A CHO cell culture method for regulating the galactosylation level of protein drugs

Abstract

The present invention relates to the field of biomedicine, and in particular to a CHO cell culture method for regulating the galactosylation level of protein drugs. The culture method includes a basic culture stage and a fed-batch culture stage. In the fed-batch culture stage, in addition to adding feed medium, L-cysteine ​​is also added; L-cysteine ​​can be added simultaneously with the feed medium in the fed-batch culture stage, and the addition amount is 1.2 to 4.8 mmol / L; it can also be added separately in the basic culture stage and the fed-batch culture stage, and the addition amount in the basic culture stage is 0.3 to 2.0 mmol / L, and the addition amount in the fed-batch culture stage is 0.6 to 1.8 mmol / L. The method of the present invention can not only improve the galactosylation level of protein drugs by adjusting the feed amount of cysteine, but also does not change other key quality attributes of the drug. The culture method is simple to operate and suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a CHO cell culture method for regulating the galactosylation level of protein drugs. Background Art

[0002] Glycosylation is an important post-translational modification of protein drugs. The two most common forms of glycosylation are O-glycans (oligosaccharides linked to hydroxyl-containing amino acids such as Ser, Thr, or Tyr) and N-glycans (oligosaccharides linked to Asn-X-Ser / Thr, where X is any amino acid except Pro). Protein glycosylation begins in the endoplasmic reticulum and Golgi apparatus. Under the action of glycosyltransferases, glycosidases, and substrates, glucose and mannose residues are removed and sugar groups such as N-acetylglucosamine (GlcNAc), sialic acid, fucose, and galactose are attached to form complex and diverse glycoforms. For antibodies, the distribution of these glycoforms directly affects their immunogenicity and biological function. Galactosylation utilizes galactose as a building block of the galactosylation chain reaction, with galactosyltransferase linking galactose to adjacent N-acetylglucosamine sugars. Galactosylation modifies the spatial conformation of the antibody Fc fragment, increasing its ability to bind to the C1q receptor, thereby enhancing complement-dependent cytotoxicity (CDC). For antibodies with CDC, adding galactosylation can enhance their CDC effect, thereby increasing their ability to kill target cells. For antibodies that lack or do not require CDC (e.g., by modifying different IgG subtypes or Fc carbohydrate sites to eliminate Fc receptor binding), controlling galactosylation during the manufacturing process is equally important. Galactosylation is highly susceptible to multiple factors, including upstream process parameters, culture scale, reactor material, site changes, and raw materials. Batch-to-batch consistency of galactosylation is also a key indicator of the controllability and stability of antibody drug production processes. Currently, galactosylation modification can be regulated by controlling pH, pCO2 or metal ion additives in upstream processes, but such regulation is usually accompanied by a decrease in the quality and yield of other proteins.

[0003] WO2012149197 uses the method of adding manganese or galactose to the complete culture medium to regulate the galactosylation level of recombinantly expressed antibodies. Low antibody galactosylation and batch-to-batch instability are common problems in monoclonal antibody drug development. A method for increasing antibody galactosylation is needed to improve galactosylation while enhancing process controllability and improving antibody quality.

[0004] When CHO cells are used as host cells to produce monoclonal antibodies or fusion protein drugs, the glycoform ratio is a key quality attribute, and different glycoform ratios can affect the stability of the drug molecule. Approximately 95% of recombinant IgGs produced by conventional CHO cells have galactose as the terminal sugar. Therefore, the level of galactosylation is a key quality attribute for monoclonal antibodies or fusion proteins expressed in CHO cells. Galactosylation is an important post-translational modification of monoclonal antibodies, affecting, for example, the controllability, stability, and batch-to-batch consistency of antibody drugs. Furthermore, it can enhance the antibody's CDC effect. However, galactosylation is difficult to control during the culture process, and differences in culture scale and subtle differences in process parameters significantly affect the level of antibody galactosylation. In existing culture processes, methods for adjusting the glycoform ratio mainly include: culture medium screening, addition of glycoform modifiers, and culture process optimization. While these methods can achieve the goal of adjusting glycoform ratios, other product quality parameters (such as charge variant ratio and phosphorylation level) are often affected during the process, thus affecting product quality control.

[0005] Based on the current problems in the preparation of monoclonal antibodies or fusion protein drugs, the inventors discovered that by adding an appropriate amount of cysteine ​​to the feed culture medium, the ratio of glycoforms G0F, G1F, and G2F can be adjusted, thereby improving the galactosylation level of the protein drug. At the same time, the controllability and reproducibility of the process are enhanced, so that the glycosylation level of the product is consistent with that of the original formulation. While achieving the goal of regulating galactosylation modification, other antibody quality parameters can remain unchanged. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. To this end, the present invention provides a CHO cell culture method for regulating the galactosylation level of protein drugs.

[0007] In its first aspect, the present invention provides a CHO cell culture method for regulating the galactosylation level of a protein drug. According to embodiments of the present invention, this method not only regulates the galactosylation level of a protein drug by adjusting the cysteine ​​feed rate, but also does not alter other critical quality attributes of the drug. This culture method is simple to operate and suitable for large-scale production.

[0008] The above technical objectives of the present invention are achieved through the following technical solutions:

[0009] A CHO cell culture method for regulating the galactosylation level of protein drugs comprises a basic culture stage and a fed-batch culture stage. In the fed-batch culture stage, L-cysteine ​​is added in addition to the feed medium.

[0010] In some embodiments of the present invention, L-cysteine ​​can be added simultaneously with the feed medium during the fed-batch culture phase, with the addition amount ranging from 1.2 to 4.8 mmol / L. In some embodiments of the present invention, the L-cysteine ​​addition amount is preferably 1.5 to 3.6 mmol / L, more preferably 1.8 to 2.1 mmol / L. During research, the inventors discovered that selecting an appropriate L-cysteine ​​addition amount can adjust the galactosylation glycoform ratio to the target level. When the L-cysteine ​​addition amount is too low, even if a glycoform modifier is added during the culture process, it is not possible to adjust the galactosylation glycoform ratio without affecting other key quality parameters of the product. When the L-cysteine ​​addition amount is higher than this concentration range, the ratio of galactosylation glycoforms is too unbalanced, with the G0F ratio being too low and the G1F and G2F ratios being too high, and the target glycoform ratio cannot be achieved, remaining consistent with the glycosylation types and ratios of the original formulation.

[0011] In some embodiments of the present invention, L-cysteine ​​can be supplemented during both the basal and fed-batch stages, with the amount added being 0.3-2.0 mmol / L during the basal stage and 0.6-1.8 mmol / L during the fed-batch stage. The inventors have discovered that supplementing L-cysteine ​​during both the basal and fed-batch stages can also improve the galactosylation level of protein drugs.

[0012] Preferably, the L-cysteine ​​is added during the fed-batch culture stage, which can make the entire culture process operation simpler and help maintain the concentration of nutrients at a certain level, thereby preventing the situation where the initial nutrient concentration is too high and the later nutrient concentration is too low, thereby ensuring the stability of the process.

[0013] In some embodiments of the present invention, L-tyrosine and L-tryptophan are also added during the fed-batch culture phase. During research, the inventors discovered that L-tyrosine and L-tryptophan, in the technical solution of the present invention, are primarily used to provide nutrients required for cell growth, but cannot be used to improve the galactosylation ratio of the drug.

[0014] In some embodiments of the present invention, the supplementary amount of L-tyrosine is 2.0-2.8 mmol / L, and the supplementary amount of L-tryptophan is 0.2-0.6 mmol / L.

[0015] In some embodiments of the present invention, the feeding culture starts on the 3rd day and is fed every 2 days until the end of the culture.

[0016] In some embodiments of the present invention, the cell culture medium may comprise serum-free and / or animal-derived products or components. 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, CHO cells can be cultured in a defined medium appropriate for the specific cells being cultured without undue experimentation.Commercially available culture media may be used, including but not limited to: Millipore's EX-CELL Advanced CHO basal medium, Gibco's ExpiCHO basal medium, Zhongshan Kangsheng CHO CD04, CD OptiCHO, Hycell, Shanghai Duoning DN Feed 1, Zhongshan Kangsheng's CHO Feed 02, GrowthA, Dynamis, Iscove's Modified Dulbecco's Medium, RPMI 1640 and Minimum Essential Medium-α (MEM-α), Dulbecco's Modification of Eagle's Medium (DMEM), DME / F12, αMEM, Basal Medium Eagle with Earle's BSS. BSS), high glucose DMEM with glutamine, high glucose DMEM without glutamine, low glucose DMEM without glutamine, DMEM:F12 1:1 with glutamine, GMEM (Glasgow's MEM), GMEM with glutamine, Grace's Complete Insect Medium, Grace's Insect Medium without FBS, Ham's F-10 with glutamine, Ham's F-12 with glutamine, IMDM with HEPES and glutamine, IMDM with HEPES and no glutamine, 15 (Leibovitz) (2X) without glutamine or phenol red, 15 (Leibovitz) without glutamine, McCoy's 5A Modified Medium

[00155] The cells may be grown in 1% CO 2 culture medium (0.1% CO 2 5-8 mL) or 1% CO 2 culture medium (0.1% CO 2 5-12 mL). The cells may be grown in 1% CO 2 culture medium (0.1% CO 2 5-8 mL) or 1% CO 2 culture medium (0.1% CO 2 5-8 mL). The cells may be grown in 1% CO 2 culture medium (0.1% CO 2 5-8 mL) or 1% CO 2 culture medium (0.1% CO 2 5-8 mL) or 1% CO 2 culture medium (0.1% CO 2 5-8 mL) or 1% CO 2 culture medium (0.1% CO 2 5-8 mL) or 1% CO 2 culture medium (0.1% CO 2 5-8 mL)Supplemental components or ingredients, including optional components, may be added to the exemplary media described above at appropriate concentrations or amounts as needed or as desired and as known and practiced by those skilled in the art using routine skills.

[0017] In some embodiments of the present invention, the culture medium in the basic culture stage is selected from any one of EX-CELL Advanced CHO, ExpiCHO and CHO CD04, more preferably ExpiCHO.

[0018] In some embodiments of the present invention, the culture medium in the fed-batch culture stage is selected from either DN Feed 1 or CHOFeed02, preferably DN Feed 1; and the supplementary amount is 4.5% to 5.5% of the total volume of the culture medium.

[0019] In some embodiments of the present invention, the protein drug can be any one of a monoclonal antibody and a fusion protein.

[0020] In some embodiments of the present invention, the L-cysteine, L-tyrosine and L-tryptophan of the present invention can be added to the culture medium or culture system in the form of a mother liquor concentrate.

[0021] In some embodiments of the present invention, the method of the present invention is used to regulate the galactosylation level of a protein drug, comprising the following steps:

[0022] (1) Cell inoculation stage; (2) Basic culture stage; (3) Fed-batch culture stage;

[0023] L-cysteine ​​needs to be supplemented during the fed-batch culture stage; the L-cysteine ​​supplemented during the fed-batch culture stage can be added simultaneously with the feed medium, with a supplement amount of 1.2 to 4.8 mmol / L, preferably 1.5 to 3.6 mmol / L, and more preferably 1.8 to 2.1 mmol / L; L-cysteine ​​can also be supplemented separately during the basal culture stage and the fed-batch culture stage, with a supplement amount of 0.3 to 2.0 mmol / L during the basal culture stage and a supplement amount of 0.6 to 1.8 mmol / L during the fed-batch culture stage.

[0024] In another aspect of the present invention, there is also provided the application of the culture method of the present invention in regulating the galactosylation level of protein drugs.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) During the fed-batch culture phase, the proportion of galactosylated glycoforms of the protein drug is adjusted to the target level by adding an appropriate amount of cysteine ​​without affecting other quality parameters of the drug, such as the charge isoform ratio and phosphorylation level;

[0027] (2) The cell culture process of the present invention is simple to operate, has a stable process, and is conducive to industrial production.

[0028] Explanation of terms

[0029] As used herein, "cell culture medium" or "culture medium" refers to a nutrient solution used to maintain, grow, proliferate, or expand cells in an artificial in vitro environment (outside a multicellular organism or tissue). Examples include basal culture media prepared to support cell growth, production culture media prepared to optimize cells for specific cell culture or to promote monoclonal antibody production, and concentrated culture media prepared by concentrating nutrients at high concentrations. Nutrients and culture medium components refer to the components that make up a cell culture medium and are used interchangeably herein.

[0030] As used herein, "feed medium" and "addition medium" may refer to a medium composed of a specific nutrient or multiple nutrients, which are concentrated components of a basal medium. Feed medium compositions and concentrations may vary depending on the cells being cultured.

[0031] The "complement-dependent cytotoxicity (CDC)" mentioned in the present invention refers to the cytotoxicity in which complement participates, i.e., the classical complement pathway is activated by the binding of specific antibodies to corresponding antigens on the cell membrane surface to form a complex, and the formed membrane attack complex exerts a lytic effect on target cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1. Glycoform ratios at different L-cysteine ​​supplementation levels.

[0033] Figure 2. Charge isomer results of L-cysteine ​​at different supplementary amounts;

[0034] Figure 3 RP-HPLC results of different supplementary amounts of L-cysteine;

[0035] Figure 4. Sugar type ratios of L-cysteine ​​in different addition methods and amounts.

[0036] Figure 5. Charge isomer results of L-cysteine ​​in different addition methods and amounts.

[0037] Figure 6 Glycoform ratios of different GAL+ addition methods and amounts;

[0038] Figure 7 GAL + Phosphorylation ratio results of different addition methods and different supplementation amounts;

[0039] Figure 8 Different concentrations of Gal + Glycoform ratio results;

[0040] Figure 9 Different concentrations of Gal+ Charge variant results;

[0041] Figure 10 Different concentrations of Gal + RP-HPLC results;

[0042] Figure 11. Results of different L-tryptophan addition methods and sugar type ratios. DETAILED DESCRIPTION

[0043] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0044] The basal medium and feed medium used in the present invention are prepared and used as shown in Table 1:

[0045] Table 1

[0046]

[0047]

[0048] Example 1 Investigation of different supplementary amounts of L-cysteine

[0049] The cell culture process is as follows:

[0050] (1) Inoculation: The culture cells are CHO cells, and the expression product is IgG4 fusion protein. The culture scale is 250ml shake flask, the liquid volume is 60ml, and the inoculation density is 8*10 5 There were 4 groups of experiments, with two parallel experiments in each group.

[0051] (2) Cultivation: The shake flask was placed in a shaker and cultured at a temperature of 37°C, a carbon dioxide concentration of 8%, and a rotation speed of 140 rpm.

[0052] (3) Feeding: Feeding was started on the third day of culture and was conducted every two days. The amounts of DN Feed1 and L-tyrosine added are shown in Table 1. The amount of L-tryptophan added was 0.4 mmol / L, and the amounts of L-cysteine ​​added to Y1, Y2, Y3, and Y4 were 1.5 mmol / L, 1.8 mmol / L, 2.1 mmol / L, and 1.2 mmol / L, respectively.

[0053] (4) After culturing for 11 days, the culture was terminated and quality parameters such as glycoform ratio and charge isomers were tested. The results are shown in Figures 1, 2, and 3. In Figure 1, the control is the test result of the original product dulaglutide.

[0054] Conclusion: According to Figures 1, 2, and 3, increasing the amount of cysteine ​​supplementation can reduce the G0F ratio, increase the G1F and G2F ratios, and improve the galactosylation level, without significantly affecting other quality parameters.

[0055] Example 2

[0056] The effect of cysteine ​​on glycoforms was verified in other protein therapeutics. CHO cells were used as culture cells, and the product expressed was a monoclonal antibody. The effects of different cysteine ​​addition methods on glycoforms were also investigated.

[0057] (1) Inoculation: The culture scale is 250 ml shake flask, the liquid volume is 60 ml, and the inoculation density is 8*10 5 There were 6 groups of experiments in total, with two parallel experiments in each group.

[0058] (2) Cultivation: The shake flask was placed in a shaker and cultured at a temperature of 37°C, a carbon dioxide concentration of 8%, and a rotation speed of 140 rpm.

[0059] (3) Feeding: Feeding was started on the third day of culture and was conducted every two days. The amounts of DN Feed 1 and L-tyrosine added are shown in Table 1. The amount of L-tryptophan added was 0.4 mmol / L. The specific method of adding L-cysteine ​​is shown in Table 2.

[0060] (4) After culturing for 7 days, the culture was terminated and quality parameters such as glycoform ratio and charge isomers were measured. The results are shown in Figures 4 and 5.

[0061] Table 2 L-cysteine ​​addition strategy

[0062]

[0063]

[0064] Note: The concentration of L-cysteine ​​in the basal culture medium is 0.6 mmol / L, and the initial addition is to be added to the basal culture medium within 1 hour after inoculation.

[0065] Conclusion: As shown in Figures 4 and 5, whether adding cysteine ​​during the fed-batch stage or adding it during both the basal and fed-batch stages can reduce the G0F ratio, increase the G1F and G2F ratios, and improve the galactosylation level of the drug, without significantly affecting other drug quality parameters.

[0066] Comparative Example 1

[0067] (1) Glycoform regulators were added at different times during the fed-batch culture phase to investigate their effects on glycosylation levels and other quality parameters.

[0068] Use Glycosylation Adjust(Gal+ , Sigma, catalog number: 14701C) to adjust the sugar ratio, Gal + It is an additive with targeted glycosylation properties that can easily achieve the ideal N-linked glycosylation target by increasing the occupancy of galactose on oligosaccharides.

[0069] The cell culture process is as follows:

[0070] (1) Inoculation: The culture cells are CHO cells, and the expression product is IgG4 fusion protein. The culture scale is 250ml shake flask, the liquid volume is 60ml, and the inoculation density is 8*10 5 There were 7 groups of experiments in total, with two parallel experiments in each group.

[0071] (2) Cultivation: The shake flask was placed in a shaker and cultured at a temperature of 37°C, a carbon dioxide concentration of 8%, and a rotation speed of 140 rpm.

[0072] (3) Feeding: Feeding was started on the third day of culture and was added every two days. The amounts of DN Feed 1 and L-tyrosine added are shown in Table 1. The amount of L-tryptophan added was 0.4 mmol / L, and the amount of L-cysteine ​​added was 1.2 mmol / L.

[0073] (4) During the culture process, Gal+ additives were added according to the strategy in Table 2.

[0074] (5) After 11 days of culture, the culture was terminated and quality parameters such as glycoform ratio and charge isomers were measured. The results are shown in Figures 6 and 7.

[0075] Table 3 Gal + Add strategy

[0076]

[0077]

[0078] According to Figures 6 and 7, the addition of Gal on the 6th day + Compared with the regimen of + The effect of reducing the G0F ratio is weak, and the phosphorylation ratio is slightly higher. Therefore, it is preferred to add Gal on the 6th day. + .

[0079] (2) Based on the above experiment (1), the addition of different concentrations of Gal on the 6th day was investigated. + Effects on glycoform ratios and other quality parameters.

[0080] (1) Inoculation: The culture cells are CHO cells, and the expression product is IgG4 fusion protein. The culture scale is 250ml shake flask, the liquid volume is 60ml, and the inoculation density is 8*10 5There were 8 groups of experiments in total, with two parallel experiments in each group.

[0081] (2) Cultivation: The shake flask was placed in a shaker and cultured at a temperature of 37°C, a carbon dioxide concentration of 8%, and a rotation speed of 140 rpm.

[0082] (3) Feeding: Feeding was started on the third day of culture and was added every two days. The amounts of DN Feed 1 and L-tyrosine added are shown in Table 1. The amount of L-tryptophan added was 0.4 mmol / L, and the amount of L-cysteine ​​added was 1.2 mmol / L.

[0083] (4) On the 6th day of culture, add Gal+ additives according to the strategy in Table 3.

[0084] (5) After 11 days of culture, quality parameters such as glycoform ratio and charge isomers were measured. The results are shown in Table 5 and Figures 8, 9, and 10. In Figure 10, the control is the result for the original product dulaglutide.

[0085] Table 4 Gal+ addition strategy

[0086]

[0087] Table 5 Experimental data

[0088]

[0089]

[0090] Conclusion: Compared with the control group Y8 and the original research results, according to the results in Figure 8, Gal + The results in Figures 9 and 10 show that as Gal + With the increase of the added amount, the proportion of the main peak decreased, the proportion of impurities in the acidic area increased, and the phosphorylation proportion increased.

[0091] Therefore, although Gal+ additives can increase the level of galactosylation, they will affect other quality parameters of the product.

[0092] Comparative Example 2

[0093] Based on the study of cysteine, the effect of tryptophan on glycosylation levels was investigated. The culture cells were also CHO cells, and the expression product was a monoclonal antibody.

[0094] (1) Inoculation: The culture scale is 250 ml shake flask, the liquid volume is 60 ml, and the inoculation density is 8*10 5 There were 7 groups of experiments in total, with two parallel experiments in each group.

[0095] (2) Cultivation: The shake flask was placed in a shaker and cultured at a temperature of 37°C, a carbon dioxide concentration of 8%, and a rotation speed of 140 rpm.

[0096] (3) Feeding: Feeding was started on the third day of culture and was continued every two days. The amount of DN Feed 1 and L-tyrosine added is shown in Table 1. The amount of L-cysteine ​​added was 1.2 mmol / L, and the specific method of adding L-tryptophan is shown in Table 6.

[0097] (4) After culturing for 7 days, the culture was terminated and the glycoform ratio was measured. The results are shown in Figure 11.

[0098] Table 6 Tryptophan addition strategy

[0099]

[0100] Conclusion: According to the results in Figure 11, different addition methods and amounts of L-tryptophan cannot regulate the galactosylation level of protein.

[0101] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean 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 can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0102] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A CHO cell culture method for regulating the galactosylation level of protein drugs, characterized in that: The culture method includes a basic culture stage and a fed-batch culture stage; in addition to the feed culture medium, L-cysteine ​​is also added in the fed-batch culture stage; the L-cysteine ​​is added in the basic culture stage and the fed-batch culture stage respectively, with the addition amount in the basic culture stage being 0.3-2.0 mmol / L and the addition amount in the fed-batch culture stage being 0.6-1.8 mmol / L; L-tyrosine and L-tryptophan are also added in the fed-batch culture stage, with the addition amount of L-tyrosine being 2.0-2.8 mmol / L and the addition amount of L-tryptophan being 0.2-0.6 mmol / L; the fed-batch culture starts on the third day, with feeding once every two days until the end of the culture.

2. The method according to claim 1, characterized in that The culture medium used in the basic culture stage is selected from any one of EX-CELL Advanced CHO, ExpiCHO and CHO CD04.

3. The method according to claim 2, characterized in that The culture medium used in the basic culture stage is selected from ExpiCHO.

4. The method according to claim 1, wherein The culture medium used in the fed-batch culture stage is selected from either DNFeed1 or CHO Feed 02; the added amount is 4.5-5.5% of the total volume of the culture medium.

5. The method according to claim 4, characterized in that The culture medium used in the fed-batch culture stage is selected from DNFeed1.

6. The method according to claim 1, characterized in that The protein drug can be any one of a monoclonal antibody or a fusion protein.

7. Use of the method according to any one of claims 1 to 6 in regulating the galactosylation level of protein drugs.

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