell.

The construction method of the fermentation cell strain in the disclosure comprises the following steps:

The host cell is CHO-K1 cell (ATCC CCL-61), the expression vector pTGS-GS (through modifying the expression vector pTGS-FRT-DHFR, remove hygromycin and DHFR genes and add GS genes) integrates the light chain gene and the heavy chain gene of the antibody to obtain the recombinant plasmid, and the recombinant plasmid can express the recombinant anti-interleukin-6 receptor (IL-6R) humanized monoclonal antibody. Stably transfecting the recombinant into CHO-K1 host cells, and screening to obtain the monoclonal cell strain for producing and expressing the tolizumab.

The amino acid sequences of the heavy chain and the light chain of the tobira monoclonal antibody are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2:

QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO：1)

DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPS RFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：2).

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

i) The seed culture medium is selected from Maxgrow, advanced CHO, expiCHO, CHO CD04, CD OptiCHO, hycell, DN Feed1, CHO Feed 02, growthA or Dynamis;

ii) cell seeding density of 0.35×10 ⁶～0.75×10⁶ cells/mL;

iii) pH is 6.8-7.0, and

Iii) the culture temperature is 36.5-37.5 ℃.

In some embodiments, the seed medium is Maxgrow a 201.

In some embodiments, the culture temperature of the seed culture stage is 37 ℃.

In some embodiments, the cell culture method for producing an antibody further comprises a harvesting stage.

In some embodiments, the antibody is harvested in the harvesting phase.

In some embodiments, the harvesting stage comprises a purification step for the antibody, specifically comprising:

1) Obtaining a clarified liquid of the fermentation broth by centrifugation;

2) Performing affinity chromatography;

3) Deep filtration is carried out;

4) Sequentially performing anion exchange chromatography and cation exchange chromatography;

5) Nanofiltration of the cation exchange chromatography product.

In some embodiments, the purification step further comprises the steps of:

and carrying out ultrafiltration on the nanofiltration product to obtain an antibody stock solution.

In some embodiments, affinity chromatography is performed with MabSelect SuRe as a packing material.

In some embodiments, depth filtration is performed using a depth filter. Preferably, depth filtration is performed using depth filter A1HC (purchased from Merck).

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

In some embodiments, nanofiltration is performed using SV4 nanofiltration membranes (PALL, boll (china) limited).

In some embodiments, ultrafiltration is performed with a tangential flow ultrafiltration membrane P2B050a25 (merck, milbo) having a molecular weight cut-off of 50 kDa.

In some embodiments, the purification step comprises, in particular, in order, 1) obtaining a clarified liquid of the fermentation broth. 2) Affinity chromatography capture was performed with MabSelect SuRe packing and incubation (first virus removal) was performed at pH 3.5.+ -. 0.1. 3) Clarification after low pH incubation with depth filter A1HC (purchased from merck) filtered most of the impurities. 4) Anion exchange chromatography with Q Sepharose Fast Flow packing and cation exchange chromatography with POROS 50HS packing, this stage being fine purification (second virus removal). 5) A third virus removal was performed using SV4 nanofiltration membrane (PALL) filtration. 6) The stock solution is concentrated by a tangential flow ultrafiltration membrane P2B050A25 (Merck Mirburg) with the molecular weight cutoff of 50kDa to obtain the recombinant anti-interleukin-6 receptor (IL-6R) humanized monoclonal antibody.

In some embodiments, the disclosure discloses 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 steps of:

a. inoculating cells in a basal medium;

b. And (3) material supplementing:

The glycoform regulator is EX-CELL Glycosylation Adjust (GAL+).

a. Inoculating cells in a basal medium, wherein the basal medium is Maxpro 301,301;

b. And (3) material supplementing:

The sugar type regulator is EX-CELL Glycosylation Adjust (GAL+), the feed medium also comprises 0.4 g/L-1.2 g/L tyrosine salt, 0.5 g/L-1.1 g/L cysteine salt and 0.8 g/L-1.5 g/L asparagine, and the concentration of the amino acid and Maxfeed 401 is calculated based on the feed volume;

The antibody is tobrazumab.

In some embodiments, in step a, cells (seed solution) are inoculated in the basal medium, wherein the seed solution is inoculated with the basal medium in a 1:1 volume ratio, into the production culture stage. In some embodiments, the medium of the production stage comprises a seed medium (e.g., maxgrow a 201) and a basal medium (e.g., maxpro a 301). In some embodiments, the medium of the production stage comprises a seed medium (e.g., maxgrow a 201), a feed medium (e.g., maxgrow a 401), and a basal medium (e.g., maxpro a 301).

b. And (3) material supplementing:

i) The method comprises the steps of supplementing a feed culture medium, wherein the feed culture medium comprises 9 g/L-25 g/L Maxfeed g/401, 0.8 g/L-1.0 g/L of tyrosine salt, 0.5 g/L-1.0 g/L of cysteine salt and 1.0 g/L-1.2 g/L of asparagine, and the concentration of each component in the feed culture medium is calculated based on the feed volume;

ii) supplementing a sugar-type regulator, wherein the sugar-type regulator is EX-CELL Glycosylation Adjust (GAL+), the adding amount of the sugar-type regulator is 0.5% -2% (v/v) of a feed medium, and the sugar-type regulator and the feed medium are supplemented simultaneously;

The culture temperature in the production stage is 34.5-37.5 ℃, and antibodies are obtained after culturing for 10-14 days, wherein the antibodies are toxamab.

b. And (3) material supplementing:

i) The method comprises the steps of supplementing a feed culture medium, wherein the feed culture medium comprises 9.51g/L or 23.78g/L Maxfeed g/L tyrosine disodium salt hydrate, 0.85g/L cysteine hydrochloride monohydrate, 1.12g/L asparagine, 1-3 g/L sodium hydroxide, 3-6 g/L disodium hydrogen phosphate and 40-70 g/L glucose, and the concentration of each component in the feed culture medium is calculated based on the volume of feed;

the culture temperature in the production stage is 34.5-37.5 ℃, and the antibody is obtained after culturing for 10-14 days;

The antibody is tobrazumab.

b. And (3) material supplementing:

i) Supplementing a feed medium, wherein the feed medium comprises 9.51g/L or 23.78g/L Maxfeed g/L tyrosine disodium salt hydrate, 0.85g/L cysteine hydrochloride monohydrate, 1.12g/L asparagine, 1.75g/L sodium hydroxide, 4.26g/L disodium hydrogen phosphate and 55g/L glucose, and the concentration of each component in the feed medium is calculated based on the volume of the feed;

ii) supplementing a sugar-type regulator, wherein the sugar-type regulator is EX-CELL Glycosylation Adjust (GAL+), the adding amount of the sugar-type regulator is 1% (v/v) of a feed medium, and the sugar-type regulator and the feed medium are supplemented simultaneously;

culturing at 35 ℃ in the production stage, and harvesting antibodies after culturing for 10-14 days;

The antibody is tobrazumab.

The disclosure discloses a cell culture method for producing tobramycin, which solves the problems of complex production process, low expression quantity, large quality control difference and the like of the prior biological similar medicine of tobramycin, and provides powerful support for the industrial production of the antibody.

Drawings

FIG. 1 is a graph showing the expression levels of antibodies under different conditions in example 1.

Detailed Description

Definition of the definition

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the procedures of cell culture, biochemistry, nucleic acid chemistry, immunology laboratories and the like as used herein are all conventional procedures widely used in the corresponding fields.

Throughout the specification and claims, the words "comprise," "have," "include," and the like are to be construed as having an inclusive rather than an exclusive or exhaustive meaning, i.e., a meaning of "including but not limited to," unless the context clearly dictates otherwise. Unless otherwise indicated, "comprising" includes "consisting of.

The term "about" or "approximately" as used herein means that the value is within an acceptable error range for the particular value being determined by one of ordinary skill in the art, the value depending in part on how the measurement or determination is made (i.e., the limits of the measurement system). For example, "about" in each implementation in the art may mean within 1 or exceeding a standard deviation of 1. Or "about" or "substantially comprising" may mean a range of up to ±20%, e.g., a pH of about 5.5 means a pH of 5.5±1.1. Furthermore, the term may mean at most one order of magnitude or at most 5 times the value, especially for biological systems or processes. Unless otherwise indicated, when a particular value is found in the present disclosure and claims, the meaning of "about" or "consisting essentially of" should be assumed to be within the acceptable error range for that particular value.

In the present disclosure, the term "antibody" is an immunoglobulin capable of specifically recognizing and binding an antigen, which encompasses a variety of antibody constructs, including but not limited to monoclonal antibodies, polyclonal antibodies, bispecific antibodies, or antibody fragments.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the population comprising each antibody is identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring antibody production by any particular method. For example, monoclonal Antibodies for use 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, hybrid mia, 14 (3): 253-260 (1995), harlow et al, antibodies: A Laboratory Manual (ColdSpring Harbor Laboratory Press,2nd ed.1988); HAMMERLING et al, in MonoclonalAntibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981), in bacteria, and methods of producing Antibodies, Recombinant DNA methods in eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567); phage display techniques (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 part or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO1998/24893, WO1996/34096, WO 1991/10741, jakobovits et al, proc. Natl. Acad. Sci. USA 90:2551 (1993), jakobovits, UK. Eh. 19964 (1993), WO 7, 19933, 1995, and 1995, and 7, 1995, and 1995, 7, and 1995, 7, 1995, and 1995, 7, and 1995, 7, and 1996, and 1995, 1996, and, 1996, and, 1996, 33, 1996, 33, and, 33, human,.

The term "cell culture medium" refers to an aqueous solution of nutrients that can be used to grow cells for a long period of time. Typically, the cell culture medium comprises an energy source, typically a carbohydrate, preferably glucose, an amino acid, preferably an amino acid 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.

The term "basal medium" is a medium used for culturing cells, which is used directly for culturing cells and is not used as an additive to other media, but various components may 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 periodically fed with glucose or other nutrients, DMEM will be considered a basal medium.

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

The term "seeding" refers to seeding cells into a medium provided for cell culture. In particular, the culture medium may be provided prior to transferring the cells, or may be provided simultaneously with the transfer of the cells into the cell bioreactor. In large scale animal cell culture, it is conventional practice to provide a culture medium in advance, and after maintaining the temperature and oxygen saturation within predetermined ranges, transfer the cells into a bioreactor.

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

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

The term "fed-batch culture" or "fed-batch culture" refers to a method of culturing cells in which additional components are provided to the culture one or more times after the start of the culture process. The components so provided typically comprise the nutritional components of the cells that are consumed during the culturing process. In the present disclosure, after the cells are inoculated into the basal medium from the end of the seed culture stage, i.e., the feed culture stage is entered, the day of inoculation of the cells is taken as day 0 of the feed culture.

The term "initial culture volume" is the culture volume after seeding cells in basal medium.

The term "antibody stock solution" refers to an antibody solution obtained by preserving an antibody obtained after purification in a buffer system conventionally used by those skilled in the art.

The present disclosure is described below with reference to specific embodiments. Those skilled in the art will appreciate that these examples are merely illustrative of the present disclosure and are not intended to limit the scope of the present disclosure in any way.

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

The present disclosure will be described below in connection with specific embodiments, to which the disclosure is not limited.

Reagents and apparatus used in the following methods are all commonly used in the art and are commercially available, unless explicitly stated otherwise, and methods used are all conventional in the art, which can be performed unambiguously by a person skilled in the art and with corresponding results based on the description of the examples.

Measurement and detection method

(1) Cell viability and Density determination by trypan blue staining with a cell viability Analyzer.

(2) Purification of tobrazumab:

The purification step comprises, in particular, 1) obtaining a clarified liquid of the fermentation broth. 2) Affinity chromatography capture was performed with MabSelect SuRe packing and incubation (first virus removal) was performed at pH 3.5.+ -. 0.1. 3) Clarification after low pH incubation with depth filter A1HC, most of the impurities were filtered. 4) Anion exchange chromatography with Q Sepharose Fast Flow packing and cation exchange chromatography with POROS 50HS packing, this stage being fine purification (second virus removal). 5) A third virus removal was performed using SV4 nanofiltration membrane (PALL) filtration. 6) Concentrating the stock solution by a tangential flow ultrafiltration membrane P2B050A25 with the molecular weight cut-off of 50kDa to obtain the tobrazumab.

(3) Determination of tobrazumab concentration:

The column equilibrium was determined by HPLC, phosphate buffer, standard curve procedure was set in the system program, and then sample was taken. The chromatographic conditions are a chromatographic column POROS ^TM A20 (specification: 2.1X105 mm,20 μm), a sample injection volume: 50. Mu.L, a flow rate: 2.00mL/min, a detection wavelength: 280nm, a column temperature: 30 ℃ and an operation time period: 9min of gradient operation, and a sample injection tray temperature: 6 ℃. The related data is recorded and processed.

(5) SEC-HPLC assay:

The detection is carried out by HPLC, i.e. the system is equilibrated with the mobile phase at a flow rate of 0.5mL/min until stable, and then the sample is introduced. Chromatographic conditions are a chromatographic column TSKgel G3000 SW _XL (specification 7.8X105 mm,5 μm), a sample volume of 20. Mu.L, a flow rate of 0.5mL/min, a detection wavelength of 280nm, a column temperature of 30℃and an operation time period of 35min at isocratic.

(6) CE-SDS assay:

The detection was performed by capillary electrophoresis, and the capillary electrophoresis systems were washed with about 1500. Mu.L of water, about 3000. Mu.L of running buffer, about 1500. Mu.L of 1M NaOH, and about 1500. Mu.L of 1M HCl, respectively, by the capillary electrophoresis systems. Before each sample injection, the sample is injected after filling with running buffer for 10min at 4.0 bar. The capillary tube length is 33cm, the effective length is 8.5cm, the detection wavelength is 214nm, the column temperature is 20 ℃, and the sample tray is 15 ℃.

(7) IEC-HPLC assay:

The method is characterized in that the HPLC method is adopted to measure, chromatographic column balancing is firstly carried out, and sample injection is carried out after balancing. The chromatographic conditions are that a chromatographic column is PropacWCX-10 (specification: 4X 250mm,10 μm), the sample injection volume is 10 mu L, the flow rate is 0.8mL/min, the detection wavelength is 214nm, the column temperature is 30 ℃, and the operation time is 60min of gradient operation.

TABLE 1 gradient run table

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

(8) N-sugar spectrometry:

The determination is carried out by HPLC method, and the sample is treated by Gly-X INSTANTPC N sugar rapid analysis Kit 96 Kit. Before detection, water and acetonitrile are used for chromatographic column balancing, and sample injection is carried out after balancing. Chromatographic conditions were XBridge Glycan BEH Amide XP Column (specification: 2.1X105 mm,2.5 μm, 130A), sample volume: 20. Mu.L, detection wavelength: λex=285 nm, λem=345 nm, flow rate of 0.3mL/min, 0.1mL/min, 0.15mL/min, column temperature of 60 ℃, sample chamber temperature of 8 ℃, run duration: gradient run 65min (see Table below), 10min later (75 min total).

Table 2 gradient run table for N-glycospectrometry

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

Example 1.2L bioreactor feed Medium optimization

Based on the original process of the company (inoculation density 1×10 ⁶cells/mL～1.5×10⁶ cells/mL, temperature 37 ℃,200rpm,40% do, ph=6.90±0.10, glucose fed to 4.0g/L daily, 10% F fed on day 3, day 6 and day 9), the feed medium was optimally analyzed on a 2L reactor, and the system examined the effect of different feed media on cell growth, etc.

All parameter settings were started from the inoculated culture, i.e. fixed during the culture. Parallel optimization was performed using 3 2L bioreactors from Electrolab, UK, and the experimental design is shown in Table 3-1.

TABLE 3-1 optimization of the experimental protocol for the feed medium

Inoculating cells into a seed culture medium for culture, mixing a seed solution and a basic culture medium in a ratio of 1:1 after seed amplification, transferring into a bioreactor for culture, and entering a production culture stage. The seed culture medium is C liquid, namely Maxgrow culture medium (purchased from Cytiva Situo), and the basal culture medium is A liquid, namely Maxpro culture medium (purchased from Cytiva Situo). The ingredients of the feed are shown in Table 3-2 below.

TABLE 3-2 ingredients of the feed

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 results are shown in the following tables 4, 5 and fig. 1. Wherein the control group is to directly purchase the finished product of the yameto (Rogowski) for chromatographic detection.

TABLE 4 optimization of expression level data for 2L tank feed Medium

TABLE 5 optimization analysis results of 2L tank feed Medium

It can be seen from Table 4 and FIG. 1 that the amount of antibody expressed under the conditions of feed conditions of F1 and F2 was significantly better than F, wherein F1 was optimal. As can be seen from Table 5, the main peak contents of TOC161002-2# (feed condition is F1) and TOC161002-6# (feed condition is F2) are equivalent to the main peak content of the original drug of elegant jejunum, and the main peak content of TOC161101-2# (feed condition is F) is significantly lower than the main peak content of the original drug of elegant jejunum.

Example 2.2 optimization of bioreactor temperature

Based on the optimization process of example 1 (inoculation density 1.00E+06 cells/mL-1.50E+06 cells/mL, temperature 37 ℃,200rpm,40% DO, pH=6.90+ -0.10, glucose fed to 4.0g/L each day, 10% F1 fed on days 3, 6 and 9), optimization analysis was performed on the culture temperature in a 2L bioreactor, and the effect of different culture temperatures on cell growth, protein expression and quality was systematically examined to determine the key process parameters of cell culture. The pH set point was 6.90.+ -. 0.10 and the dissolved oxygen set point was 40% and all parameter settings were fixed from the start of the inoculation culture, i.e. during the culture. Parallel optimization was performed using 3 2L bioreactors from Electrolab, UK, and the experimental design is shown in Table 6.

TABLE 6 2L tank culture temperature optimization experiment scheme

The fermentation broth was harvested and centrifuged to obtain a supernatant, which was measured by affinity high performance liquid chromatography and analyzed as shown in table 7 below. The results show that the temperature has little effect on the expression level and antibody quality, but the 35 ℃ temperature reduction can slightly increase the percent of the main peak of CE. Therefore, the culture temperature is preferably 35 ℃.

TABLE 7

Example 3.2 optimization of sugar type modulators for bioreactors

Based on the optimization process of the embodiment 1 and the embodiment 2, TOC161201#2 is selected, namely, the inoculation density is 1.00E+06 cells/mL, the temperature is 35 ℃ and 200rpm, the DO concentration is 40%, the pH=6.90+/-0.10, the glucose is fed to 4.0g/L every day, 10% F1 liquid is fed to 3,6 and 9 days, sugar type regulators are fed simultaneously during feeding, the concentration of sugar type regulator EX-CELL Glycosylation Adjust (GAL+) (purchased from sigma company, model 14701C) is optimized on a 2L reactor, the influence of sugar type regulator EX-CELL Glycosylation Adjust (GAL+) with different concentrations on cell growth and antibody expression and quality is examined systematically, and key process parameters of cell culture are determined.

Parallel optimization was performed using 3 2L bioreactors, and the experimental design is shown in Table 8.

TABLE 8 optimized culture process of sugar type regulators with different concentrations

Note that the amount of sugar type modifier was calculated based on the feed volume.

The fermentation broth was harvested and centrifuged to obtain a supernatant, which was measured by affinity high performance liquid chromatography and analyzed as shown in table 9 below. From the data analysis in Table 9, it is found that when the concentration of the sugar type regulator is 2% (v/v) of the fed volume, the G0F% value is significantly decreased, and therefore, when the concentration of the sugar type regulator is 1% (v/v) or 1.5% (v/v) of the fed volume, it is preferable.

TABLE 9

Example 4.300L pilot scale production Process parameter control in bioreactor

The process TOC170101-1# obtained by optimizing the above example was used as the process in the production culture stage of this example, namely, the culture temperature was 35 ℃, dissolved oxygen was 40%, pH was 6.9, the reactor rotation speed was 200rpm, 10% F1 solution was fed on days 3,6 and 9, the concentration of sugar-type regulator EX-CELL Glycosylation Adjust (GAL+) was 1% (calculated with respect to the fed volume), and glucose was fed to 4.0g/L per day. And in the seed culture stage, gradual amplification and amplification culture are adopted, 20mL, 80mL, 320mL, 5L and 30L of amplification culture are adopted, the culture temperature is 36.5-37.5 ℃, the pH is 6.9-7.1, the seed culture medium is Maxgrow-201, when the seed cell density in the 30L bioreactor is 1.5X10 ⁶～3.0×10⁶ cells/mL, the seed culture is inoculated into the 300L bioreactor, the culture in the production culture stage is carried out, and the antibody is harvested after the culture is carried out for 10-14 days.

This example was run on a 300L cell culture scale (lot 0162817002). Product verification and analysis show that the average expression level of the antibody is about 2.05+/-0.01 g/L, and the purity of SEC-HPLC exceeds 98 percent.

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 results are shown in tables 10 and 11 below. Wherein the control group is to directly purchase the finished product of the yameto (Rogowski) for chromatographic detection.

IEC-HPLC analysis revealed that the acid peak, main peak and basic peak of the pilot sample were consistent with those of the original ground drug, ofmeiluo. The CE-SDS result analysis revealed that the non-reduced CE-SDS main peak and HIL peak of the pilot sample were consistent with those of the original ground drug, ocimum gratissimum. Analysis of the N-glycospectrometry results revealed that the glycoform of the pilot sample remained consistent with that of the original ground drug, dimefon.

Table 10 pilot production analysis data for 300l scale

Table 11 data from pilot production sugar analysis at 300l scale

In conclusion, the 300L (harvest volume 210L) pilot plant production data show that the process is stable and reliable, the product quality meets the quality standard, and the quality comparison research result shows that the product prepared by the process disclosed by the invention and the product prepared by the process disclosed by the inventionThe quality is highly similar, demonstrating the feasibility of the process of the disclosed product.

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 to 2×10 ⁶ cells/mL; preferably, the cell seeding density in step a is 1.0×10 ⁶ cells/mL to 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 cell culture 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 is 0.35×10 ⁶ to 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.