Method for regulating and controlling high-polymannose type in antibody glycoform and application of method

Abstract

The invention relates to the technical field of biology, in particular to a method for regulating and controlling polymannose in antibody glycoforms and application of the method. The method comprises the following steps: inoculating cells expressing an antibody into a culture medium for culturing, and regulating and controlling the glycoform of the antibody in the culture process; the method for regulating and controlling the glycoform of the antibody comprises the following steps: during culture, adding a pH regulator to control the pH of a culture system; when the cells expressing the antibody grow to a plateau phase, supplementing a carbon source into a culture system; the pH regulator comprises lactic acid; the carbon source comprises lactic acid. The method can effectively reduce the proportion of high polymannose in the antibody glycoform, the pH regulator and the carbon source are easy to purchase, the method is simple and easy to operate, the concentration of the pH regulator and the carbon source and the addition amount of the pH regulator and the carbon source can be properly adjusted according to requirements, and the method is suitable for regulation and control of mannosylation in the antibody drug development process. Especially the proportion of Man 5.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular, to a method for regulating high-mannose type in antibody glycoforms and its application. Background Art

[0002] In the field of biopharmaceuticals, antibody drugs are widely used in disease treatment. High-mannose type is a common immature glycoform in antibody N-glycosylation modification. The high-mannose type expressed by CHO cells in antibodies is mainly penta-mannose type (Man5). A high proportion of Man5 increases the clearance efficiency of antibodies in the human body, thereby reducing the residence time of the drug in the human body, increasing the dosing frequency or requiring an increase in the dosing dose, weakening the competitiveness of the drug in the market. Therefore, the Man5 level is often an important quality parameter of antibody drugs, and reducing the Man5 level is often the goal pursued in the development of new drugs or biosimilars.

[0003] In view of this, the present invention is specifically proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for regulating high-mannose type in antibody glycoforms and its application. The method for regulating high-mannose type in antibody glycoforms is suitable for the regulation of mannosylation during the development of antibody drugs, especially the proportion of Man5.

[0005] In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:

[0006] One aspect of the present invention relates to a method for regulating high-mannose type in antibody glycoforms, comprising the following steps:

[0007] Inoculate cells expressing an antibody into a culture medium for cultivation, and regulate the antibody glycoform during the cultivation process;

[0008] The method for regulating the antibody glycoform includes: when performing the cultivation, adding a pH regulator to control the pH of the culture system; and / or, when the cells expressing the antibody grow to the plateau phase, supplementing a carbon source to the culture system;

[0009] Wherein, the pH regulator includes: lactic acid; the carbon source includes: lactic acid.

[0010] The method for regulating high-mannose type in antibody glycoforms can effectively reduce the proportion of high-mannose type in antibody glycoforms, and the used pH regulator and carbon source are easy to purchase. This method is simple and easy to operate, and the concentration of the added pH regulator and carbon source, as well as the addition amount of the pH regulator and carbon source, can be appropriately adjusted according to needs. It is suitable for the regulation of mannosylation during the development of antibody drugs, especially the proportion of Man5.

[0011] Another aspect of the present invention also relates to a method for preparing a protein drug, which includes the method for regulating high-mannose in the antibody glycoform as described above.

[0012] In the method for preparing the protein drug, the glycoform ratio of the prepared antibody drug is reasonable, especially the ratio of high-mannose decreases.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] The method for regulating high-mannose type in the antibody glycoform provided by the present invention can reasonably regulate high-mannose type in the antibody glycoform by adding lactic acid as an additional carbon source and pH regulator during the cell culture process for expressing the protein, in combination with adjusting the culture temperature. During the normal cell culture process, lactic acid is also one of the metabolic intermediates and can be metabolized by cells subsequently, having no obvious impact on cell growth and metabolism, but can achieve the effect of reducing the ratio of high-mannose. The lactic acid used in the present invention is easy to purchase, the provided method is simple and easy to operate, and the addition concentration can be appropriately adjusted according to requirements, being suitable for the regulation of high-mannosylation during the drug development process, especially the ratio of Man 5. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0016] Figure 1 It is the viable cell density diagram of Example 1;

[0017] Figure 2 It is the viable cell density diagram of Example 2;

[0018] Figure 3 It is the viable cell density diagram of Example 3;

[0019] Figure 4 It is the viable cell density diagram of Example 4;

[0020] Figure 5 It is the viable cell density diagram of Example 5;

[0021] Figure 6 It is the lactic acid metabolism diagram of Example 1;

[0022] Figure 7 It is the lactic acid metabolism diagram of Example 4;

[0023] Figure 8 It is the lactic acid metabolism diagram of Example 5. Detailed implementation mode

[0024] The technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific implementation modes. However, those skilled in the art will understand that the following described embodiments are part of the embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0025] One aspect of the present invention relates to a method for regulating high-mannose type in antibody glycans, including the following steps:

[0026] Inoculate the cells expressing the antibody into a culture medium for culture, and regulate the antibody glycan during the culture process;

[0027] The method for regulating the antibody glycan includes: adding a pH regulator to control the pH of the culture system during the culture; and / or, when the cells expressing the antibody grow to the plateau phase, supplementing a carbon source to the culture system;

[0028] Wherein, the pH regulator includes: lactic acid; the carbon source includes: lactic acid.

[0029] The method for regulating high-mannose type in antibody glycans can effectively reduce the proportion of high-mannose type in antibody glycans, and the used pH regulator and carbon source are easy to purchase. This method is simple and easy to operate, and the concentration of the added pH regulator and carbon source, as well as the addition amount of the pH regulator and carbon source, can be appropriately adjusted according to needs, and is suitable for the regulation of mannosylation during the development of antibody drugs, especially the proportion of Man 5.

[0030] The method for regulating high-mannose type in antibody glycans controls the pH of the culture system by using a pH regulator cascade during the culture process of cells expressing the antibody, so as to regulate the pH of the cell culture medium in the cell culture while regulating the antibody glycan.

[0031] The method for regulating the high-mannose type in antibody glycoforms can achieve the effect of reducing high-mannose by supplementing the carbon source to the culture system when the cells grow to the plateau phase and start to consume the carbon source in the culture system. The regulation of glycoforms is mainly achieved by adding lactic acid to prevent the cells from consuming too much lactic acid during the culture process. Usually, after the cells grow to the plateau phase, the consumption of lactic acid will increase. If the consumption of lactic acid is too much, the cells will tend to generate pyruvate from alanine through the action of alanine dehydrogenase, and ammonium ions will be produced in this process. High levels of ammonium ions may cause an increase in Man5 and the production of high-mannose. Supplementing lactic acid after the plateau phase aims to maintain a dynamic equilibrium in this process, as high levels of ammonium ions may cause the production of high-mannose type antibodies.

[0032] Further, adding the pH regulator controls the pH of the culture system to 6.60 - 7.30, including but not limited to any point value among 6.60, 6.70, 6.80, 6.90, 7.10, 7.20, or 7.30 or the range value between any two of them. The culture pH is determined according to the growth characteristics of the cells expressing the antibody.

[0033] Further, when the cells expressing the antibody grow to the plateau phase, after 1 day of supplementing the carbon source to the culture system, the carbon source content in the culture system ≥ 0.15 g / L, including but not limited to any point value among 0.15 g / L, 0.35 g / L, 0.55 g / L, 0.75 g / L, 0.95 g / L, 1.05 g / L, or 1.25 g / L or the range value between any two of them. When the cells grow to the plateau phase, they start to consume lactic acid in the system, and at this time, supplementing a certain amount of carbon source to the culture system is beneficial for regulating the antibody glycoform.

[0034] Further, the culture method of the cells expressing the antibody includes any one of batch culture, fed-batch culture, or perfusion culture.

[0035] Further, when the fed-batch culture method is adopted, when the cells expressing the antibody grow to the plateau phase and the lactic acid content in the culture system is detected to be < 1.0 g / L (such as any point value among 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, or 0.99 g / L or the range value between any two of them), the carbon source is supplemented to the culture system.

[0036] Further, when the perfusion culture method is adopted, after the cells expressing the antibody grow to the plateau phase, the carbon source is supplemented to the culture system after each medium change.

[0037] In some specific embodiments, the perfusion culture includes the following steps:

[0038] The cells expressing the antibody are cultured by perfusion in a shake flask, and when the cell density reaches (4.0 - 8.0)×10 6 Cells / mL and is greater than 8.0×10 6 Cells / mL, centrifugal medium exchange is performed using the growth medium. After the medium exchange, the carbon source is supplemented into the culture system. One day after the carbon source is supplemented, the content of the carbon source in the culture system is ≥0.5 g / L.

[0039] Further, the conditions for centrifugal medium exchange are centrifugation at 1000 rpm for 5 min, and the medium exchange volume is 10 mL.

[0040] In some specific embodiments, the fed-batch culture includes the following steps:

[0041] The cells expressing the antibody are cultured by fed-batch in a shake flask. When the cells expressing the antibody grow to the plateau phase, the carbon source is supplemented into the culture system. One day after the carbon source is supplemented, the content of the carbon source in the culture system is ≥0.15 g / L.

[0042] In some specific embodiments, the fed-batch culture includes the following steps:

[0043] The cells expressing the antibody are cultured by fed-batch in a bioreactor. During the culture process, the pH regulator is controlled in relation to the pH of the culture system. When the pH of the culture system exceeds the upper limit of the set value of 7.30, the pH regulator is automatically added to lower the pH value.

[0044] Further, when the fed-batch culture method is adopted, the method for regulating the glycoform of the antibody further includes: when the cell density of the cells expressing the antibody is ≥13.0×10 6 Cells / mL, the culture temperature is adjusted to 31.0 - 35.0 °C (for example, it can be but is not limited to any one of the point values of 31.0 °C, 32.0 °C, 33.0 °C, 34.0 °C or 35.0 °C or the range values between any two of them).

[0045] High-mannose glycans are the result of imperfect protein modification in cells. Generally, when the temperature is lowered, the cells produce more protein. At this time, if the temperature is increased, the protein synthesis rate is slowed down, thus providing time for sufficient protein modification, and therefore reducing the proportion of high-mannose glycans of the protein.

[0046] Further, before adjusting the culture temperature, CO 2 is used to regulate the pH of the culture system, and after adjusting the culture temperature, the pH regulator is used to regulate the pH of the culture system.

[0047] Further, the cells expressing the antibody include, but are not limited to: CHO cells. The CHO cells involved in the present invention refer to Chinese hamster ovary cells.

[0048] Further, the culture time of the cells expressing the antibody is 10 to 30 days, including but not limited to any point value among 10 days, 15 days, 20 days, 25 days or 30 days or the range value between any two of them.

[0049] Further, the initial culture temperature of the cells expressing the antibody is 34.5 to 37 °C, including but not limited to any point value among 34.5 °C, 35 °C, 35.5 °C, 36 °C, 36.5 °C or 37 °C or the range value between any two of them.

[0050] Further, when culturing the cells expressing the antibody by fed-batch culture method, the initial culture temperature is 36 to 37 °C.

[0051] Further, when culturing the cells expressing the antibody by perfusion culture method, the initial culture temperature is 34.5 to 35.5 °C.

[0052] In the present invention, the inoculation day is the 0th day, and the subsequent days are postponed accordingly.

[0053] In the present invention, a 250 mL shake flask is used to culture the cells, and the fed-batch method is adopted for culture.

[0054] Further, the culture volume of the shake flask is 60 mL.

[0055] Another aspect of the present invention also relates to a method for preparing a protein drug, including the method for regulating the high-mannose oligosaccharide in the antibody glycoform.

[0056] For the method for preparing the protein drug, the glycoform ratio of the prepared antibody drug is reasonable, especially the ratio of high-mannose oligosaccharide decreases.

[0057] The culture medium involved in the present invention is as follows:

[0058] Basal medium: Add 4 mM glutamine to CHO CD04, or add 4 mM glutamine to Actipro;

[0059] The feeding medium includes: Cell Boost 7a and Cell Boost 7b;

[0060] Growth medium: Add 4 mM glutamine to ExcelPro CHO Perfusion Growth Medium 01;

[0061] Production medium: 4 mM glutamine was added to ExcelPro CHO Perfusion Production Medium 01.

[0062] The embodiments of the present invention will be described in detail below in conjunction with the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained commercially.

[0063] Example 1

[0064] The method for regulating the high-mannose type in the antibody glycoform provided in this example, during the cell culture process of expressing monoclonal antibodies, adopts the fed-batch culture method. The control group uses CO 2 to regulate the pH of the culture system, and the experimental group uses diluted lactic acid (Merck, product number 252476) to regulate the pH of the culture system, including the following steps:

[0065] (1) Using CHO CD04 + 4 mM glutamine as the basal medium and Cell Boost 7a and Cell Boost 7b as the feeding media;

[0066] (2) Inoculate CHO cells into a 3-L reactor at an inoculation density of 0.5×10 6 Cells / mL. The culture volume of the reactor is 1.5 L, and the inoculation medium is CHO CD04 + 4 mM glutamine.

[0067] (3) The initial cell culture temperature is 36.5 °C, the stirring speed is 350 rpm, and CO is introduced into the culture system 2 to regulate the pH of the culture system to be maintained within the range of 6.90 ± 0.30, and the DO is controlled at 50%;

[0068] (4) When the cell density reaches 15.0×10 6 Cells / mL, adjust the culture temperature, cool down to 32.0 °C. In the experimental group, cancel CO 2 to regulate the pH of the culture system, and use a lactic acid solution with a concentration of 300 g / kg to regulate the pH of the culture system to be maintained within the range of 6.90 ± 0.30. When the pH is higher than 7.20, the lactic acid solution is slowly added to the reactor until the pH drops within the set range, and then stop adding the lactic acid solution. Repeat this process. The addition amount of the 300 g / kg lactic acid solution at the end of the experiment is more than 5 g;

[0069] (5) Feeding was carried out on the 3rd, 5th, 7th, 10th and 12th days, with the feeding amount of Cell Boost 7a being 4.0% and the feeding amount of Cell Boost 7b being 0.4%;

[0070] (6) Cells were harvested on the 14th day of culture, and antibody protein purification was carried out after centrifugation.

[0071] (7) The glycan quality of the antibody protein was determined.

[0072] It can be seen from Figure 1 that in the fed-batch culture of this example, the cells were not significantly affected after the addition of lactic acid before the 11th day. After the 11th day, the addition of lactic acid slowed down the cell growth, but did not change the cell growth trend.

[0073] It can be seen from Figure 6 that using lactic acid cascade pH, the residual lactic acid content in the culture system can be increased and the pH can be decreased during the culture process.

[0074] Example 2

[0075] The method for regulating the high-mannose type in the antibody glycan provided in this example adopts a perfusion culture method during the cell culture process for expressing bispecific antibodies. The control group was cultured normally. On the basis of the control group, when the cells grew to the plateau phase, a carbon source was supplemented to the culture system. The specific steps are as follows:

[0076] (1) ExcelPro CHO Perfusion Growth Medium 01 + 4 mM glutamine was used as the growth medium, and ExcelPro CHO Perfusion Production Medium 01 + 4 mM glutamine was used as the production medium;

[0077] (2) CHO cells were inoculated into a 50 mL shake flask at an inoculation density of 2.0×10 6 Cells / mL. The culture volume of the shake flask was 10 mL, and the inoculation medium was the growth medium;

[0078] (3) The temperature of the shaker for cell culture was 35.0 °C, the shaker speed was 190 rpm, the orbital diameter was 50 mm, the CO 2 concentration was 5.0%, and the tilt angle was 70 degrees;

[0079] (4) When the cell culture density reached 4.0 - 8.0×10 6 Cells / mL, at this time the cells were in the logarithmic growth phase, and the growth medium was used for centrifugal medium exchange. Centrifugation was carried out at 1000 rpm for 5 minutes, and the medium exchange volume was 10 mL;

[0080] (5) When the cell culture density is greater than 8.0×10 6 Cells / mL, centrifugation and medium replacement are carried out using the production medium. Centrifuge at 1000 rpm for 5 minutes, and the medium replacement volume is 10 mL;

[0081] (6) In the experimental group, a carbon source (lactic acid solution) with a concentration of 300 g / kg is supplemented to the culture system every day after centrifugation and medium replacement. At this time, the cells are in the plateau phase, so that the lactic acid residue amount on the next day is ≥0.5 g / L, and the total addition amount of the 300 g / kg lactic acid solution after the culture is completed is 0.13 g; the control group does not add lactic acid solution;

[0082] (7) Harvest the cells after culturing for 14 days, and carry out antibody protein purification after centrifugation.

[0083] (8) Measure the glycan quality of the antibody protein.

[0084] It can be seen from Figure 2 that during the perfusion culture process of this example, adding lactic acid has no significant effect on cell growth.

[0085] Example 3

[0086] The method for regulating high-mannose type in antibody glycan provided in this example adopts fed-batch culture during the cell culture process of expressing bispecific antibody. The control group is cultured normally. On the basis of the control group, in the experimental group, when the cells are cultured to a certain number, the culture temperature is adjusted, and a carbon source is supplemented to the culture system. The specific steps are as follows:

[0087] (1) Use Actipro + 4 mM glutamine as the basal medium, and Cell Boost 7a and Cell Boost 7b as the feeding media;

[0088] (2) Inoculate CHO cells into a 250 mL shake flask at an inoculation density of 0.5×10 6 Cells / mL. The culture volume in the shake flask is 60 mL, and the inoculation medium is Actipro + 4 mM glutamine;

[0089] (3) The initial cell culture temperature is 36.5 °C, the shaker speed is 110 rpm, the orbital diameter is 50 mm, and the CO 2 concentration is 5.0%;

[0090] (4) When the cell density reaches 13.0×10 6 Cells / mL, adjust the culture temperature and cool it down to 32.0 °C;

[0091] (5) Feeding was carried out on the 3rd, 5th, 7th and 10th days, with the feeding amount of Cell Boost 7a being 4.0% and the feeding amount of Cell Boost 7b being 0.4%; feeding was carried out on the 12th day, with the feeding amount of Cell Boost 7a being 5.0% and the feeding amount of Cell Boost 7b being 0.5%;

[0092] (6) The lactic acid content was detected. In the experimental group, a carbon source (lactic acid solution) with a concentration of 300 g / kg was supplemented into the culture system after cooling. At this time, the cells were in the plateau phase, so that the residual lactic acid amount on the next day was ≥ 0.15 g / L, and the total addition amount of the 300 g / kg lactic acid solution was 0.37 g after the culture ended. The control group did not perform any operations;

[0093] (7) The cells were harvested on the 14th day of culture, and antibody protein purification was carried out after centrifugation;

[0094] (8) The glycan quality of the antibody protein was determined.

[0095] It can be seen from Figure 3 that during the fed-batch culture process of this example, adding lactic acid would slightly slow down the late-stage cell growth rate.

[0096] Example 4

[0097] The method for regulating the high-mannose type in the antibody glycan provided in this example adopts fed-batch culture during the cell culture process for expressing bispecific antibodies. The control group was cultured normally. On the basis of the control group, the experimental group added a pH regulator to regulate the pH to the culture system, and at the same time, both the control group and the experimental group supplemented a carbon source to the culture system when the cells grew to the plateau phase. The specific steps are as follows:

[0098] (1) Actipro + 4 mM glutamine was used as the basal medium, and Cell Boost 7a and Cell Boost 7b were used as the feeding media;

[0099] (2) CHO cells were inoculated into a 3-L reactor at an inoculation density of 0.5 × 10 6 Cells / mL. The culture volume of the reactor was 1.5 L, and the inoculation medium was Actipro + 4 mM glutamine;

[0100] (3) The initial temperature for cell culture was 36.5 °C, the stirring speed was 350 rpm, the pH range was 6.90 ± 0.25, and the DO was controlled at 50%. The control group introduced CO 2 into the culture system to regulate the pH of the culture system, and the experimental group used a 120 g / kg lactic acid solution as a pH regulator to regulate the pH of the culture system;

[0101] (4) When the cell density reached 14.0 × 106 Adjust the culture temperature to 32.0 °C when the cell density reaches [[X]] cells / mL. When the pH of the experimental group is higher than 7.15, a 120 g / kg lactic acid solution is slowly added to the reactor until the pH drops within the set range, then stop adding the 120 g / kg lactic acid solution, and repeat this cycle;

[0102] (5) Feed supplements on the 3rd, 5th, 7th, and 10th days. The feeding amount of Cell Boost 7a is 4.0%, and the feeding amount of Cell Boost 7b is 0.4%. Feed supplements on the 12th day. The feeding amount of Cell Boost 7a is 5.0%, and the feeding amount of Cell Boost 7b is 0.5%;

[0103] (6) Detect the lactic acid content. When the lactic acid content is lower than 1.0 g / L, supplement the culture system with a carbon source (lactic acid solution) with a concentration of 120 g / kg. At this time, the cells are in the stationary phase, so that the residual lactic acid amount on the next day is ≥ 0.15 g / L. After the culture ends, the total addition amount of the 120 g / kg lactic acid solution in the control group is 6.5 g, and the total addition amount of the 120 g / kg lactic acid solution in the experimental group is 44.5 g;

[0104] (7) Harvest the cells after culturing for 14 days, and perform antibody protein purification after centrifugation;

[0105] (8) Determine the glycoform quality of the antibody protein.

[0106] It can be seen from Figure 4 that during the fed-batch culture process of this example, adding lactic acid will slightly slow down the cell growth, but will not change the cell growth trend.

[0107] It can be seen from Figure 7 that during the culture process, the more lactic acid remains in the culture system, the lower the pH.

[0108] Example 5

[0109] The method for regulating the high-mannose type in the antibody glycoform provided in this example adopts fed-batch culture during the cell culture process of expressing bispecific antibodies. The control group is cultured normally, and a 120 g / kg lactic acid solution is used as a pH regulator to control the pH of the culture system. At the same time, a carbon source is supplemented when the cells grow to the stationary phase. The experimental group adjusts the temperature after cooling on the basis of the control group. The specific steps are as follows:

[0110] (1) Use Actipro + 4 mM glutamine as the basal medium and Cell Boost 7a and Cell Boost 7b as the feeding media;

[0111] (2) At a seeding density of 0.5×10 6Inoculate CHO cells at a density of Cells / mL into a 3-L bioreactor with a culture volume of 1.5 L. The inoculation medium is Actipro + 4 mM glutamine;

[0112] (3) The temperature for initial cell culture is 36.5 °C, the stirring speed is 350 rpm, DO is controlled at 50%, the pH range is 6.90 ± 0.25, and a 120 g / kg lactic acid solution is used as the pH regulator to control the pH of the culture system;

[0113] (4) When the cell density reaches 14.0×10 6 Cells / mL, adjust the culture temperature. The temperature of the experimental group is reduced to 34.0 °C, and the temperature of the control group is reduced to 32.0 °C. When the pH of the experimental group is higher than 7.15, the 120 g / kg lactic acid solution is slowly added to the bioreactor in a cascading manner until the pH is reduced within the set range, and then the addition of lactic acid is stopped, and so on in a cycle;

[0114] (5) Feed supplements are carried out on the 3rd, 5th, 7th, and 10th days. The feeding amount of Cell Boost 7a is 4.0%, and the feeding amount of Cell Boost 7b is 0.4%; on the 12th day, the feeding amount of Cell Boost 7a is 5.0%, and the feeding amount of Cell Boost 7b is 0.5%;

[0115] (6) Detect the lactic acid content. When the lactic acid content is lower than 1.0 g / L, a carbon source (lactic acid solution) with a concentration of 120 g / kg is supplemented to the culture system so that the lactic acid residue amount the next day is ≥ 0.15 g / L. After the culture ends, the total addition amount of the 120 g / kg lactic acid solution in the control group is 45.6 g, and the total addition amount of the 120 g / kg lactic acid solution in the experimental group is 44.5 g;

[0116] (7) Harvest the cells after culturing for 14 days, and perform antibody protein purification after centrifugation;

[0117] (8) Determine the quality of the antibody protein glycoform.

[0118] It can be seen from Figure 5 that during the fed-batch culture process of this example, adding lactic acid and different cooling temperatures have no significant impact on cell culture.

[0119] It can be seen from Figure 8 that during the culture process, the more lactic acid remains in the culture system, the lower the pH.

[0120] The detection results of the antibody glycoform ratios of the experimental groups and control groups in each example are shown in Table 1.

[0121] Table 1 Antibody glycoform ratios

[0122]

[0123] As can be seen from Table 1, using lactic acid as a pH regulator and a carbon source during cell culture, and adjusting the culture temperature can effectively reduce the proportion of high-mannose, indicating that the technical solution of the present invention can effectively reduce high-mannose, especially reduce the proportion of Man 5.

[0124] Although the present invention has been illustrated and described with reference to specific embodiments, it should be understood that the above embodiments are only used to illustrate the technical solution of the present invention and are not intended to limit it; those of ordinary skill in the art should understand that without departing from the spirit and scope of the present invention, the technical solutions described in the foregoing embodiments may be modified, or some or all of the technical features may be equivalently replaced; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention; therefore, this means that all such replacements and modifications that fall within the scope of the present invention are included in the appended claims.

Claims

1. A method for regulating the high polymannose type in antibody glycoforms, characterized in that: The following steps are involved: The cells expressing the antibody are inoculated into a culture medium for cultivation, and the antibody glycoform is regulated during the cultivation process; The method for regulating antibody glycoforms comprises: adding a pH regulator to control the pH of the culture system during the culture; and / or, when the antibody-expressing cells grow to a stationary phase, adding a carbon source to the culture system; Wherein, the pH regulator includes: lactic acid; the carbon source includes: lactic acid.

2. The method for regulating high polymannose type in antibody glycoforms according to claim 1, characterized in that: The pH regulator is added to control the pH of the culture system to be 6.60-7.

30.

3. The method for regulating high polymannose type in antibody glycoforms according to claim 1, characterized in that: When the cells expressing the antibody grow to a stationary phase, the carbon source is supplemented to the culture system for 1 day, and the carbon source content in the culture system is ≥0.15 g / L.

4. The method for regulating high polymannose type in antibody glycoforms according to claim 1, characterized in that: The culture method of the antibody-expressing cells includes: any one of batch culture, fed-batch culture or perfusion culture.

5. The method for regulating high polymannose type in antibody glycoforms according to claim 1, characterized in that: When the culture method adopts fed-batch culture, when the cells expressing the antibody grow to a stationary phase and the lactic acid content in the culture system is detected to be less than 1.0 g / L, the carbon source is added to the culture system.

6. The method for regulating high polymannose type in antibody glycoforms according to claim 1, characterized in that: When the culture method adopts perfusion culture, after the cells expressing the antibody grow to the stationary phase, the carbon source is added to the culture system after each fluid change.

7. The method for regulating high polymannose type in antibody glycoforms according to claim 1, characterized in that: The initial culture temperature of the antibody-expressing cells is 34.5-37°C.

8. The method for regulating high polymannose type in antibody glycoforms according to any one of claims 1 to 7, characterized in that: When the culture method adopts fed-batch culture, the method for regulating antibody glycoforms further comprises: when the cell density of the antibody expression is ≥13.0×10 6 Cells / mL, and then adjust the culture temperature.

9. The method for regulating high polymannose type in antibody glycoforms according to claim 8, characterized in that: The culture temperature was adjusted to 31.0-35.0°C.

10. A method for preparing a protein drug, characterized in that: The invention comprises the method for regulating high polymannose in antibody glycoforms as described in any one of claims 1 to 9.

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