A method for reducing the level of high mannose glycoproteins produced by a mammalian cell expression system and uses thereof

Abstract

The present application relates to a method for reducing the level of high mannose type glycoprotein produced by a mammalian cell expression system and its application, the method comprising any one of the following ways or a combination of the two: (a) adding N-acetyl-D-mannosamine to the subculture medium of the previous generation of mammalian cell expression system before inoculation, then inoculating into the production medium after cultivation, and continuing cultivation; (b) adding N-acetyl-D-mannosamine to the culture medium during cultivation after inoculating the mammalian cell expression system from the subculture medium into the production medium. This method can not only effectively reduce the level of high mannose type glycosylation in the produced glycoprotein, but also does not affect the protein yield, and the cell growth and metabolism are also not affected, and other quality results such as protein purity, acid-alkali peak, etc. will not change significantly, so it is significant for the research and preparation of new antibody drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of protein biological product preparation, and relates to a method for reducing the level of high-mannose type glycoprotein produced by a mammalian cell expression system and application thereof, in particular to a method for reducing the level of high-mannose type glycoprotein produced by a mammalian cell expression system and application thereof in the production and preparation of protein biological products. BACKGROUND

[0002] At present, many biological agents produced are protein therapeutics, mainly glycoproteins in the form of antibodies, antibody-based fusion proteins and antibody drug conjugates. These drugs have many important characteristics that change their impact on the human body, and small deviations can lead to serious consequences, so the quality control of these biological agents is crucial to pharmaceutical companies, which makes it necessary to correctly analyze the key quality attributes (CQA), such as glycosylation. Protein glycosylation is a post-translational modification that is crucial to the function, immunogenicity and efficacy of recombinant glycoprotein therapeutics. Glycosylation has been shown to affect various physicochemical properties of glycoprotein therapeutics, including protein folding, solubility, binding affinity, stability and half-life.

[0003] CHO cells (Chinese hamster ovary cells) are one of the most widely used cell lines in mammalian cell expression systems, and are involved in the production of more than 70% of recombinant biopharmaceutical proteins. CHO cells have many advantages, for example: they are fibroblasts, a non-secretory cell, which itself secretes very little CHO endogenous proteins, thus being very advantageous for the isolation and purification of target proteins; suitable for large-scale bioreactor suspension culture; adapted to grow in chemically defined media to ensure reproducibility between different culture batches; and more importantly, they can produce proteins with complex glycosylation, post-translational modification, which is similar to human-produced proteins.

[0004] Yu M et.al (2012) characterized the in vitro bioactivity of antibodies with different levels of Man 5 and Man 8 / 9, and the results showed that high mannose glycoforms seemed to increase ADCC activity, decrease CDC activity, increase binding affinity to FcyRIIIA, and decrease binding affinity to FcyRIIA and IIB. In addition, Yu M et.al also studied the PK of antibodies with homogeneous Man 5 or Man 8 / 9 glycan in mice, and found that the clearance rate of antibodies with Man 5 and Man 8 / 9 glycoforms was 3 times faster than that of complex fucosylated glycoforms. Turner MR et.al (2012) reported that in the subcutaneous (SC) environment lacking serum a-mannosidase, the binding of antibodies with high mannose glycoforms to DC-SIGN and dectin-2 on skin dendritic cells might trigger the immunogenicity of SC administered proteins. Gorovits B et.al (2013) reported that the N-glycan on the Asn297 amino acid within the CH2 domain of the Fc region of LgG / ADC Fc region contained high mannose residues, which had a higher binding affinity to MR, and might cause MR uptake driven potential off-target toxicity.

[0005] Therefore, high content of high mannose glycoforms will increase the efficacy of antibody drugs (mainly enhance ADCC effect), but will reduce the half-life of the drug (accelerate the clearance rate in vivo) and have strong immunogenicity. In the early stage of new antibody drug development, it is generally considered acceptable to control the content of mannose glycoform Man 5 to below 10% (usually 5%). Human IgG usually contains a small amount of Man 5 glycoform, and since its impact on clearance rate, immunogenicity and efficacy is uncertain, high levels of high mannose in therapeutic antibodies can be a problem.

[0006] There are a large number of literatures focusing on methods to increase the content of high mannose glycoforms, such as adding the a-mannosidase I inhibitor kifunensine, the cell culture medium supplement raffinose, adjusting the ratio of mannose to glucose in the fed medium, and adding the Golgi pH neutralizer monensin. However, there is less available information on mechanisms for reducing the level of high mannose glycoforms, of which there are mainly four strategies as follows: first, select cell strains with high protein expression and high activity of Golgi N-acetylglucosamine transferase-I (GnTI) (i.e. low Man 5 level) during the cloning and screening stage; second, change the cell culture mode, such as using IPC (Intensified Perfusion Culture) and IFB (Intensified Fed Batch) processes, but the processes are relatively complex; third, adjust the cell culture conditions, such as low osmotic pressure and NH4 +The level is high, but its effect on reducing the high-mannose level is relatively limited; fourth, a certain amount of Mn is added during the cultivation stage. 2+ Accelerating GnTI turnover can reduce the high-mannose form, but its effectiveness may be influenced by cell line type, supplementation level, other culture conditions, and cell culture medium components. Fifth, studies have shown that shortening the culture cycle can reduce high-mannose levels, but this is accompanied by a decrease in antibody expression. Furthermore, in the development of biosimilars, developers are making significant efforts to modulate the high-mannose form to achieve biosimilarity with the original drug. Therefore, developing more strategies to reduce high-mannose levels is highly valuable. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a method for reducing the level of high-mannose glycoproteins produced by mammalian cell expression systems and its application. Specifically, it provides a method for reducing the level of high-mannose glycosylation proteins produced by mammalian cell expression systems and its application in the production and preparation of protein-based biopharmaceuticals. This method not only effectively reduces the high-mannose glycosylation level in the produced glycoproteins without affecting protein yield, cell growth and metabolism, or other quality parameters such as protein purity and pH levels, but also does not significantly alter these properties. Therefore, it is significant for the research and development and preparation of new antibody drugs.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a method for reducing the level of high-mannose glycoproteins produced by mammalian cell expression systems, the method comprising any one or a combination of two of the following:

[0010] (a) Add N-acetyl-D-mannosamine to the passage medium of the previous generation of mammalian cell expression system, culture it, and then inoculate it into the production medium and continue culturing.

[0011] (b) After the mammalian cell expression system is inoculated from the passage medium to the production medium, N-acetyl-D-mannosamine is added to the medium during the culture process.

[0012] The present application creatively discovers that by adding N-acetyl-D-mannosamine in the culture system of the mammalian cell expression system in the above two ways, the expressed protein of the mammalian cell is not affected, the level of high-mannose glycoprotein is effectively reduced, and other qualities such as protein purity, acid-alkali peak, etc. are not adversely affected. Compared with the strategy for reducing the level of high-mannose glycoprotein disclosed in the prior art, the method is simpler and easier to operate, the effect of reducing the level of high-mannose glycoprotein is more significant, and it is basically not affected by cell strain type, supplement level, other culture conditions and cell culture medium components.

[0013] Preferably, in mode (a), the N-acetyl-D-mannosamine is added and cultured for 2-4 days before inoculation into the production medium.

[0014] The 2-4 days may be, for example, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, etc., and other specific point values within this numerical range can also be selected, which will not be repeated here.

[0015] In the method of the present application, mode (a) is to add N-acetyl-D-mannosamine in the culture system when the previous generation of the mammalian cell expression system is cultured, and then inoculate into the production medium after 2-4 days of culture, and continue to be cultured in a conventional manner.

[0016] Preferably, in mode (b), the time point of adding N-acetyl-D-mannosamine is 0-11 days after inoculation into the production medium, preferably 0-7 days, more preferably 0-5 days.

[0017] The 0-11 days may be, for example, 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, etc., and other specific point values within this numerical range can also be selected, which will not be repeated here.

[0018] In the method of the present application, mode (b) is to subculture in a conventional manner, and then add N-acetyl-D-mannosamine in the culture system after 0-11 days of inoculation into the production medium, and culture the production target protein. The present application also discovers that different addition times have no obvious effect on viable cell density, cell viability, lactic acid metabolism, have no obvious effect on antibody expression amount, have no obvious effect on antibody purity, have no obvious effect on acid peak, main peak, and alkaline peak, but the effect of reducing the level of high-mannose glycoprotein is more significant when added in 0-5 days.

[0019] Preferably, in mode (a) or mode (b), the concentration of N-acetyl-D-mannosamine in the culture system is 10-100 mM, preferably 10-80 mM, more preferably 10-40 mM.

[0020] The 10-100 mM can be, for example, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, and other specific point values in the range can be selected, which will not be repeated here.

[0021] The present application also creatively finds that when the added N-acetyl-D-mannosamine concentration is 10-100 mM, the level of high-mannose glycoprotein can be reduced; when it is higher than 100 mM, the cell growth and antibody expression amount will be inhibited to a certain extent; when the concentration is lower than 10 mM, the level of high-mannose glycoprotein produced by mammalian cells cannot be significantly reduced. When the concentration is 10-40 mM, the relationship between the protein expression amount, the level of high-mannose glycoprotein, and the cell growth and metabolism state can be better balanced.

[0022] Preferably, in mode (a) or mode (b), the culture mode after inoculation into the production medium is to culture in a shaking bed at 35.0-37.0°C (for example, 35.0°C, 35.5°C, 36.0°C, 36.5°C, 37.0°C, etc.), and when the cell density reaches (10-16) × 10 6 cells / mL (for example, 10 × 10 6 cells / mL, 12 × 10 6 cells / mL, 15 × 10 6 cells / mL, etc.), the temperature is lowered to 32.5-34.5°C (for example, 32.5°C, 33.0°C, 33.5°C, 34.0°C, 34.5°C, etc.) for further culture.

[0023] Other specific point values in the above range can be selected, which will not be repeated here.

[0024] The method of the present application can better balance the relationship between the protein expression amount, the level of high-mannose glycoprotein, and the cell growth and metabolism state after the cell density reaches (10-16) × 10 6 cells / mL and the temperature is lowered to 32.5-34.5°C.

[0025] More preferably, in mode (a) or mode (b), the culture mode after inoculation into the production medium is to culture in a shaking bed at 35.5-36.5°C, and when the cell density reaches (10-16) × 10 6 cells / mL, the temperature is lowered to 33.0-34.0°C for further culture.

[0026] Preferably, the mammalian cell expression system comprises CHO cells, NS0 cells, or SP2 / 0 cells, and more preferably, CHO cells.

[0027] The mammalian cell expression system in the method of the present application is more preferably CHO cell because CHO cell has more advantages than other mammalian cells. Firstly, CHO cell itself rarely secretes endogenous protein, thus being very advantageous for the separation and purification of target protein. Secondly, CHO cell is suitable for large-scale bioreactor suspension culture. Thirdly, CHO cell is suitable for growth in a chemically defined medium to ensure the repeatability between different culture batches. Fourthly, CHO cell can produce protein with complex glycosylation and post-translational modification, which is more similar to the protein produced by human.

[0028] Preferably, the glycoprotein includes a natural glycoprotein or a glycoprotein expressed by inserting exogenous DNA into a host cell through genetic engineering.

[0029] Preferably, the glycoprotein expressed by inserting exogenous DNA into a host cell through genetic engineering includes a monoclonal antibody, a bispecific antibody, an antibody conjugated drug or an Fc fusion protein.

[0030] Preferably, the subculture medium includes CD CHO medium.

[0031] Preferably, the production medium includes ActiPro TM medium.

[0032] The subculture medium and the production medium used in the method of the present application can be achieved by using conventional subculture medium and production medium in the art.

[0033] In the present application, in the method (a) or the method (b), the inoculation into the production medium is followed by the addition of a feed medium during the culture process.

[0034] Preferably, the feed medium includes CB7a / CB7b medium.

[0035] In a second aspect, the present application provides use of the method for reducing the level of high-mannose type glycoprotein produced by a mammalian cell expression system in the production of a protein-based biological preparation according to the first aspect.

[0036] Since high content of high-mannose type will increase the efficacy of antibody (mainly enhance ADCC effect), but will reduce the half-life of the drug (accelerate the clearance rate in vivo) and have strong immunogenicity, the method for reducing the level of high-mannose type glycoprotein produced by a mammalian cell expression system of the present application can be applied in the research and development of new antibody drugs, helping to control the content of high-mannose type glycoprotein to be below 10%.

[0037] In a third aspect, the present application provides a new use of N-acetyl-D-mannosamine, i.e. the use of N-acetyl-D-mannosamine in the preparation of a reagent for reducing the level of high-mannose glycoprotein produced by a mammalian cell expression system.

[0038] There is no report in the prior art that N-acetyl-D-mannosamine is used to reduce the level of high-mannose glycoprotein, and therefore the present application also develops a new use of N-acetyl-D-mannosamine.

[0039] Preferably, the mammalian cell expression system comprises CHO cells, NS0 cells or SP2 / 0 cells, and more preferably CHO cells.

[0040] Preferably, the glycoprotein comprises a natural glycoprotein or a glycoprotein expressed by inserting foreign DNA into a host cell through genetic engineering.

[0041] Preferably, the glycoprotein expressed by inserting foreign DNA into a host cell through genetic engineering comprises a monoclonal antibody, a bispecific antibody, an antibody conjugated drug or an Fc fusion protein.

[0042] Compared with the prior art, the present application has the following beneficial effects:

[0043] The present application creatively finds that adding N-acetyl-D-mannosamine in the culture system of a mammalian cell expression system in the above two ways can effectively reduce the level of high-mannose glycoprotein without affecting the expressed protein of the mammalian cell, and will not adversely affect other qualities (such as protein purity, acid-base peak, etc.). Compared with the strategies for reducing the level of high-mannose glycoprotein disclosed in the prior art, the method is simpler and easier to operate, the effect of reducing the level of high-mannose glycoprotein is more significant, and it is basically not affected by cell strain type, supplement level, other culture conditions and cell culture medium components. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 is a graph showing the effect of different concentrations of N-acetyl-D-mannosamine on antibody expression;

[0045] Figure 2 is a graph showing the effect of different concentrations of N-acetyl-D-mannosamine on the expression level of high-mannose glycoprotein Man 5;

[0046] Figure 3 is a graph showing the effect of different concentrations of N-acetyl-D-mannosamine on Man 5 / Titer;

[0047] Figure 4 is a graph showing the effect of different concentrations of N-acetyl-D-mannosamine on viable cell density;

[0048] Figure 5 Figure 6 is a graph of the effect of different N-acetyl-D-mannosamine concentrations on cell viability;

[0049] Figure 6 Figure 7 is a graph of the effect of different N-acetyl-D-mannosamine concentrations on lactate metabolism;

[0050] Figure 7 Figure 8 is a graph of the effect of different N-acetyl-D-mannosamine addition times on antibody expression;

[0051] Figure 8 Figure 9 is a graph of the effect of different N-acetyl-D-mannosamine addition times on high-mannose glycoprotein Man 5 expression level;

[0052] Figure 9 Figure 10 is a graph of the effect of different N-acetyl-D-mannosamine addition times on Man 5 / Titer;

[0053] Figure 10 Figure 11 is a graph of the effect of different N-acetyl-D-mannosamine addition times on viable cell density;

[0054] Figure 11 Figure 12 is a graph of the effect of different N-acetyl-D-mannosamine addition times on cell viability;

[0055] Figure 12 Figure 13 is a graph of the effect of different N-acetyl-D-mannosamine addition times on lactate metabolism;

[0056] Figure 13 Figure 14 is a graph of the effect of different culture temperatures on antibody expression;

[0057] Figure 14 Figure 15 is a graph of the effect of different culture temperatures on high-mannose glycoprotein Man 5 expression level;

[0058] Figure 15 Figure 16 is a graph of the effect of different culture temperatures on Man 5 / Titer;

[0059] Figure 16 Figure 17 is a graph of the effect of different culture temperatures on viable cell density;

[0060] Figure 17 Figure 18 is a graph of the effect of different culture temperatures on cell viability;

[0061] Figure 18 Figure 19 is a graph of the effect of different culture temperatures on lactate metabolism. DETAILED DESCRIPTION

[0062] The technical solutions of the present application are further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations of the present application.

[0063] The mammalian cell expression system involved in the following content is a cell strain capable of stably expressing IgG1 monoclonal antibody, which is obtained by transfecting CHO-K1 cells with a double expression vector containing IgG1 coding genes, and the specific operation is as follows: the double expression vector contains gene sequences encoding monoclonal antibody light chain and heavy chain and different screening marker genes, and then is transfected into CHO-K1 cells by electroporation in a certain ratio, and then is passaged in a culture medium containing a screening pressure. Further, a single cell is obtained by Single Cell Printer (SCP) sorting combined with single cell imaging technology. The used CHO-K1 cell strain is preserved in the applicant's laboratory.

[0064] The production medium involved in the following content is ActiPro TM , purchased from HyClone Company; the subculture medium is CDCHO medium, purchased from Gibco Company; the feed medium is CB7a / CB7b medium, purchased from HyClone Company; and N-acetyl-D-mannosamine is purchased from New Zealand Pharmaceuticals (NZP) Company.

[0065] Preparation Example

[0066] In this preparation example, a 1M N-acetyl-D-mannosamine stock solution is prepared:

[0067] (1) A preparation container is prepared, and the weight is recorded. Purified water is added to the container;

[0068] (2) N-acetyl-D-mannosamine powder is added, and mixed for 15 min until completely dissolved;

[0069] (3) The volume is adjusted with purified water, and then filtered to remove bacteria;

[0070] (4) The prepared 1M N-acetyl-D-mannosamine stock solution is divided and stored in sterile centrifuge tubes, labeled, and stored in a -80°C refrigerator for standby.

[0071] Example 1

[0072] In this example, the effects of the addition concentration of N-acetyl-D-mannosamine on the growth and metabolism of mammalian cell expression system cells, the expression amount of antibodies, the purity of antibodies, the level of high-mannose glycoprotein Man 5, and the acid peak, main peak and basic peak are explored:

[0073] Specific operation: The mammalian cell expression system (cell strain capable of stably expressing IgG1 monoclonal antibody) was taken out from the liquid nitrogen tank, quickly thawed in a 37.0°C constant temperature bath, and suspended in a shake flask containing CD CHO medium, and then the shake flask was placed in a 36.5°C, 125 rpm shaker for culture. Then every 2-3 days, the cells were subcultured once at a density of 0.15 x 10 6 cells / mL. About 14 days later, the cells were inoculated into 250 mL shake flasks containing ActiPro 6 medium at a density of 0.40 x 10 TM cells / mL, the culture volume was 50 mL, and the shake flask was placed in a 36.5°C, 125 rpm shaker for culture. After the cell density reached about 10 x 10 6 cells / mL, it was transferred to a 33.0°C, 125 rpm shaker.

[0074] The experiment was divided into 10 groups, as shown in the table below, a total of 10 shake flasks, of which SF No.01 was used as a control group, SF No.02-07 added corresponding concentrations of N-acetyl-D-mannosamine, of which SF No.02-03 was added on the 5th day of culture, SF No.04-07 was added three times on the 5th day, 7th day, and 9th day of culture, respectively. SF No.08-10 added manganese ions (Mn 2+ ) or uridine (of which SF No.08 was added on the 0th day of culture, and SF No.09-10 was added on the 3rd day of culture), as a positive control group. On the 14th day, the antibody expression was detected and the harvest was centrifuged, then purified by Protein A, and then sent to the analysis department for quality detection. All test data were taken as 1 for the control group SF No.01.

[0075] SF No. N-acetyl-D-mannosamine (mM) Manganese ions (μΜ) Uridine (mM) 01 0 - - 02 10 - - 03 20 - - 04 40 - - 05 60 - - 06 80 - - 07 100 - - 08 - 5 - 09 - - 5 10 - - 10

[0076] The test results are as follows:

[0077] (1) The antibody expression results are shown in Table 1 and Figure 1 , the addition of 10-40 mM N-acetyl-D-mannosamine (ManNAc) had no effect on antibody expression (Titer), and the addition of 60-100 mM N-acetyl-D-mannosamine resulted in a 14%-20% reduction in antibody expression. 5 μM manganese ions or 5 mM uridine had no effect on antibody expression, but 10 mM uridine affected antibody expression, reducing antibody expression by 21%.

[0078] Table 1

[0079]

[0080] (2) High mannose glycoprotein Man 5 expression level results as shown in Table 2, Figure 2 , Figure 3 addition of 10-100 mM N-acetyl-D-mannosamine can reduce the level of Man 5, wherein the concentration of 20 mM-100 mM can reduce Man 5 by 44%-65%. In addition, after the addition of N-acetyl-D-mannosamine, G0F is significantly improved, it is speculated that N-acetyl-D-mannosamine as a precursor of N-acetylneuraminic acid / sialic acid (Neu5Ac), after addition, promotes the synthesis of glycoforms in the direction of maturity, thereby reducing the level of Man 5. 5 μM manganese ions or 5 mM uridine cannot effectively reduce Man 5, and the addition of 10 mM uridine can reduce Man 5 by 36%.

[0081] Table 2

[0082]

[0083]

[0084] Note #1: The absolute value of Man 5 of SF No. 01 is 7.7%.

[0085] (3) Antibody purity results are shown in Table 3. From the results of SDS-Caliper-NR and SEC-UPLC, the addition of different concentrations of N-acetyl-D-mannosamine has no effect on antibody purity.

[0086] Table 3

[0087]

[0088]

[0089] (4) The iCIEF results of each group are shown in Table 4. From the iCIEF results, the addition of different concentrations of N-acetyl-D-mannosamine has no effect on acidic peaks, main peaks, and basic peaks.

[0090] Table 4

[0091]

[0092] (5) The effects on viable cell density, cell viability, and lactate metabolism are shown in Figure 4 , Figure 5 , Figure 6 From the aspects of cell growth and metabolism, compared with the control group, the addition of 10-100 mM N-acetyl-D-mannosamine has no effect on viable cell density, cell viability, and lactate metabolism. The addition of 10 mM uridine can inhibit cell growth.

[0093] In summary, the addition of 10-100 mM N-acetyl-D-mannosamine can reduce the level of Man 5; when the concentration is controlled at 10-40 mM, it has no effect on antibody expression; when the concentration is at 20-40 mM, it can significantly reduce Man 5 and maintain the antibody expression at a level comparable to the control group. After adding 100 mM uridine, Man 5 is reduced by 62%, but the antibody expression is reduced by 20%.

[0094] Example 2

[0095] This example investigates the effect of the addition time point of N-acetyl-D-mannosamine on the growth and metabolism of mammalian cell expression system, antibody expression, antibody purity, high mannose glycoprotein Man 5 level, and the effects of acidic peak, main peak, and basic peak:

[0096] Specific operation: The mammalian cell expression system (cell strain capable of stably expressing IgG1 monoclonal antibody) was taken out from the liquid nitrogen tank, quickly thawed in a 37.0°C constant temperature bath, and suspended in a shake flask containing CD CHO medium, then the shake flask was placed in a 36.5°C, 125 rpm shaker for culture. Then every 2-3 days, the cells were passaged once at a density of 0.15×10 6 cells / mL. After about 14 days, the cells were inoculated into 250 mL shake flasks containing ActiPro 6 medium at a density of 0.40×10 TM cells / mL, the culture volume was 50 mL, and the shake flask was placed in a 36.5°C, 125 rpm shaker for culture. After the cell density reached about 10×10 6 cells / mL, it was transferred to a 33.0°C, 125 rpm shaker.

[0097] The experiment was divided into 5 groups, as shown in the table below, a total of 5 shake flasks, of which SF No.01 was used as the control group, SF No.02 was added at N-1 stage, and inoculated into ActiPro TM medium after three days, SF No.03-05 were inoculated into ActiPro TM medium and added with 20 mM N-acetyl-D-mannosamine at 0th day, 5th day, and 11th day of culture, respectively. At 14th day, the antibody expression was detected, and after centrifugation of the harvest, Protein A one-step purification was performed, and then the quality detection was performed by the analysis department. All test data were taken as 1 for the control group SF No.01.

[0098]

[0099] The test results are as follows:

[0100] (1) The antibody expression levels are shown in Table 5 and Figure 7 As shown, different addition times had no significant effect on antibody expression levels.

[0101] Table 5

[0102]

[0103] (2) The expression levels of the high-mannose glycoprotein Man 5 are shown in Table 6 and Figure 8 , Figure 9 As shown, during the N-1 stage of seed culture, adding 20 mM N-acetyl-D-mannosamine on day 0 or day 5 after inoculation effectively reduced Man 5 levels by 41%-65%. However, adding it on day 11 after inoculation did not significantly reduce Man 5 levels.

[0104] Table 6

[0105]

[0106] Note #1: The absolute value of Man 5 for SF No.01 is 6.6%.

[0107] Table 2 shows that the overall high-mannose pattern is consistent with the trend of Man 5 levels. The cells used in Examples 1 and 2 were the same clones, thus predicting the overall high-mannose pattern in Table 6 and the trend of Man 5 levels.

[0108] The trends at level 5 are consistent.

[0109] (3) The antibody purity results are shown in Table 7. Different addition times have no effect on antibody purity.

[0110] Table 7

[0111]

[0112] (4) The iCIEF results of each group are shown in Table 8. From the iCIEF results, it can be seen that the addition of N-acetyl-D-mannosamine at different time points has no effect on the acid peak, the main peak, or the basic peak.

[0113] Table 8

[0114]

[0115] (5) The results of the effects on live cell density, cell viability, and lactate metabolism are as follows: Figure 10 , Figure 11 , Figure 12 As shown, from the perspective of cell growth and metabolism, compared with the control group, different N-acetyl-D-mannosamine addition times had no significant effect on cell density, cell viability, and lactic acid metabolism.

[0116] In summary, the addition of 20 mM N-acetyl-D-mannosamine at different stages did not significantly affect antibody expression levels (within 10% change) compared to the control group. When the addition of N-acetyl-D-mannosamine was at the time of inoculation into the passage medium, at day 0 after inoculation, at day 5 after inoculation, it could significantly reduce the level of Man 5, but when added at day 11 of culture, it could not significantly reduce the level of Man 5.

[0117] Example 3

[0118] This example investigates the effects of cell culture temperature on cell growth and metabolism, antibody expression levels, antibody purity, high mannose glycoprotein Man 5 levels, and acidic peak, main peak, and basic peak after the addition of N-acetyl-D-mannosamine:

[0119] Specific operation: The mammalian cell expression system (cell strain capable of stably expressing IgG1 monoclonal antibody) was taken out from the liquid nitrogen tank, quickly thawed in a 37.0°C constant temperature bath, and suspended in a shake flask containing CD CHO medium, then the shake flask was placed in a 36.5°C, 125 rpm shaker for culture. Then every 2-3 days, the cells were passaged once at a density of 0.15 x 10 6 cells / mL. After about 14 days, the cells were inoculated into 250 mL shake flasks containing ActiPro 6 medium at a density of 0.40 x 10 TM cells / mL, the culture volume was 50 mL, and the culture was carried out in a 36.5 or 35.5°C, 125 rpm shaker. After the cell density reached about 10 x 10 6 cells / mL, it was transferred to a 33.0°C, 31.0°C, 29.0°C, 34.0°C, or 35.0°C, 125 rpm shaker.

[0120] The experiment was divided into 5 groups, as shown in the table below, a total of 5 shake flasks, of which No. SF No. 01 was the control group, without the addition of N-acetyl-D-mannosamine, SF No. 02-05 were all added with 20 mM N-acetyl-D-mannosamine at the 5th day of inoculation into ActiPro TM medium. On day 14, the antibody expression levels were detected and the harvest was centrifuged, then purified by Protein A one-step purification, and then sent to the analysis department for quality detection. All test data were taken as 1 for the control group SF No. 01.

[0121]

[0122]

[0123] Note: According to the platform historical experience, the initial culture temperature of 36.5°C is reduced to 34.0°C or above, which has a high probability to reduce the cell density and cell viability in the later stage, thereby reducing the antibody expression. Therefore, this condition is not investigated in this example.

[0124] The test results are as follows:

[0125] (1) The antibody expression results are shown in Table 9 and Figure 13 As shown in Table 9 and

[0126] Table 9

[0127]

[0128] (2) The high-mannose glycoprotein Man 5 expression level results are shown in Table 10 and Figure 14 , Figure 15 As shown in Table 10 and

[0129] Table 10

[0130]

[0131] Note #1: The absolute value of Man 5 of SF01 is 6.6%.

[0132] As can be seen from Table 2, the total high-mannose type and Man 5 level trends are consistent, and the cells used in Example 1, Example 2, and Example 3 are the same clone. Therefore, it is predicted that the total high-mannose type and Man 5 level trends are consistent in Table 10.

[0133] (3) The antibody purity results are shown in Table 11. From the SDS-Caliper-NR and SEC-UPLC results, different culture temperatures have no effect on the purity of the antibody. Because the expression amount of SF No.04 antibody is too low, the sample is not sent for quality analysis.

[0134] Table 11

[0135]

[0136] (4) iCIEF results of each group are shown in Table 12. From the iCIEF results, different culture temperatures have no effect on acidic peaks, main peaks, and basic peaks. SF No. 04 antibody expression is too low, and quality analysis is not performed.

[0137] Table 12

[0138]

[0139] (5) The effects of viable cell density, cell viability, and lactic acid metabolism are shown in Tables 13, 14, and 15, respectively. From the cell growth and metabolism, compared with the control group, different N-acetyl-D-mannosamine addition times have no significant effect on viable cell density, cell viability, and lactic acid metabolism. Figure 16 、 Figure 17 、 Figure 18

[0140] In summary, compared with no addition of N-acetyl-D-mannosamine, in the condition of adding 20 mM N-acetyl-D-mannosamine, the initial temperature is 35.5-36.5°C, and the temperature is reduced to 33.0-34.0°C, the antibody expression is consistent with the control, and Man 5 is significantly reduced.

[0141] The applicant states that the present application is described by the above examples to illustrate a method for reducing the level of high-mannose glycoprotein produced by a mammalian cell expression system and its application, but the present application is not limited to the above examples, that is, it does not mean that the present application must rely on the above examples to be implemented. Those skilled in the art should understand that any improvement on the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. all fall within the protection scope and disclosure scope of the present application.

[0142] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above embodiments. Within the technical concept scope of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0143] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined by any suitable means without contradiction. In order to avoid unnecessary repetition, the present application does not further describe various possible combination manners.​

Claims

1. A method for reducing the level of high mannose type glycoprotein Man 5 produced by a mammalian cell expression system, characterized in that, The method comprises any one of the following modes or a combination of both: (a) adding N-acetyl-D-mannosamine to the subculture medium of the previous generation of the mammalian cell expression system before inoculation, culturing, and then inoculating into a production medium for further culturing; (b) adding N-acetyl-D-mannosamine to the culture medium during the culturing process after inoculating the mammalian cell expression system from the subculture medium into the production medium; In mode (b), the time point of adding N-acetyl-D-mannosamine is 0-5 days after inoculation into the production medium; In mode (a) or mode (b), the concentration of N-acetyl-D-mannosamine in the culturing system is 20-40 mM; In the method (a) or the method (b), the culture method after inoculation into the production medium is culturing in a 35.0-37.0℃ shaker, and when the cell density reaches (10-16) ×10 6 cells / mL, the temperature is lowered to 32.5-34.5℃ for continued culturing.

2. The method for reducing the level of Man 5 of high mannose type glycoprotein produced by a mammalian cell expression system according to claim 1, wherein, In mode (a), the culturing after adding N-acetyl-D-mannosamine is 2-4 days before inoculation into the production medium.

3. The method for reducing the level of Man 5 of high mannose type glycoprotein produced by a mammalian cell expression system according to claim 1, wherein, In the method (a) or the method (b), the culture method after inoculation into the production medium is culturing in a 35.5-36.5℃ shaker, and when the cell density reaches (10-16) ×10 6 cells / mL, the temperature is lowered to 33.0-34.0℃ for continued culturing.

4. The method for reducing the level of Man 5 of high mannose type glycoprotein produced by a mammalian cell expression system according to claim 1, wherein, The mammalian cell expression system comprises CHO cells, NS0 cells, or SP2 / 0 cells.

5. The method for reducing the level of Man 5 of high mannose type glycoprotein produced by a mammalian cell expression system according to claim 4, wherein, The mammalian cell expression system is CHO cells.

6. The method for reducing the level of Man 5 of high mannose type glycoprotein produced by a mammalian cell expression system according to claim 1, wherein, The glycoprotein comprises a natural glycoprotein or a glycoprotein expressed by inserting foreign DNA into a host cell through genetic engineering.

7. The method for reducing the level of Man 5 of high mannose type glycoprotein produced by a mammalian cell expression system according to claim 6, wherein, The glycoprotein expressed by inserting foreign DNA into a host cell through genetic engineering comprises a monoclonal antibody, a bispecific antibody, an antibody conjugated drug, or an Fc fusion protein.

8. The method for reducing the level of Man 5 of high mannose type glycoprotein produced by a mammalian cell expression system according to claim 1, wherein, The subculture medium comprises CD CHO medium.

9. The method for reducing the level of Man 5 of high mannose type glycoprotein produced by a mammalian cell expression system according to claim 1, wherein, The production medium comprises ActiPro™ medium.

10. The method for reducing the level of Man 5 of high mannose type glycoprotein produced by a mammalian cell expression system according to claim 1, wherein, In mode (a) or mode (b), a feed medium is supplemented during the culturing process after inoculation into the production medium.

11. The method for reducing the level of Man 5 of high mannose type glycoprotein produced by a mammalian cell expression system according to claim 10, wherein, The feed medium comprises CB7a / CB7b medium.

12. Use of the method for reducing the level of high-mannose glycoprotein Man 5 produced by a mammalian cell expression system in the production of an antibody.

13. Use of N-acetyl-D-mannosamine in the preparation of a preparation for reducing the level of high-mannose glycoprotein Man 5 produced by a mammalian cell expression system; The use comprises any one of the following modes or a combination of both: (a) adding N-acetyl-D-mannosamine to the subculture medium of the previous generation of the mammalian cell expression system before inoculation, culturing, and then inoculating into a production medium for further culturing; (b) adding N-acetyl-D-mannosamine to the culture medium during the culturing process after inoculating the mammalian cell expression system from the subculture medium into the production medium; In mode (b), the time point of adding N-acetyl-D-mannosamine is 0-5 days after inoculation into the production medium; In mode (a) or mode (b), the concentration of N-acetyl-D-mannosamine in the culturing system is 20-40 mM; In the method (a) or the method (b), the culture method after inoculation into the production medium is culturing in a 35.0-37.0℃ shaker, and when the cell density reaches (10-16) ×10 6 cells / mL, the temperature is lowered to 32.5-34.5℃ for continued culturing.

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