Liquid medium for reducing oxidation in recombinant proteins expressed in CHO cells

Abstract

The present invention provides a liquid culture medium for reducing oxidation of recombinant proteins expressed in CHO cells, a culture method, and a method for reducing oxidation of recombinant proteins expressed in CHO cells. The liquid culture medium comprises: a basal culture medium, a feed culture medium and minoxidil. Adding minoxidil to the culture medium effectively reduces oxidative impurities formed during the synthesis of fusion proteins and antibody drugs in CHO cells, and does not affect the synthesis of fusion proteins and antibody drugs and their quality attributes. The culture medium has low cost, simple operation, and is suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of biological technology, and in particular, the present invention relates to a CHO cell liquid culture medium, a CHO cell culture method, and a method for reducing oxidation of recombinant proteins expressed in CHO cells. Background Art

[0002] Monoclonal antibodies and other biological drugs have been widely used in the treatment of tumors, inflammation, metabolic diseases, etc., and have achieved good therapeutic effects. Due to the complexity of the structure of biological drugs, they usually need to be synthesized in biological hosts. Host cell lines used for the expression of recombinant mAbs or fusion protein drugs include CHO, NS0, Sp2 / 0, HEK293 and PER.C6. Because CHO cells have post-translational modifications similar to human proteins during protein synthesis, and are easily adapted to serum-free culture media, they can grow in suspension in defined culture media, which promotes the improvement and amplification of bioprocesses, and has been proven to be a reliable and safe host. Therefore, CHO is the most commonly used host cell in the production of recombinant protein drugs.

[0003] During the protein synthesis process of CHO cells, the synthesis of protein products is closely related to the culture conditions in addition to the clone itself. Post-translational modifications in the protein synthesis process are crucial to its biological activity and stability. In the protein synthesis process, common post-translational modifications include oxidation, phosphorylation, deamination, C-terminal resection, glycosylation, etc. The oxidation of amino acids will change the secondary and tertiary structures of biological macromolecules, which may lead to changes in their biological activity, shortened half-life, and the formation of aggregates. In the process of protein synthesis, amino acid residues are oxidized to form oxidative impurities due to the catalytic action of related enzymes or the action of reactive oxygen generated by cell metabolism.

[0004] Hazeltine LB et al. found that by adjusting the concentrations of manganese ions, copper ions, cysteine ​​and tryptophan in the culture medium, tryptophan oxidation can be reduced (Biotechnol Prog, 2016). At the same time, Yun Z et al. found that adding glutathione and iron ion chelators can reduce the level of reactive oxygen in cells (J Biosci Bioeng, 2003). There are also literature reports that adding lipoic acid, methionine, N-acetylcysteine ​​and cysteine ​​can adjust the level of reactive oxygen in cells, thereby reducing protein oxidation (Exp Toxicol Pathol, 2008). Hou Y et al. published their latest research in the journal Biotechnol J in 2019: By increasing the concentrations of nicotinamide and cysteine ​​in the culture medium, the hydroxylation of lysine during protein synthesis can be alleviated. These methods will more or less affect the expression of the product or its quality attributes. For example, adding metal ions to the culture medium will reduce oxidation, but it will also change the pH and osmotic pressure of the culture medium and affect cell growth. Summary of the invention

[0005] This application is based on the inventor's discovery and understanding of the following facts and problems:

[0006] In the prior art, fusion proteins and antibody drugs are easily formed during the synthesis process in CHO cells. Based on the discovery of the above problems, the inventors proposed a liquid culture medium for reducing oxidation of recombinant proteins expressed in CHO cells, and found that adding minoxidil to the culture medium during CHO cell culture can reduce oxidation impurities without affecting cell growth and product synthesis, and without affecting other quality attributes of the target product, and still has a reducing effect on oxidation impurities of fusion proteins and antibody drugs when the culture medium and cell clones change. The present invention is suitable for solving the problem of oxidation impurities formed during the synthesis process of fusion proteins and antibody drugs in CHO cells.

[0007] In a first aspect of the present invention, the present invention provides a liquid culture medium for reducing oxidation of recombinant proteins expressed in CHO cells. The culture medium comprises a culture medium additive, wherein the culture additive is minoxidil, and the liquid culture medium can reduce the formation of oxidative impurities in protein synthesis.

[0008] In some embodiments, the liquid culture medium further comprises a basal medium and a feed medium.

[0009] In some embodiments, minoxidil is prepared into a stock solution and added once within 0-10 days after cell inoculation or added multiple times in stages during the culture process.

[0010] In some embodiments, the concentration of minoxidil used in the present invention is 0.1-1 mM.

[0011] In some embodiments, the concentration of minoxidil used in the present invention is 0.6 mM.

[0012] In some embodiments, the concentration of minoxidil used in the present invention is 0.3 mM.

[0013] In some embodiments, the basal medium of the present invention is Dynamis or EXCELL Advanced CHOmedium; and the feed medium is CellBoost or EXCELL Advanced Feed1.

[0014] In some embodiments, the basal medium of the present invention is Dynamis; and the feed medium is CellBoost.

[0015] In some embodiments, the basal medium of the present invention is EXCELL Advanced CHO medium; and the feed medium is EXCELL Advanced Feed1.

[0016] In a second aspect of the present invention, the present invention provides a method for culturing CHO cells using the liquid culture medium of the present invention. According to the culture method of the embodiment of the present invention, the addition of minoxidil to the culture medium can reduce the oxidation of amino acids in the target protein or monoclonal antibody, reduce the content of oxidative impurities, and will not significantly affect other properties such as the expression amount, glycoform, and charge heterogeneity of the target protein or monoclonal antibody.

[0017] In some embodiments, the liquid culture medium is used to culture CHO cells to obtain a fusion protein or monoclonal antibody with less oxidative impurities. The culture process is batch culture, fed-batch culture or perfusion culture. The culture is carried out in a flask, a stirred reactor or a wave reactor at a temperature of 30-37°C.

[0018] In some embodiments, before the culturing, the method further comprises inoculating the CHO cells in the basal medium.

[0019] In some specific embodiments, the inoculum size is 0.2 to 1.2*10 6 The above inoculation density is more suitable for the growth of CHO cells and the synthesis of target products.

[0020] In some embodiments, the fusion protein is an Fc fusion protein.

[0021] In some specific embodiments, the fusion protein is an Fc fusion protein of GLP-1.

[0022] In some specific embodiments, the monoclonal antibody is adalimumab.

[0023] In the third aspect of the present invention, the present invention proposes the use of the culture medium or culture method in reducing oxidative impurities in fusion proteins and antibody drugs.

[0024] In a fourth aspect of the present invention, the present invention provides a method for reducing oxidation of recombinant proteins expressed in CHO cells, the method comprising adding minoxidil during cell culture, or culturing CHO cells using the culture medium or culture method of the present invention.

[0025] Detailed description

[0026] The present invention will list the literature corresponding to the determined specific content in detail. The present invention is expected to cover all options, variants and equivalents, which may be included in the field of the prior invention as defined in the claims. Technicians in the relevant field will recognize many methods and materials similar or equivalent to those described herein, which can be applied to the practice of the present invention. The present invention is in no way limited to the description of methods and materials. There are many documents and similar materials that are different from or contradict the present application, including but not limited to the definition of terms, the usage of terms, the technology described, or the scope controlled by the present application.

[0027] The "LC-MS trypsin profile analysis" used in the present invention is a technology that applies liquid chromatography-mass spectrometry to the study of proteins. The analysis principle is to add a reducing agent to the analysis sample under denaturing conditions to open all cross-linked disulfide bonds inside the fusion protein, and then add an alkylating agent to block all free sulfhydryl groups, so that the fusion protein molecules exist in the form of free extended peptide chains in the solution. Trypsin is added to enzymatically hydrolyze the fully extended peptide chains into small peptide segments, and then LC-MS analysis is performed. The content of oxidative impurities and glycoforms are detected by analyzing the molecular weight difference of each peptide segment between the experimental group and the control group samples.

[0028] The "Protein A" used in the present invention is a Staphylococcus aureus cell wall protein with a molecular weight of 42KD. Natural Protein A has 5 IgG binding domains and a non-Fc binding domain of unknown function. The binding domain can specifically bind to the Fc segment of a variety of immunoglobulins and binds very weakly to the Fab region or light chain. The non-Fc binding domain can bind to some foreign proteins, resulting in insufficient purity of the eluted IgG. Therefore, in reality, recombinant Protein A is mostly used, and its non-specific binding is significantly reduced.

[0029] The "Protein A affinity chromatography column" used in the present invention is widely used for column chromatography separation and purification of various antibodies. Its principle is to combine Protein A with agarose gel in a certain way to prepare an affinity filler for antibody purification. After affinity chromatography, high-purity antibodies can be purified from samples such as ascites, serum and culture fluid. The detection wavelength is 280nm and the column temperature is 25°C.

[0030] The "capillary zone electrophoresis" used in the present invention is used for charge isomer detection. The analysis process is as follows: the sample is collected by Protein A capture, 150 μg of the sample is concentrated in a 10KDa ultrafiltration tube, and iodoacetamide solution is added, mixed, and then placed in a 70°C water bath for 15 minutes, and then centrifuged to obtain the supernatant for detection using a capillary instrument.

[0031] The "Trypan blue staining method" used in the present invention is used for cell density detection. The analysis process is: take 1 mL of cell suspension in an EP tube, mix it evenly, take 20 μL and mix it evenly with 0.2% Trypan blue solution, take 20 μL in a test plate, and use a cell viability analyzer to detect the density of live cells.

[0032] The "Dynamis" used in the present invention is a commercial culture medium for culturing CHO cells, purchased from Thermofisher.

[0033] The "EXCELL Advanced CHO medium" used in the present invention is a commercial culture medium for culturing CHO cells, purchased from Merck.

[0034] The "CellBoost 7a" used in the present invention is a commercial culture medium for culturing CHO cells, purchased from GE.

[0035] The "EXCELL Advanced Feed 1" used in the present invention is a commercial culture medium for culturing CHO cells, purchased from Merck.

[0036] The "CHO ​​cell" used in the present invention is the abbreviation of Chinese hamster ovary cell.

[0037] The term "monoclonal antibody" or "mAb" used in the present invention is an abbreviation for monoclonal antibody.

[0038] "mM" used in the present invention is an abbreviation for the concentration unit mmol / L.

[0039] "M" used in the present invention is the abbreviation of the concentration unit mol / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The effect of adding minoxidil on the content of oxidative impurities in Example 1 is shown.

[0041] Figure 2 The effects of adding minoxidil on other aspects in Example 1: A: cell growth; B: expression level; C: glycoform; D: charge heterogeneity.

[0042] Figure 3 The effect of adding minoxidil on the content of oxidative impurities in Example 2 is shown.

[0043] Figure 4 The effects of adding minoxidil on other aspects in Example 2: A: cell growth; B: expression level; C: glycoform; D: charge heterogeneity.

[0044] Figure 5The effect of adding minoxidil on the content of oxidative impurities in Example 3 is shown.

[0045] Figure 6 The effects of adding minoxidil on other aspects in Example 3: A: cell growth; B: expression level; C: glycoform; D: charge heterogeneity.

[0046] Figure 7 This is the effect of adding minoxidil on the content of oxidative impurities in Example 4.

[0047] Figure 8 The effects of adding minoxidil on other aspects in Example 4: A: cell growth; B: expression level; C: glycoform; D: charge heterogeneity.

[0048] Fig. 9 The effect of adding minoxidil on the content of oxidative impurities in Example 5 is shown.

[0049] Fig.10 The effects of adding minoxidil on other aspects in Example 5: A: cell growth; B: expression level; C: glycoform; D: charge heterogeneity. DETAILED DESCRIPTION

[0050] The following embodiments of the present invention are described in detail. The following embodiments are exemplary and intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0051] Example 1

[0052] 1. Pour the basic culture medium Dynamis into a 1L triangular flask with a liquid volume of 300mL. After sterilization and cooling, cultured CHO cell seeds were placed at 6*10 5 / mL was transferred into a conical flask.

[0053] 2. Add minoxidil at a final concentration of 0.3 mM and culture at 37°C, 8% CO2, and 140 rpm. Use the control without adding minoxidil.

[0054] 3. After 3 days of culture, daily feeding began. The feeding medium was CellBoost7a. After 13 days of culture, the culture was stopped. The fermentation broth was centrifuged at 1000g for 20 minutes. The supernatant was taken and the product (GLP-1 and Fc fusion protein) was captured by ProteinA column. ProteinA capture parameters: the eluent was pH 4.4-4.6 0.1M HAc-NaAc, the elution flow rate was 0.1CV / min, and the elution time was 15min. The eluate containing the target protein was collected and the expression level of the target protein was detected.

[0055] 4. LC-MS trypsin profile analysis of oxidative impurities in the product, such as Figure 1As shown, it can be seen that after adding minoxidil, the content of oxidative impurities in the final product was significantly reduced. The glycoforms of the product were further analyzed using LC-MS trypsin profiles, and the cell density was detected by trypan blue staining to obtain the effects of adding minoxidil on cell number, product expression, product charge heterogeneity, and product glycoforms. The results are as follows: Figure 2 As shown in A, B, C, and D. Figure 1 and Figure 2 It can be seen that after adding minoxidil, the content of oxidative impurities in the final product was significantly reduced, and there was no obvious effect on cell growth and other product quality attributes.

[0056] Example 2

[0057] 1. Pour the basic culture medium Dynamis into a 1L triangular flask with a liquid volume of 300mL. After sterilization and cooling, the cultured CHO cell seeds are placed at 8*10 5 / mL was transferred into a conical flask.

[0058] 2. Add minoxidil at a final concentration of 0.3 mM and culture at 30°C, 8% CO2, and 140 rpm. Use the control without adding minoxidil.

[0059] 3. After 3 days of culture, daily feeding began, and the feeding medium was CellBoost 7a. After 15 days of culture, the culture was stopped, the fermentation broth was centrifuged at 1000g for 20 minutes, the supernatant was taken, and the product (adalimumab) was captured by Protein A column. Protein A capture parameters: the eluent was pH 4.4-4.3 0.1M HAc-NaAc, the elution flow rate was 0.1CV / min, and the elution time was 10 minutes. The eluate containing the target antibody was collected and the expression level of the target antibody was detected.

[0060] 4. LC-MS trypsin profile analysis of oxidative impurities in the product, such as Figure 3 As shown, it can be seen that after adding minoxidil, the content of oxidative impurities in the final product was significantly reduced. The glycoforms of the product were further analyzed using LC-MS trypsin profiles, and the cell density was detected by trypan blue staining to obtain the effects of adding minoxidil on cell number, product expression, product charge heterogeneity, and product glycoforms. The results are as follows: Figure 4 As shown in A, B, C, and D. Figure 3 and Figure 4 It can be seen that after adding minoxidil, the content of oxidative impurities in the final product was significantly reduced, and there was no significant effect on cell growth and other product quality attributes. This shows that its anti-oxidative effect is also suitable for the production of monoclonal antibodies.

[0061] Example 3

[0062] 1. Load the basic culture medium Dynamis into a 50L reactor with a liquid volume of 30L. Add the cultured CHO cell seeds at a rate of 8*10 5 / mL was introduced into the reactor, and the dissolved oxygen (DO) was controlled at 30-50%, pH 6.8-7.4, and temperature 37°C.

[0063] 2. Add minoxidil to a final concentration of 0.6 mM, and use the control without adding minoxidil.

[0064] 3. After 3 days of culture, daily feeding began. The feeding medium was CellBoost 7a. On the 12th day, the culture was stopped and the fermentation broth was collected and centrifuged. The centrifugation parameters were 1000g, 4°C, and 20min. The supernatant was taken and the product (fusion protein of GLP-1 and Fc) was captured by ProteinA column. ProteinA capture parameters: the eluent was pH4.4-4.6 0.1M HAc-NaAc, the elution flow rate was 0.1CV / min, and the elution time was 15min. The eluate containing the target protein was collected and the expression level of the target protein was detected.

[0065] 4. LC-MS trypsin profile analysis of oxidative impurities in the product, such as Figure 5 As shown, it can be seen that after adding minoxidil, the content of oxidative impurities in the final product was significantly reduced. The glycoforms of the product were further analyzed using LC-MS trypsin profiles, and the cell density was detected by trypan blue staining to obtain the effects of adding minoxidil on cell number, product expression, product charge heterogeneity, and product glycoforms. The results are as follows: Figure 6 As shown in A, B, C, and D. Figure 5 and Figure 6 It can be seen that when the culture process is scaled up to a 50L reactor, adding minoxidil still has the effect of reducing oxidative impurities.

[0066] Example 4

[0067] 1. Load the basic culture medium EXCELL Advanced CHO medium into a 50L reactor with a liquid volume of 35L. 5 / mL was introduced into the reactor, and the dissolved oxygen (DO) was controlled at 30-50%, pH 6.8-7.4, and temperature 37°C.

[0068] 2. Add minoxidil to a final concentration of 0.3 mM, and use the control without adding minoxidil.

[0069] 3. After 3 days of culture, daily feeding began, and the feeding medium was EXCELL Advanced Feed 1. Culture was carried out under conditions of dissolved oxygen (DO) 30-50%, pH 6.8-7.4, and 37°C until the 6th day, minoxidil was added at a final concentration of 0.3mM, and culture was continued until the 14th day. The fermentation broth was collected and centrifuged. The centrifugation parameters were 1000g, 4°C, and 20min. The supernatant was taken and the product (GLP-1 and Fc fusion protein) was captured by Protein A chromatography column. Protein A capture parameters: the eluent was pH4.4-4.6 0.1M HAc-NaAc, the elution flow rate was 0.1CV / min, and the elution time was 15min. The eluent containing the target protein was collected and the expression level of the target protein was detected.

[0070] 4. LC-MS trypsin profile analysis of oxidative impurities in the product, such as Figure 7 As shown, it can be seen that after adding minoxidil, the content of oxidative impurities in the final product was significantly reduced. The glycoforms of the product were further analyzed using LC-MS trypsin profiles, and the cell density was detected by trypan blue staining to obtain the effects of adding minoxidil on cell number, product expression, product charge heterogeneity, and product glycoforms. The results are as follows: Figure 8 As shown in A, B, C, and D. Figure 7 and Figure 8 It can be seen that adding minoxidil still has the effect of reducing oxidative impurities when culturing with different culture media.

[0071] Example 5

[0072] 1. Pour the basic culture medium Dynamis into a 1L triangular flask with a liquid volume of 300mL, and add the cultured CHO cell seeds at a rate of 10*10 5 / mL were introduced into the reactor.

[0073] 2. Add minoxidil at a final concentration of 0.9 mM and culture at 30°C, 8% CO2, and 140 rpm. Use the control without adding minoxidil.

[0074] 3. After 3 days of culture, daily feeding began, and the feeding medium was CellBoost 7a. On the 15th day of culture, the culture was stopped, and the fermentation broth was collected and centrifuged. The centrifugation parameters were 1000g, 4°C, and 20min. The supernatant was taken and the product (adalimumab) was captured by ProteinA chromatography column. ProteinA capture parameters: the eluent was pH 4.0-4.3, 0.1M HAc-NaAc, the elution flow rate was 0.1CV / min, and the elution time was 10min. The eluate containing the target antibody was collected and the expression level of the target antibody was detected.

[0075] 4. LC-MS trypsin profile analysis of oxidative impurities in the product, such as Fig. 9 As shown, it can be seen that after adding minoxidil, the content of oxidative impurities in the final product was significantly reduced. The glycoforms of the product were further analyzed using LC-MS trypsin profiles, and the cell density was detected by trypan blue staining to obtain the effects of adding minoxidil on cell number, product expression, product charge heterogeneity, and product glycoforms. The results are as follows: Fig.10 As shown in A, B, C, and D. Fig. 9 and Fig.10 It can be seen that after adding 0.9 mM minoxidil, the oxidative impurities in the final product were significantly reduced, and there was no significant effect on cell growth.

[0076] In the description of this specification, the description with reference to the terms "some embodiments", "some specific embodiments", "examples", "specific examples", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0077] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Use of minoxidil in preparing a liquid culture medium for reducing oxidation of recombinant proteins expressed in CHO cells, wherein the liquid culture medium can reduce the formation of oxidative impurities in protein synthesis, and the protein is a fusion protein or a monoclonal antibody; the concentration of minoxidil in the culture medium is 0.1mM-1mM.

2. The use according to claim 1, characterized in that The liquid culture medium further comprises a basal culture medium and a feed culture medium; the basal culture medium is Dynamis or EXCELL Advanced CHO medium; and the feed culture medium is CellBoost or EXCELL Advanced Feed1.

3. The use according to claim 1, characterized in that The minoxidil is prepared into a stock solution, which is added once within 0-10 days after cell inoculation or added multiple times in stages during the culture process.

4. The use according to claim 1, characterized in that The concentration of minoxidil in the culture medium was 0.6 mM.

5. The use according to claim 1, characterized in that: The concentration of minoxidil in the culture medium was 0.3 mM.

6. The use according to claim 1, characterized in that: The concentration of minoxidil in the culture medium was 0.9 mM.

7. The use according to claim 1, characterized in that The fusion protein is an Fc fusion protein.

8. The use according to claim 1, characterized in that The fusion protein is the Fc fusion protein of GLP-1; the monoclonal antibody is adalimumab.

9. A method for reducing oxidation of recombinant proteins expressed in CHO cells, characterized in that Minoxidil was added during the cell culture process, and the concentration of minoxidil in the culture medium was 0.1 mM-1 mM.

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