CN-121975721-A - Additive composition for CHO cell culture and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an additive composition for CHO cell culture and application thereof. The composition is carboxymethyl-beta-cyclodextrin 0.1-0.5g/L, asparagine 0.4-1.2g/L, and sodium pyruvate 0.02-0.1g/L. By adding the composition, the antibody yield can be effectively improved, and the glycosylation modification proportion of G0F can be improved, so that a simple, convenient and effective yield and quality control method is provided for antibody drug research and development and production.

Inventors

• JIANG YIFAN
• LI CHENXIN
• Jiao hongyan
• JIA YU
• Huo Huixin
• XUE WEN
• LI ZIYI
• XU FENGQIN
• Yun Linying
• YANG LIPING
• GENG YA

Assignees

• 华北制药集团新药研究开发有限责任公司

Dates

Publication Date

20260505

Application Date

20260129

Claims (9)

1. 1. An additive composition for CHO cell culture, which is characterized by comprising carboxymethyl-beta-cyclodextrin, asparagine and sodium pyruvate, wherein the concentration of each component is 0.1-0.5g/L of carboxymethyl-beta-cyclodextrin, 0.4-1.2g/L of asparagine and 0.02-0.1g/L of sodium pyruvate.
2. 2. Additive composition according to claim 1, characterized in that the additive composition concentration is between 0.2 and 0.3g/L carboxymethyl- β -cyclodextrin, between 0.6 and 1.0g/L asparagine, between 0.05 and 0.1g/L sodium pyruvate.
3. 3. The additive composition of claim 1, wherein the additive composition has a concentration of carboxymethyl-beta-cyclodextrin of 0.2g/L, asparagine of 1.0g/L, sodium pyruvate of 0.05g/L.
4. 4. A CHO cell culture medium comprising the additive composition of any one of claims 1-3 and a basal medium.
5. 5. The culture medium according to claim 4, wherein the basal medium is one or more of CHO MaxA medium and CHO MaxC medium.
6. 6. Use of the additive composition of any one of claims 1-3 or the culture medium of any one of claims 4-5 in CHO cell culture.
7. 7. The use according to claim 6, wherein the CHO cell is a CHO-K1 cell or CHO-S cell.
8. 8. The use according to claim 7, wherein the specific step of CHO cell culture comprises: (1) Preparing a culture medium, namely adding the additive composition into a prepared basic culture medium according to the concentration of the additive composition, and filtering and sterilizing the basic culture medium by a filter of 0.22 mu m for later use; (2) Cell resuscitating, namely rapidly thawing the CHO cells frozen by liquid nitrogen in a 37 ℃ water bath, and adding the CHO cells into a shake flask containing a subculture medium; (3) Culturing cells by placing the shake flask in a shake table with the concentration of 36.5-37 degrees C, CO 2 and the rotation speed of 110-130 rpm and the humidity of 75-80 percent; (4) Subculturing, namely, subculturing every 2-4 days, maintaining the logarithmic growth phase of the cells, wherein the subculture density is 0.2-1.0X10 6 cells/mL; (5) Inoculating, namely inoculating cells into shake flasks containing the basic culture medium containing the additive composition according to an inoculation density of 0.8-1.0+/-0.1X10 6 cells/mL, wherein the shake flask liquid filling amount of 125mL is 30mL, and the shake flask liquid filling amount of 250mL is 60mL; (6) Culturing in incubator at 36.5 deg.c and rotation speed of shaking table of 110-130rpm with CO 2 concentration of 6-8% and humidity of 75-80%; (7) The feeding strategy comprises the steps of starting from the culture to the 3 rd day, adding a feeding culture medium on the 5 th, 7 th, 9 th and 11 th days, adding 3% of a feeding culture medium A on the 3 rd day, 0.3% of a feeding culture medium B on the 3 rd day, adding 6% of a feeding culture medium A on the 5 th, 7 th, 9 th and 11 th days, adding 0.6% of a feeding culture medium B on the 5 th, 7 th, 9 th and 11 th days, adding glucose in the culture process, and keeping the strategy of less than 5g/L and 5g/L, wherein the feeding culture medium A is MaxFA, and the feeding culture medium B is MaxFB; (8) Harvesting, continuing to culture until day 14, harvesting cells, and centrifugally collecting supernatant for antibody purification and subsequent detection.
9. 9. The use according to claim 8, wherein the assay comprises an N-glycation profile assay, and wherein the supernatant is purified prior to the assay by HPLC liquid phase.

Description

Additive composition for CHO cell culture and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to an additive composition for CHO cell culture and application thereof. Background The monoclonal antibody has the advantages of high specificity, remarkable treatment effect and the like due to the characteristics of the monoclonal antibody, and is widely applied to the treatment of various disease fields such as tumors, autoimmune diseases, infectious diseases and the like. Along with the continuous development of biotechnology, recombinant monoclonal antibodies have become core products in the field of biopharmaceuticals. Chinese Hamster Ovary (CHO) cells are taken as host cells, occupy most of the proportion in the production of monoclonal antibodies, have obvious advantages of stable integration of exogenous genes, can realize accurate post-translational modification (approaching to human beings) of antibody proteins and high-density suspension culture in serum-free, and nearly 70% of varieties are preferred to the expression system in recombinant antibody medicines on the market worldwide at present. The expression level of the antibody is an index of important attention to the research and development of antibody medicines and the production cost, and the glycosylation level and the glycoform distribution directly influence the quality characteristics of the antibody, such as biological activity, immunogenicity, half-life, stability and the like. The common glycoforms of the antibody are G0F, G1F, G F, wherein the high-low ratio of the G0F glycoform is directly related to the key properties of ADCC (antibody dependent cellular cytotoxicity) effect, in vivo clearance rate and the like of the antibody, so that the key quality attributes are the key quality attributes, and the regulation is often receiving general attention. In addition, in the research and development and production of biological similar drugs, the G0F glycoform proportion needs to be accurately regulated so as to ensure that the curative effect and the safety of the drug have extremely high similarity with those of the original drug. Researchers develop a lot of researches on how to increase the antibody yield of CHO cells and how to perform glycoform regulation, and the main means of the research include optimization of expression vectors, gene editing and transformation of cell strains, regulation of culture process parameters (such as temperature, pH and dissolved oxygen), improvement of culture medium components and the like. The gene editing technology involves the problems of over-high replacement cost, uncontrollable research and development period and the like under the condition that cell strains are determined. The process parameter regulation is easily affected by fluctuation of a culture system, so that the sugar type proportion among different batches may have poor consistency. The fixed addition of commercial sugar type regulating reagent has the problems of insufficient regulating force, reduced antibody yield, uncontrolled cell state and the like. In addition, the prior art is difficult to realize 'yield improvement and glycosylation optimization and meeting the requirements', and the like. The culture medium is added with small molecular compounds to adjust the yield and quality of production, and the method is a preferred strategy for process optimization in industrial production due to simple operation, controllable cost and easy large-scale amplification. The addition of various substances has been reported to have the potential to increase antibody production, such as sodium butyrate, deoxyuridine, ionone, and 3-hydroxy anthranilic acid, etc. The regulating reagent for antibody glycosylation modification includes manganese ion, uridine, thioglycerol, etc. However, the existing additives have the disadvantages of poor regulation and control pertinence, low antibody yield or other key quality attribute abnormality caused by improving the target glycoform proportion, complex formulation of part of the additives, poor consistency among batches, adverse industrial production and the like. Therefore, it is of great importance to provide an additive composition for CHO cell culture that can achieve "increasing the expression yield of CHO cell antibodies" and "increasing the G0F glycosylation ratio" simultaneously. Disclosure of Invention The present invention has been made in an effort to overcome the disadvantages of the prior art and to provide a composition for improving antibody expression while reducing the G0F ratio of glycoform modification, and the inventors have found that the G0F ratio can be increased and antibody yield can be significantly improved by adding an appropriate amount of the composition to a base. In order to achieve the above purpose, the invention adopts the following technical scheme: An additive composition for CHO cell culture com