Method for regulating and controlling multiple glycoforms of cell expression product

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for simultaneously regulating and controlling multiple glycoforms of a cell expression product. The method comprises the step of adding a glycoform regulator in a cell culture process, wherein the glycoform regulator comprises one or more of galactose, manganese chloride, tris (hydroxymethyl) aminomethane, bis (2-hydroxyethyl) amino-tris (hydroxymethyl) methane and uridine. According to the method, antibody glycosylation modification can be effectively adjusted, particularly, the galactosylation rate, the mannosylation rate and the fucosylation rate can be regulated and controlled, on the basis, growth metabolism of cells, the expression quantity of antibodies and charge heterogeneity and purity are not affected, operation is easy, cost is low, and the method is suitable for regulating and controlling various similar biological drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for regulating various glycoforms of cell expression products. BACKGROUND

[0002] In the production of therapeutic antibodies, post-translational modifications caused by different physicochemical factors increase the heterogeneity of antibody structure and function. Among the many post-translational modifications of proteins, glycosylation is a very important one. The site of glycosylation, the type and abundance of glycoforms can affect the stability of the molecule and its interaction with Fc receptors in vivo, thereby affecting the effectiveness, safety and quality stability of the product, and therefore glycosylation is generally considered a key quality attribute of monoclonal antibody drugs.

[0003] Most therapeutic antibodies and other recombinant therapeutic proteins (including fusion proteins) are expressed by CHO cells, and N-glycosylation is the most common form of glycosylation, which is usually synthesized in the endoplasmic reticulum (ER) and Golgi apparatus. Antibody structure, activity and function are closely related to the glycosylation modification of antibodies. Antibody glycosylation modification can maintain the spatial conformation of the antibody and stabilize the structure of the antibody, while some specific glycoforms are also closely related to the half-life, immunogenicity, anti-inflammatory effect and antibody function of the antibody. However, due to the complex molecular structure and large molecular weight of the antibody, a large number of modifications can occur during production and storage, resulting in a variety of isomers of the antibody, and the combination of these isomers leads to charge heterogeneity, glycosylation modification heterogeneity, etc. of the antibody.

[0004] Charge heterogeneity is an important quality attribute in the production process of monoclonal antibodies, and charge heterogeneity produces acid-base peaks, especially acid peaks, which seriously affect the stability and biological function of monoclonal antibodies, and further affect their tissue penetration and pharmacokinetics. Factors leading to charge heterogeneity include various chemical degradation mechanisms (such as oxidation, deamidation, isomerization and cleavage) and addition reactions (glycosylation addition or covalent addition) occurring at different positions of the protein. Among them, the alkaline peak is mainly derived from the unevenness of the C-terminal lysine, methionine oxidation or aspartic acid conversion to succinimide, while the acidic peak is generally derived from N-sugar terminal sialylation modification, deamidation of amino acid residues, etc. The cumulative effect of these events occurring on the molecule leads to changes in the structure and conformation of the molecule, has a significant impact on the isoelectric pH of the molecule, and has a potential impact on the function of the protein.

[0005] In general, different glycoforms and charge isomers have a significant impact on therapeutic effect. However, antibodies, fusion proteins and other therapeutic proteins are usually a mixture of multiple variants, each antibody or recombinant protein has its own characteristics, and it is particularly important to regulate and analyze product-related variants.

[0006] The existing method for changing glycosylation is mainly through the genetic regulation level and the cell culture process to change the adjustment. The genetic level regulation is to change the antibody glycosylation type or glycosylation level by modifying the related glycosylation enzyme of the host cell, but the method has a long cycle, and due to its safety, complexity and stability, it needs a long time to verify and determine, which greatly increases the research and development time and cost. The method of controlling the antibody charge heterogeneity by controlling the cell culture process is also very challenging.

[0007] Considering the complexity of glycosylation biosynthesis and mammalian cell culture, it is still difficult to regulate specific glycosylation quickly and effectively in the development of new drugs and biological similar drugs. Therefore, it is of great significance to provide a precise and efficient biosynthesis method for simultaneously regulating the glycosylation of cell expression products. SUMMARY

[0008] The purpose of the present application is to provide a method for regulating the glycosylation of cell expression products, especially a method for simultaneously regulating the glycosylation, purity and charge heterogeneity of cell expression products, which is suitable for the regulation of galactosylation rate, mannosylation rate and fucosylation rate in the development process of antibody drugs.

[0009] In one aspect of the present application, the method for regulating the glycosylation of cell expression products comprises adding a glycosylation regulator during cell culture, wherein the glycosylation regulator comprises one or more of galactose, manganese chloride (MnCl2), Tris, Bis-Tris and uridine.

[0010] In a preferred aspect of the present application, the glycosylation is galactosylation, mannosylation and fucosylation.

[0011] In a preferred aspect of the present application, the glycosylation regulator is selected from one or both of galactose and Tris.

[0012] In a preferred aspect of the present application, the glycosylation regulator is a combination of galactose and Tris.

[0013] In a preferred aspect of the present application, the concentration of galactose is 2-12 mM; preferably 4-10 mM; for example, it can be 4 mM, 5 mM, 6 mM, 8 mM, 10 mM or any point value within the range thereof; more preferably 8 mM.

[0014] In a preferred aspect of the present application, the concentration of Tris is 0.5-1.5 mM; preferably 0.5-1.0 mM; for example, it can be 0.5 mM, 0.75 mM, 1 mM or any point value within the range thereof; more preferably 0.75 mM.

[0015] In a preferred embodiment of the present invention, when the regulator is a combination of galactose and Tris, the concentration of galactose is 2-12 mM, and the concentration of Tris is 0.5-1.5 mM; preferably, the concentration of galactose is 4-10 mM, and the concentration of Tris is 0.5-1.0 mM; for example, it can be a combination of 4 mM galactose + 1 mM Tris, 5 mM galactose + 0.5 mM Tris, 6 mM galactose + 0.5 mM Tris, 8 mM galactose + 0.5 mM Tris, 8 mM galactose + 0.75 mM Tris, or any point value within the range.

[0016] In a preferred embodiment of the present invention, when the regulator is a combination of galactose and Tris, its concentration is 8 mM galactose + 0.5 mM Tris or 8 mM galactose + 0.75 mM Tris.

[0017] In a preferred embodiment of the present invention, when the regulator is a combination of galactose and Tris, its concentration is 8 mM galactose + 0.75 mM Tris.

[0018] In a preferred embodiment of the present invention, the initial culture density of the fed batch of cells is (0.5±0.1)×

[0019] 10 6 cells / mL.

[0020] In a preferred embodiment of the present invention, the initial culture density of the fed batch of cells is 0.5×10 6 cells / mL.

[0021] In a preferred embodiment of the present invention, the initial culture conditions of the fed-batch culture of the cells are 5%±2% CO2, 80%±20% humidity and 36.5°C±0.5°C temperature.

[0022] In a preferred embodiment of the present invention, the initial culture conditions of the cells in a shake flask fed batch (Fed Batch) are 5%±2% CO2, 80%±20% humidity, 36.5°C±0.5°C temperature and 125±10 rpm rotation speed.

[0023] In a preferred embodiment of the present invention, the initial culture conditions of the cells in a shake flask fed batch culture (Fed Batch) are 5% CO2, 80% humidity, 36.5°C temperature and 125 rpm rotation speed.

[0024] In a preferred embodiment of the application, the bioreactor Fed Batch initial culture conditions for the cells are 6.9 ± 0.2 pH, 10-90% DO, 36.5°C ± 0.5°C temperature, and 180 ± 10 rpm rotation speed.

[0025] In a preferred embodiment of the application, the bioreactor Fed Batch initial culture conditions for the cells are 6.9 pH, 5% CO2, 80% humidity, 40% DO, 36.5°C temperature, and 180 rpm rotation speed.

[0026] In a preferred embodiment of the application, the method comprises lowering the culture temperature of the cells to 32.0°C ± 0.5°C on day 4.

[0027] In a preferred embodiment of the application, the method comprises lowering the culture temperature of the cells to 32.0°C on day 4.

[0028] In a preferred embodiment of the application, the method comprises adjusting the type and amount of addition of the expansion medium, the Fed Batch culture base medium, and the feed medium.

[0029] In a preferred embodiment of the application, the expansion medium is CD CHO.

[0030] In a preferred embodiment of the application, the base medium is selected from HSM12, CD CHO 031, or Dynamis.

[0031] In a preferred embodiment of the application, the base medium is HSM12.

[0032] In a preferred embodiment of the application, the feed medium is selected from one or several of CD Feed 008, CD Feed 009, CellBoost 7a, and Cell Boost 7b.

[0033] In a preferred embodiment of the application, the feed medium is Cell Boost 7a and Cell Boost 7b.

[0034] In a preferred embodiment of the application, the feed medium is Cell Boost 7a and Cell Boost 7b, and the ratio of Cell Boost 7a:Cell Boost 7b is 10:1.

[0035] In a preferred embodiment of the application, the method comprises adding the feed medium and the sugar type modulator on the feed day.

[0036] In a preferred embodiment of the application, the cell can be derived from a mammal, such as a human and non-human primate, as well as a rabbit, a mouse, a goat, a pig and other mammalian species. In a preferred embodiment, the mammal is a mouse.

[0037] In a preferred embodiment of the application, the cell is a CHO cell.

[0038] In a preferred embodiment of the application, the cell is a CHO-K1, CHO DG44 and CHO-S cell, preferably a CHO-K1 cell.

[0039] In a preferred embodiment of the application, the expression product is a recombinant protein, preferably an antibody, more preferably a bispecific monoclonal antibody, further preferably an IgG4 type bispecific monoclonal antibody.

[0040] In a preferred embodiment of the application, the bispecific monoclonal antibody of IgG4 type is Emicizumab.

[0041] The beneficial effects of the present application are:

[0042] The method of the present application can effectively regulate the glycosylation modification of antibodies, especially can regulate the galactosylation rate, mannosylation rate and fucosylation rate, and on this basis has no effect on the growth metabolism of cells, the expression amount of antibodies and charge heterogeneity and purity, is simple to operate, low in cost, and suitable for regulating various biological similar drugs.

[0043] Related definitions:

[0044] In the present application, unless otherwise specified, all ranges, including ranges defined between two specified end values, include the specified end values. For example, the range between 4-10 represents the range between 4 and 10, including 4 and 10.

[0045] In the present application, when the length of time, the period or the interval is expressed in days, and the time point is expressed as a day or a day, the length of time or the time point is counted or distinguished by day (day), and the numerical value does not require to represent a multiple of 24 hours.

[0046] In the present application, the CHO cell refers to Chinese hamster ovary cell.

[0047] Manganese chloride: MnCl2.

[0048] Tris: Tris(hydroxymethyl)aminomethane.

[0049] Bis-Tris: Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane.

[0050] Galactose: Galactose.

[0051] VCD: viable cell density.

[0052] VCD End : Viable cell density at harvest.

[0053] CE-HPLC: Charge variants were detected by ion-exchange high performance liquid chromatography.

[0054] SE-HPLC: Size exclusion high performance liquid chromatography can be used to detect antibody purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The graph shows the antibody live cell density and cell viability results for the galactose groups No. 1 to 3.

[0056] Figure 2 The graph shows the antibody live cell density and cell viability results for the MnCl2 groups No. 4 to 6.

[0057] Figure 3 The graph shows the live cell density and cell viability results of the antibodies in the Tris groups No. 7 to 9.

[0058] Figure 4 The graph shows the antibody live cell density and cell viability results for Bis-Tris groups No. 10 to 12.

[0059] Figure 5 This is a graph showing the antibody live cell density and cell viability results for the uridine group Nos. 13 to 15.

[0060] Figure 6 The graphs show the antibody live cell density and cell viability results for No. 17 (Tris-added group) and No. 19 (Tris and galactose-added group).

[0061] Figure 7 The graph shows the lactate metabolism results of No. 17 (Tris-added group) and No. 19 (Tris and galactose-added group). DETAILED DESCRIPTION

[0062] Below in conjunction with specific embodiment, the present invention is further elaborated in detail, but those skilled in the art will understand that the embodiment described below is a part of embodiment of the present invention, rather than all embodiments, is only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise fall within the scope of protection of the present invention. The experimental method used in the following examples, unless otherwise specified, the experimental method of the specific conditions not specified in the examples, usually according to conventional conditions, the materials, reagents etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0063] 1. Experimental materials

[0064] The experimental cells were CHO-K1 cells expressing a bispecific monoclonal antibody of IgG4 type. The expansion medium CDCHO medium (abbreviated as M1), the fed batch (Fed Batch) base medium HSM12 medium (abbreviated as M2), and the Dynamis medium (abbreviated as M3) were all purchased from Gibco, and the fed medium Cell Boost 7a and Cell Boost 7b (abbreviated as FM1 and FM2, wherein FM1 / FM2 is 10:1) were purchased from Sigma. Manganese chloride (MnCl2), galactose (galactose), and tris(hydroxymethyl)aminomethane (Tris) were purchased from Sigma. Glucose was purchased from Shenguo Bioengineering (Shanghai) Co., Ltd., and the reference product was purchased from commercially available Eculizumab injection.

[0065] 2. Cell culture: seed expansion, shake flask Fed Batch culture, and 2L reactor Fed Batch culture

[0066] Seed expansion: one vial of frozen cells was recovered in a 125mL shake flask, the initial culture volume was 25mL, the expansion medium was M1, and the incubator parameters were 5% CO2, humidity 80%, temperature 36.5°C, and rotation speed 125rpm. The cells were cultured and subcultured every 3 days, and the density after subculture was about 0.3×10 6 cells / mL, until the target seed amount was reached.

[0067] Shake flask Fed Batch culture: the expanded cells were inoculated in a 250mL shake flask at a density of about 0.5×10 6 cells / mL, the initial culture volume was 50mL, the base medium was M2, the incubator parameters were 5% CO2, humidity 80%, and rotation speed 125rpm, and the initial culture temperature was 36.5°C. The culture was incubated to the 4th day, and the temperature was lowered according to the temperature lowering strategy. The feed was added in a fed batch manner, and the feed strategy was to add 4%, 5%, 5%, 5%, and 4% of the initial volume of FM1 on the 3rd, 6th, 8th, 10th, and 12th days, respectively, and to add FM2 in an amount of 10% FM1. Starting from the 3rd day, the glucose concentration was detected using a biosensor, and the glucose concentration was supplemented to 8g / L when it was lower than 6g / L on non-feed days, and to 10g / L when it was lower than 6g / L on feed days. The culture was harvested on the 14th day.

[0068] 2L reactor Fed Batch culture: the cells were inoculated in a 2L reactor at a density of about 0.5×10 6The cells were inoculated into Sartorius 2L reactors at a cell density of 1.5-2.5 x 106cells / mL, and the initial culture volume was 1400 mL. The base medium was M2 or M3. The reactor parameters are shown in the table below, and the feeding and sugar type adjustment were performed according to the above-mentioned shake flask batch feeding culture step. The addition of the sugar type regulator was performed according to the scheme.

[0069] Table 1. Reactor setting parameters

[0070]

[0071]

[0072] 3. Detection method: protein purification, yield and quality analysis

[0073] The clarified cell culture supernatant was subjected to protein A affinity chromatography purification by AT Protein A Diamond Plus filler. The purified sample was subjected to product quality detection.

[0074] The antibody yield was detected by ultraviolet-visible spectrophotometry. The absorbance of the test sample solution at a wavelength of 280 nm was determined, and the protein content of the test sample solution was calculated with an extinction coefficient (L / g-cm-1) of 1.452.

[0075] The antibody purity was detected by size exclusion chromatography (SE-HPLC) using a TOSOH TSKgel G3000 SWXL molecular sieve chromatographic column, a mobile phase of 0.1 mol / L phosphate-0.1 mol / L sodium chloride buffer, pH 6.8, a flow rate of 0.8 mL per minute, a sample injection amount of 10 μL, and detection at a wavelength of 280 nm.

[0076] The charge isoform was detected by weak cation chromatography (CE-HPLC) using a Thermo ProPac WCX-10 (4 x 250 mm) weak cation chromatographic column, gradient elution with A (9.6 mmol / L Tris, 6.0 mmol / L Piperazine, 11.0 mmol / L Imidazole, pH 6.0) and B (9.6 mmol / L Tris, 6.0 mmol / L Piperazine, 11.0 mmol / L Imidazole, 150 mmol / L Sodium Chloride, pH 9.9) as the mobile phase, and detection at a wavelength of 280 nm.

[0077] N-Glycan analysis: The N-glycan was released by PNGase F glycosidase specific enzyme, and the released sugar chain was labeled by 2-aminobenzamide (2-AB) for glycosylation analysis. The glycosylation analysis used an instrument of ultra performance liquid chromatography-fluorescence detector (UPLC-FLD), and the chromatographic column was Acquity UPLC Glycan BEH Amide (1.7 μm, 2.1*150 mm) of Waters company, with 50 mM ammonium formate and acetonitrile as the mobile phase, pH 4.5, the injection volume was 20 μL, gradient elution for 70 minutes. The excitation wavelength was 265 nm, and the emission wavelength was 425 nm

[0078] Example 1 Optimization of pH and cooling strategy

[0079] Referring to the foregoing cell culture conditions, different reaction conditions were selected for experiments on a 2L reactor. The Fed Batch base medium was M3, the initial culture temperature was 36.5°C, and the optimal pH culture conditions and cooling strategy were selected according to the quality results. The specific process parameters and detection results are shown in Tables 2-4.

[0080] Table 2 Process parameters

[0081] Group pH Cooling strategy 1.1 7.0±0.2 D4 cooled to 32.0°C 1.2 6.9±0.2 D4 cooled to 32.0°C 1.3 6.9±0.2 D5 cooled to 32.0°C 1.4 6.9±0.2 D4 cooled to 33.0°C 1.5 6.9±0.2 D4 cooled to 31.0°C

[0082] Table 3 SE-HPLC results of different processes

[0083]

[0084]

[0085] Table 4 CE-HPLC results of different processes

[0086] Group JJ (%) Acid peak (%) Main peak (%) Base peak (%) 1.1 30.1 7.2 53.9 6.7 1.2 25.9 3.5 61.1 9.4 1.3 22.8 5.3 51.2 20.7 1.4 32.8 11.2 29.9 26.1 1.5 20.6 2.8 70.0 10.6

[0087] Table 5 N-Glycan results of different processes

[0088] Group Mannosylation (%) Galactosylation (%) Fucosylation (%) 1.2 1.5 23.6 90.6 1.3 1.2 22.2 92.6 1.4 1.8 18.9 91.7 1.5 1.3 32.6 90.3 Reference 4.7 16.2 85.9

[0089] The above table is the quality result of the sample after affinity of the cell culture supernatant. The SE-HPLC results show that there is no difference in the purity of each process, and the monomer content is more than 95%; the CE-HPLC results show that the main peak content of process parameters 1.2 and 1.5 is higher; but the N-Glycan result of process parameter 1.5 shows that the galactosylation rate is higher.

[0090] Example 2 Optimization of glycoform process

[0091] 1) Referring to the foregoing steps, the cell seed amplification stage was completed.

[0092] 2) Refer to the previous step to complete the shake flask Fed Batch culture, the Fed Batch base medium is M2, and the culture is performed to the 4th day. The cooling strategy is to cool to 32.0°C.

[0093] Different types and contents of sugar-type modulators are added according to the following groups:

[0094] 400mM galactose stock solution, 500μM MnCl2stock solution, 100mM Tris stock solution, 100mM Bis-Tris stock solution and 1M uridine stock solution are respectively prepared. The addition mode is to add on the 3rd, 6th, 8th, 10th and 12th day of the feeding addition day, and the amount of each addition is shown in the following table. The control group is the blank control group.

[0095] Table 6 Addition mode of sugar-type modulators

[0096]

[0097]

[0098] Harvesting is performed on the 14th day of culture. Sampling is performed every day for cell counting, metabolic analysis, etc. The cell harvest is centrifuged at 7000rpm for 15min, filtered with a 0.22μM filter to obtain the cell culture supernatant, and then purified, analyzed for protein expression amount and quality.

[0099] 3) Result analysis

[0100] Table 7 N-Glycan results of different groups

[0101] Group Mannosylation (%) Galactosylation (%) Fucosylation (%) Control 1.1 3.4 92.3 1 1.4 4.8 92.2 2 1.2 6.2 92.5 3 1.1 6.3 92.6 4 1.4 11.6 91.1 5 0.8 17.0 91.7 6 0.8 24.2 91.0 7 2.7 3.1 89.9 8 5.0 3.3 85.9 9 11.2 3.0 71.4 10 10.3 3.1 75.7 11 16.8 2.8 57.6 12 16.4 2.4 34.3 13 2.0 3.1 91.2 14 6.7 3.8 82.5 15 8.3 5.3 80.2 Reference 4.7 16.2 85.9

[0102] Figure 1 The cell peak density values are consistent, the cell viability at the time of harvesting is more than 95%, and the trend is consistent; it is shown that the addition of galactose does not affect the growth, metabolism and expression of the cells; unlike the galactose group, the addition of high-concentration MnCl2inhibits the proliferation of the cells, as shown in Figure 2 , the VCD End of the control group is 26.0×10 6 cells / mL, and the VCD 6 of the 0.40μM MnCl2experimental group is only 19.0×10 6 cells / mL. Figure 3 and Figure 4 show that different concentrations of Tris and Bis-Tris have no effect on the growth of the cells. Figure 5The high concentration of uridine inhibited cell growth, and the cell viability was only 78% at harvest; the expression level of the low concentration uridine group was 6.6 g / L, and the expression levels of the medium and high concentration uridine groups were only 3.8 and 1.9 g / L, indicating that the cell expression was inhibited with the increase of uridine concentration.

[0103] Example 3 Confirmation of the effect of sugar type regulator on antibody in a bioreactor scale-up

[0104] The cell seed amplification stage was completed according to the foregoing steps, the Fed Batch base medium was M2, and the cell culture pH and cooling strategy in the 2L reactor were performed according to process 1.2 in Example 1. A 500 mM Tris mother liquor was prepared.

[0105] Different types and contents of sugar type regulators were added on the 3rd, 6th, 8th, 10th, and 12th days of the feeding day according to the following groups, and the control group was the blank control group:

[0106] Table 8 Addition method of sugar type regulator

[0107] Group Tris (mM) Galactose (mM) Control / / 16 0.5 / 17 0.75 / 18 0.50 8.0 19 0.75 8.0

[0108] Harvesting was performed at the 14th day of culture, the cell harvest was centrifuged at 7000 rpm for 15 min, and the cell culture supernatant was obtained by filtering with a 0.22 μM filter, and then purification and protein expression and quality analysis were performed.

[0109] Result analysis:

[0110] Table 9 Effect of different groups on antibody yield and purity

[0111] Group Yield (g / L) High molecular weight (%) Monomer (%) Control 7.4 4.5 95.5 16 7.3 4.6 95.4 17 7.2 3.8 96.2 18 7.3 3.4 96.6 19 7.4 4.0 96.0

[0112] Table 10 Effect of different groups on antibody heterogeneity

[0113] Group JJ (%) Acid peak (%) Main peak (%) Base peak (%) QQ (%) Control 28.1 3.7 57.3 8.5 2.3 16 27.5 3.8 58.1 8.5 2.3 17 27.6 3.7 58.0 8.6 2.1 18 27.7 3.9 57.6 8.6 2.2 19 29.4 2.9 58.7 8.3 0.8

[0114] Table 11 N-Glycan results of different groups

[0115] Group Mannosylation (%) Galactosylation (%) Fucosylation (%) Control 2.2 8.5 90.0 17 4.5 9.7 84.4 18 3.5 19.3 86.1 19 4.8 15.2 84.4 Reference 4.7 16.2 85.9

[0116] Figure 6 and Figure 7 It is shown that the simultaneous addition of Tris and galactose does not affect cell growth and lactate metabolism; Table 9 shows that the simultaneous addition of Tris and galactose does not affect the yield and purity of the product; Table 10 shows that the simultaneous addition of Tris and galactose does not affect the formation of acid-base variants; Table 11 shows that, compared with the Tris addition group alone, the galactose and fucose glycosylation rates are not affected, and Tris and galactose do not have an interaction.

[0117] Although the present application has been described and illustrated with a certain degree of particularity, it is understood that the present application has been made by way of examples only and that numerous changes in the details of execution can be made by those skilled in the art without departing from the spirit and scope of the application. It is therefore intended to cover in the appended claims all such changes and modifications that come within the scope of the application.

Claims

1. A method of modulating the glycoforms of a cell expressed product, comprising, The sugar type is galactosylation, mannosylation and fucosylation; the method comprises adding a sugar type regulator during cell culture, wherein the sugar type regulator is selected from one or more of galactose, tris-hydroxymethyl aminomethane and bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane.

2. The method of claim 1, wherein, The sugar type regulator is selected from one or both of galactose and tris-hydroxymethyl aminomethane; preferably a combination of galactose and tris-hydroxymethyl aminomethane.

3. The method of claim 2, wherein, The concentration of galactose is 2-12 mM; preferably 4-10 mM; more preferably 8 mM; the concentration of tris-hydroxymethyl aminomethane is 0.5-1.5 mM; preferably 0.5-1.0 mM; more preferably 0.75 mM.

4. The method of claim 2, wherein, The concentration of galactose is 2-12 mM and the concentration of tris-hydroxymethyl aminomethane is 0.5-1.5 mM; preferably, the concentration of galactose is 4-10 mM and the concentration of tris-hydroxymethyl aminomethane is 0.5-1.0 mM.

5. The method of claim 2, wherein, The concentration is 8 mM galactose + 0.5 mM tris-hydroxymethyl aminomethane or 8 mM galactose + 0.75 mM tris-hydroxymethyl aminomethane; preferably, the concentration is 8 mM galactose + 0.75 mM tris-hydroxymethyl aminomethane.

6. The method of claim 1, wherein, The initial culture density of the cells in fed-batch is (0.5±0.1)xlO 6 cells / mL; the initial culture conditions of the cells in fed-batch are 5%±2% CO2, 80%±20% humidity and 36.5°C±0.5°C temperature.

7. The method of claim 6, wherein, The initial culture conditions for shake flask fed-batch are 5%±2% CO2, 80%±20% humidity, 36.5℃±0.5℃ temperature and 125±10 rpm rotation speed; preferably 5% CO2, 80% humidity, 36.5℃ temperature and 125 rpm rotation speed; Alternatively, the initial culture conditions for bioreactor fed-batch are 6.9±0.2 pH, 10-90% DO, 36.5℃±0.5℃ temperature and 180±10 rpm rotation speed; preferably 6.9 pH, 40% DO, 36.5℃ temperature and 180 rpm rotation speed.

8. The method of claim 1, wherein, The method comprises lowering the culture temperature of the cells to 32.0℃±0.5℃ on day 4.

9. The method of claim 1, wherein, The cells are CHO cells; preferably CHO-K1, CHODG44 and CHO-S cells; more preferably CHO-K1 cells.

10. The method of claim 1, wherein, The expression product is a recombinant protein, preferably an antibody, more preferably a bispecific monoclonal antibody, further preferably an IgG4 type bispecific monoclonal antibody.

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