Culture method for reducing polymer content in CHO (Chinese hamster ovary) cell bispecific antibody

Abstract

The invention relates to a culture method for reducing the polymer content in a CHO cell bispecific antibody, CHO cells are inoculated into a culture medium for cell culture, a regulator is added in the cell culture process, culture is performed at the temperature of 35-37.5 DEG C so as to reduce the polymer content of the CHO cell secreted bispecific antibody, and the regulator is selected from cystine or cysteine. According to the method, in a cell culture process flow, the temperature and the additive amount of the regulator can be regulated according to requirements, so that the polymer content of the bispecific antibody is regulated, the growth and expression of cells are not influenced, the generation of the polymer in the bispecific antibody is inhibited, the polymer content is obviously reduced, and the cell culture efficiency is improved. The product quality of the bispecific antibody is obviously improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and in particular relates to a culture method for reducing the multimer content in bispecific antibodies of CHO cells. Background Art

[0002] Chinese Hamster Ovary (CHO) cells are the most widely used host cell for producing antibodies and protein drugs. Over 70% of marketed therapeutic protein drugs are produced in CHO cells. Compared to other mammalian cell expression systems, CHO cells offer advantages such as human-like post-translational modifications, a well-defined history, rapid growth, and high expression levels, leading to their widespread use.

[0003] Bispecific antibodies exhibit unique therapeutic potential by simultaneously targeting two antigens or epitopes. Their core advantages include: 1. Targeted Immune Cell Killing: Bispecific antibodies can directly recruit immune effector cells (such as T cells) to tumor cells, activating their killing function. For example, blinatumomab, which targets CD3 and CD19, significantly improves complete remission rates by bridging T cells with B-cell leukemia cells. Similar mechanisms are also used in drugs targeting molecules such as CD3 / CD20 and BCMA. For example, teclistamab has achieved a 63% objective response rate in multiple myeloma.

[0004] 2. Dual signaling pathway blockade and synergistic effects: Dual antibodies can simultaneously inhibit two signaling pathways, reducing tumor drug resistance. For example, Kangfang Bio's cardunilimab (a dual PD-1 / CTLA-4 antibody) significantly prolonged survival in cervical cancer, while amivantamab (an EGFR / c-MET dual antibody) achieved a 40% response rate in non-small cell lung cancer.

[0005] 3. Reduce off-target toxicity: Through highly specific dual-target binding, damage to normal tissues is reduced. For example, Zanidatamab binds to both HER2 domains, enhancing targeting and promoting HER2 internalization on the cancer cell surface, thereby reducing off-target risk.

[0006] 4. Significant cost-effectiveness: The therapeutic dose of bispecific antibodies is usually 1 / 100 to 1 / 2000 of that of traditional antibodies, and no complex combination drug regimen is required, significantly reducing development and treatment costs.

[0007] Chinese hamster ovary cells are the main host for the production of bispecific antibodies, but there are many quality problems in the expression process: the bispecific antibody needs two different heavy chains (HC) and light chains (LC) to be correctly paired, and the effective component is a monomer; but CHO cells are prone to produce homodimers or misassembled products, and incorrect pairing can lead to the production of multimers. Multimers have strong immunogenicity, which significantly affects the effectiveness and safety of bispecific antibodies.

[0008] Chinese patent CN114230669A discloses a method for producing bispecific antibodies, which comprises (1) constructing cells for producing bispecific antibodies and screening; (2) culturing the cells obtained in step (1), obtaining a culture solution and performing separation and purification to obtain bispecific antibodies; the cells include CHO cells, and the culture method includes fed-batch culture or perfusion culture. The process mentions that multimers seriously affect the yield and protein purity during purification, but the means is only to remove multimers, not to reduce the production of multimers from the source.

[0009] Therefore, it is urgent to reduce the content of multimers in bispecific antibodies by optimizing the cell culture process. SUMMARY

[0010] The main purpose of the present application is to provide a culture method for reducing the content of multimers in bispecific antibodies secreted by CHO cell expression system, which can significantly reduce the content of multimers in bispecific antibodies.

[0011] The present application realizes the above-mentioned purpose through the following technical scheme: a culture method for reducing the content of multimers in bispecific antibodies secreted by CHO cell expression system, comprising the following steps: S1, inoculating CHO cells into a culture medium for cell culture, and maintaining a culture temperature of 35-37.5℃; S2, adding a regulator during cell culture, the regulator being selected from cystine or cysteine; S3, adding a feed medium and the regulator once every 1-3 days; S4, continuing to culture for 9-13 days to end the culture.

[0012] Specifically, the culture medium is a mixture of one or more of CD CHO medium, Advanced medium, Dynamis medium and Actipro medium.

[0013] Specifically, the feed medium is Effendent Feed TM A + Effendent Feed TM B +A mixture of one or more of feed medium or Cell boost7a feed medium.

[0014] Specifically, during the cell culture process, the regulator is added at a rate of 50-100 mg / (L·day).

[0015] Specifically, during the cell culture process, the sugar concentration in the cell culture medium is maintained at 1 g / L to 8 g / L.

[0016] Specifically, the bispecific antibody is a bispecific antibody with a symmetrical structure.

[0017] Specifically, the feed culture medium added each time accounts for 1% to 5% of the total volume of the culture medium.

[0018] Compared with the prior art, the beneficial effects of this culture method are: This method allows the multimer content of bispecific antibodies to be adjusted as needed during the cell culture process by adjusting the temperature and the amount of regulator added, without affecting cell growth and expression. This culture method inhibits the formation of multimers in bispecific antibodies, significantly reducing the multimer content and significantly improving the product quality of bispecific antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a histogram of the polymer content of each group in Examples 1 to 7 of the present invention; Figure 2 This is the SEC profile of the polymer content in Example 1 of the present invention; Figure 3 This is the SEC profile of the polymer content in Example 2 of the present invention; Figure 4 8 is a histogram of the polymer content of each group in Examples 8 to 13 of the present invention; DETAILED DESCRIPTION

[0020] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, the following detailed description of the specific embodiments of the present invention is provided in conjunction with specific embodiments and accompanying drawings. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0021] A method for reducing the multimer content of a bispecific antibody secreted by a CHO cell expression system, comprising the following steps: S1. Inoculate CHO cells into culture medium for cell culture, and maintain the culture temperature at 35-37.5°C.

[0022] The bispecific antibody obtained in the culture method is a symmetrical structure bispecific antibody.

[0023] The culture medium is a mixture of one or more of CD CHO medium, Advanced medium, Dynamis medium and Actipro medium.

[0024] During the cell culture process, the sugar concentration in the cell culture solution is maintained at 1g / L to 8g / L.

[0025] S2, adding a regulator during the cell culture process.

[0026] The regulator is selected from cystine or cysteine, and the addition rate of the regulator is 50 to 100 mg / (L·day).

[0027] S3, adding a feed medium and the regulator every 1 to 3 days.

[0028] The feed medium is Effendent Feed TM A + The feed medium is Effendent Feed TM B + The feed medium is a mixture of one or more of Effendent Feed

[0029] Each time the feed medium accounts for 1% to 5% of the total volume of the culture medium.

[0030] S4, continuing to culture for 9 to 13 days, and ending the culture.

[0031] In order to verify the effect of the method, a plurality of examples are set up for experimental verification, and the conditions of each example are shown in Table 1.

[0032] Table 1 Experimental scheme of examples 1 to 7 Example culture medium additive Post-feed culture temperature 1 Dynamis Cystine: 200 mg / L / day 33℃ 2 Dynamis Cystine: 50 mg / L / day 37℃ 3 Dynamis Cystine: 100 mg / L / day 37℃ 4 Advanced Cystine: 50 mg / L / day 37℃ 5 Advanced Cystine: 100 mg / L / day 37℃ 6 Actipro Cystine: 50 mg / L / day 37℃ 7 Actipro Cystine: 100 mg / L / day 37℃

[0033] In the above examples, reagents and instruments are selected according to conventional selection in the art unless otherwise specified. The experimental methods not specified in the examples are usually carried out according to conventional conditions, such as the conditions described in the literature, books or the methods recommended by the kit manufacturers.

[0034] Materials used in the experiment: CHO cells (Chinese hamster ovary cells) were purchased from Suzhou Youyi Biotechnology Co., Ltd. through commercial channels. Dynamis was purchased from Thermo Fisher, Cellboost 7a and Actipro were purchased from Cytiva Company, and Advanced was purchased from Merck Company.

[0035] The feed medium was Cellboost 7a and Feed B. The Feed B was a solution containing 20 g / L tryptophan and 40 g / L tyrosine.

[0036] The cells were cultured in 125 mL shake flasks with breathable caps (Zhejiang Biotech, Cat. No. FL125) and incubated in a shaker at 37°C and 5% CO2 for 14 days at a speed of 120–130 rpm.

[0037] After 72 h of culture, the control group (Example 1) was cooled to 33°C for culture.

[0038] From Day 3 to Day 13, 2% Cellboost7a and 0.2% Feed B were added daily.

[0039] The specific statistical test methods are as follows: Determination of antibody expression: A TSK-gel G2000SWxl column was used with a mobile phase of 50 mmol / L phosphate buffer, pH (6.8-7.4), flow rate (0.6 mL / min), and column temperature (45°C). 50 μL of sample was injected.

[0040] The test results are as follows Figure 1 As shown; Table 2 Experimental schemes for Examples 8 to 13 Example Feed medium additive Post-feed culture temperature 8 Cellboost 7a Cystine: 200 mg / L / day 33℃ 9 Cellboost 7a Cystine: 50 mg / L / day 33℃ 10 Cellboost 7a Cystine: 50 mg / L / day 37℃ 11 Cellboost 7a Cysteine: 50 mg / L / day 37℃ 12 <![CDATA[Effendent Feed TM A+]]> Cystine: 50 mg / L / day 37℃ 13 <![CDATA[Effendent Feed TM B+]]> Cystine: 50 mg / L / day 37℃

[0041] The reagents and instruments used in the above examples are conventional in the art unless otherwise specified. Experimental methods without specific conditions specified in the examples are generally carried out under conventional conditions, such as those described in literature or books, or methods recommended by the kit manufacturer.

[0042] Materials used in the experiment: CHO cells (Chinese hamster ovary cells) were purchased from Suzhou Youyi Biotechnology Co., Ltd. through commercial channels.

[0043] The culture medium was Dynamis, purchased from Thermo Fisher Scientific. TM A+, Effendent Feed TM B+ was purchased from Thermo Fisher Scientific. Cellboost 7a was purchased from Cytiva.

[0044] The cells were cultured in 125 mL shake flasks with breathable caps (Zhejiang Biotech, Cat. No. FL125) and incubated in a shaker at 37°C and 5% CO2 for 14 days at a speed of 120–130 rpm.

[0045] After 72 h of culture, the control group (Example 1) was cooled to 33°C for culture.

[0046] The cells were cultured from Day 3 to Day 13, and supplemented with 2% feed and 0.2% Feed B every day. The Feed B was a solution containing 20 g / L tryptophan and 40 g / L tyrosine.

[0047] Examples 1 to 7 are actually the cases where the cultivation of steps S1 and S2 is completed, while Examples 8 to 13 are the cases where steps S3 and S4 are continued to be cultivated in Examples 2 to 7.

[0048] according to Figure 1 It can be found that although the polymer content of Examples 2 to 6 fluctuates slightly, it is significantly lower than the polymer content of Example 1. The additive used in Examples 1 to 7 is cystine, and the polymer content conforms to the characteristic that the more the additive is used, the higher the polymer content.

[0049] according to Figure 2~Figure 3 It can be found that when the culture temperature and the amount of additives added are different, Example 1 with a low culture temperature and a high amount of additives is more likely to produce polymers.

[0050] according to Figure 4 It can also be seen that the feed medium used in Examples 8-9 is the same as that used in Examples 10-11, but the temperature in the former is lower, resulting in a much higher multimer content than in the latter two. The additive dosage in Example 8 is higher than that in Example 9, but the resulting increase in multimer content is not significant. The additives used in Examples 10 and 11 differ, but the effect is not significantly different. Different feed media were used in Examples 12 and 13, and the multimer content was slightly increased relative to Example 10, but the effect was not as significant as that in Example 10.

[0051] In summary, this method can adjust the temperature and the amount of regulator added as needed during the cell culture process to achieve the regulation of the multimer content of the bispecific antibody without affecting the growth and expression of cells. It inhibits the production of multimers in the bispecific antibody, significantly reduces the multimer content, and significantly improves the product quality of the bispecific antibody.

[0052] For those skilled in the art, several variations and improvements can be made without departing from the inventive concept of the present invention, and all of these fall within the scope of protection of the present invention.

Claims

1. A method for reducing the content of multimers in bispecific antibodies in CHO cells, characterized in that: The steps include: S1. CHO cells were inoculated into the culture medium for cell culture and maintained at a culture temperature of 35-37.5°C. S2. adding a regulator during the cell culture process, wherein the regulator is selected from cystine or cysteine; S3, adding feed medium and the regulator every 1 to 3 days; S4. Continue culturing for 9 to 13 days and then terminate the culture.

2. The culture method according to claim 1, wherein: The culture medium is a mixture of one or more of CD CHO culture medium, Advanced culture medium, Dynamis culture medium and Actipro culture medium.

3. The culture method according to claim 1, wherein: The feed medium is Effendent Feed TM A + Feed medium, Effendent Feed TM B + A mixture of one or more of feed medium or Cell boost7a feed medium.

4. The culture method according to claim 1, wherein: During the cell culture process, the regulator is added at a rate of 50-100 mg / (L·day).

5. The culture method according to any one of claims 1 to 4, characterized in that: During the cell culture process, the sugar concentration in the cell culture medium was maintained at 1g / L~8g / L.

6. The culture method according to claim 1, wherein: The bispecific antibody is a bispecific antibody with a symmetrical structure.

7. The culture method according to claim 1, wherein: The feed medium added each time accounts for 1% to 5% of the total volume of the culture medium.

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