A method for regulating antibody glycoforms and uses thereof

Abstract

The present application relates to the technical field of biotechnology, in particular to a method for regulating antibody glycoform and application thereof.The method for regulating antibody glycoform comprises the following steps: adding a glycoform regulator in the process of culturing cells expressing antibodies; the glycoform regulator comprises at least one of fucose, ammonium chloride, N-acetylglucosamine and 2-deoxy-2-fluoro-D-galactose.The method for regulating antibody glycoform can significantly increase the proportion of G0F and reduce the proportions of G1F and G2F, the glycoform regulator used is easy to purchase, the method is simple and easy to operate, the adding concentration can be appropriately adjusted according to requirements, and the method is suitable for the regulation of galactosylation in the development process of antibody drugs, in particular the proportions of G0F, G1F and G2F.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular, to a method for regulating antibody glycoform and application thereof. BACKGROUND

[0002] In the process of biological drug development, process change often occurs, and it is crucial to maintain the comparability of quality before and after the process change. In addition, in the process of biological similar drug development, it is also crucial to maintain the comparability of quality between the similar drug and the original drug. The glycoform distribution of antibodies is a key quality attribute of antibody drugs, so it is crucial to maintain the consistency of glycoform distribution before and after the change or for the similar drug to maintain consistency with the original drug.

[0003] In the existing cell culture process, the methods for adjusting the proportion of glycoform mainly include: medium screening, culture process optimization (pH, DO, temperature optimization, etc.). In the process of biological drug development, after the existing culture medium and cell culture process optimization, there may be some differences in the glycoform distribution compared with the previous process. In addition, in the process of biological similar drug development, there may be differences in the glycoform distribution compared with the original drug.

[0004] Therefore, the present application is proposed. SUMMARY

[0005] The purpose of the present application is to provide a method for regulating antibody glycoform, which is suitable for the regulation of galactosylation in the process of antibody drug development, especially the proportion of G0F, G1F and G2F.

[0006] In order to achieve the above purpose of the present application, the following technical solution is adopted:

[0007] A method for regulating antibody glycoform, comprising the following steps:

[0008] Adding a glycoform regulator in the process of culturing cells expressing antibodies;

[0009] The glycoform regulator comprises at least one of fucose, ammonium chloride, N-acetylglucosamine and 2-deoxy-2-fluoro-D-galactose.

[0010] The method for regulating antibody glycoform achieves the effect of significantly increasing the proportion of G0F and reducing the proportion of G1F and G2F by adding a glycoform regulator to the culture medium used in the process of culturing cells expressing antibodies. The glycoform regulator used in the present application is easy to purchase, the method provided is simple and easy to operate, and the addition concentration can be appropriately adjusted according to the demand, which is suitable for the regulation of galactosylation in the process of drug development, especially the proportion of G0F, G1F and G2F.

[0011] Preferably, the process of culturing cells expressing antibodies comprises:

[0012] The antibody-expressing cells are cultured in a growth medium to a density of 4×10 6 ~ 8×10 6 cells / mL, a first centrifugal medium exchange is performed using the growth medium; the cells are cultured to a density of greater than 8×10 6 cells / mL, a second centrifugal medium exchange is performed using a production medium containing the glycoform modulator.

[0013] Preferably, the added concentration of fucose is 16-24 mM.

[0014] Preferably, the added concentration of ammonium chloride is 6-14 mM.

[0015] Preferably, the added concentration of acetylglucosamine is 6-14 mM.

[0016] Preferably, the added concentration of deoxy-2-fluoro-D-galactose is 50-150 µM.

[0017] Preferably, the antibody-expressing cells are cultured for 14-16 days.

[0018] Preferably, the antibody-expressing cells are cultured in any one of batch culture, fed-batch culture or perfusion culture.

[0019] Preferably, the antibody-expressing cells are CHO cells.

[0020] A method for preparing an antibody drug, comprising the method for regulating antibody glycoforms.

[0021] Compared with the prior art, the present application has the following advantages:

[0022] (1) The method for regulating antibody glycoforms provided by the present application achieves the effect of significantly increasing the G0F proportion and decreasing the G1F and G2F proportions by adding a glycoform modulator to the culture medium used in the culture process of protein-expressing cells. The glycoform modulator used in the present application is easy to purchase, the provided method is simple and easy to operate, and the added concentration can be appropriately adjusted according to the needs, which is suitable for the regulation of galactosylation in the drug development process, especially the proportions of G0F, G1F and G2F. DETAILED DESCRIPTION

[0023] The technical solutions of the present application will be described clearly and completely in combination with specific embodiments, but those skilled in the art will understand that the following described embodiments are part of the embodiments of the present application, not all the embodiments, and are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0024] A method for regulating antibody glycoforms, comprising the steps of:

[0025] adding a glycoform regulator during the culture process of the antibody-expressing cells;

[0026] The glycoform regulator comprises at least one of fucose, ammonium chloride, N-acetyl-D-(+)-glucosamine (GlcNAc), and 2-deoxy-2-fluoro-D-galactose (2FG).

[0027] The method for regulating antibody glycoforms achieves the effect of significantly increasing the proportion of G0F and reducing the proportions of G1F and G2F by adding a glycoform regulator to the culture medium of the antibody-expressing cells. The glycoform regulator used in the present application is easy to purchase, the method provided is simple and easy to operate, and the addition concentration can be appropriately adjusted according to the needs, which is suitable for the regulation of galactosylation, especially the proportions of G0F, G1F and G2F, in the process of drug development.

[0028] Preferably, the culture process of the antibody-expressing cells comprises:

[0029] The antibody-expressing cells are cultured in a growth medium to a density of 4×10 6 ~ 8×10 6 When the cells are cultured to a density of greater than 8×10 6 Cells / mL, a second centrifugal medium exchange is performed using a production medium containing the glycoform regulator.

[0030] Preferably, when the cells are cultured to a density of greater than 4×10 6 Cells / mL, a centrifugal medium exchange is performed using a growth medium, the centrifugal condition is 1000 rpm for 5 min, and the medium exchange volume is 10 mL.

[0031] Preferably, the cells are cultured to a density of greater than 8 x 10 6 cells / mL, centrifuged at 1000 rpm for 5 min, and the volume of the replacement medium is 10 mL.

[0032] The present application does not make specific limitations on the specific types of growth medium and production medium. The growth medium is preferably Hyclone Actipro medium, and the production medium is preferably Hyclone Actipro medium plus 5% CellBoost 7a and 0.5% Cell Boost 7b.

[0033] Preferably, the seeding density of the antibody-expressing cells is 0.8 x 10 6 ~ 1.2 x 10 6 cells / mL.

[0034] Preferably, the temperature of the shaker for cell culture is 35 ± 0.5°C, the rotation speed of the shaker is 190 ± 5 rpm, the track diameter is 50 mm, the CO2 concentration is 5 ± 0.5%, and the angle of inclination is 75 degrees.

[0035] In the present application, 50 mL of a shake flask is used to culture the cells, and the cells are cultured by perfusion.

[0036] Preferably, the culture volume of the shake flask is 10 mL.

[0037] Preferably, the added concentration of fucose is 16-24 mM.

[0038] In some specific embodiments, the added concentration of fucose can be, for example, but is not limited to, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, or 24 mM.

[0039] Preferably, the added concentration of ammonium chloride is 6-14 mM.

[0040] In some specific embodiments, the added concentration of ammonium chloride can be, for example, but is not limited to, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, or 14 mM.

[0041] Preferably, the added concentration of acetylglucosamine is 6-14 mM.

[0042] In some specific embodiments, the added concentration of acetylglucosamine can be, for example, but is not limited to, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, or 14 mM.

[0043] Preferably, the concentration of the added deoxy-2-fluoro-D-galactose is 50-150 μM.

[0044] In some embodiments, the concentration of the added deoxy-2-fluoro-D-galactose can be, but is not limited to, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, or 150 μM.

[0045] Preferably, the culturing time of the antibody-expressing cells is 14-16 days.

[0046] In the present application, the day of inoculation is day 0, and the subsequent days are sequentially extended.

[0047] Preferably, the antibody is a monoclonal antibody.

[0048] Preferably, the culturing method of the antibody-expressing cells comprises any one of batch culture, fed-batch culture, or perfusion culture.

[0049] Preferably, the type of the antibody-expressing cells comprises CHO cells.

[0050] In the present application, the CHO cells refer to Chinese hamster ovary cells.

[0051] A method for preparing a protein drug, comprising the method for regulating the glycoform of an antibody.

[0052] The method for preparing a protein drug produces an antibody drug with a reasonable ratio of glycoforms, particularly a ratio of G0F, G1F, and G2F.

[0053] Embodiments of the present application will be described in detail below with reference to specific examples and comparative examples.

[0054] Example 1

[0055] The method for regulating the glycoform of an antibody provided in this example regulates the ratio of G0F, G1F, and G2F in the glycoform of an antibody by adding 100 μM 2FG to the production medium, and the specific steps are as follows:

[0056] (1) Hyclone Actipro is used as the growth medium, and Hyclone Actipro medium plus 5% CellBoost 7a and 0.5% Cell Boost 7b is used as the production medium.

[0057] (2) The CHO cells are inoculated in a 50 mL shake flask at an inoculation density of 1.0 ± 0.2 × 10 6 cells / mL, the culture volume in the shake flask is 10 mL, and the inoculation medium is the growth medium.

[0058] (3) The temperature of the shaker for cell culture is 35±0.5℃, the rotation speed of the shaker is 190±5rpm, the track diameter is 50mm, the CO2 concentration is 5±0.5%, and the angle of inclination is 75 degrees.

[0059] (4) When the cell density is greater than 4×10 6 cells / mL, centrifugal replacement is performed using the growth medium, 1000rpm centrifugation for 5 minutes, and the replacement volume is 10mL.

[0060] (5) When the cell density is greater than 8×10 6 cells / mL, centrifugal replacement is performed using the production medium containing sugar type regulator, 1000rpm centrifugation for 5 minutes, and the replacement volume is 10mL.

[0061] (6) The cells are harvested at 14 days of culture, and antibody protein purification is performed after centrifugation.

[0062] (7) The quality of the antibody protein sugar type is determined.

[0063] Example 2

[0064] The method for regulating the antibody sugar type provided in this example regulates the ratio of G0F, G1F and G2F in the antibody sugar type by adding 20mM Fucose to the production medium alone, and the specific steps are referred to Example 1.

[0065] Example 3

[0066] The method for regulating the antibody sugar type provided in this example regulates the ratio of G0F, G1F and G2F in the antibody sugar type by adding 10mM NH4Cl to the production medium alone, and the specific steps are referred to Example 1.

[0067] Example 4

[0068] The method for regulating the antibody sugar type provided in this example regulates the ratio of G0F, G1F and G2F in the antibody sugar type by adding 10mM GlcNAc to the production medium alone, and the specific steps are referred to Example 1.

[0069] Example 5

[0070] The method for regulating the antibody sugar type provided in this example regulates the ratio of G0F, G1F and G2F in the antibody sugar type by adding 100μM 2FG and 10mM NH4Cl to the production medium in combination, and the specific steps are referred to Example 1.

[0071] Example 6

[0072] The method for regulating the glycoform of the antibody provided in the embodiment regulates the ratio of G0F, G1F and G2F in the glycoform of the antibody by adding 100 μM 2FG and 20 mM Fucose into the production medium in combination, and the specific steps refer to Embodiment 1.

[0073] Embodiment 7

[0074] The method for regulating the glycoform of the antibody provided in the embodiment regulates the ratio of G0F, G1F and G2F in the glycoform of the antibody by adding 100 μM 2FG and 10 mM GlcNAc into the production medium in combination, and the specific steps refer to Embodiment 1.

[0075] Embodiment 8

[0076] The method for regulating the glycoform of the antibody provided in the embodiment regulates the ratio of G0F, G1F and G2F in the glycoform of the antibody by adding 10 mM NH4Cl and 20 μM Fucose into the production medium in combination, and the specific steps refer to Embodiment 1.

[0077] Embodiment 9

[0078] The method for regulating the glycoform of the antibody provided in the embodiment regulates the ratio of G0F, G1F and G2F in the glycoform of the antibody by adding 10 mM NH4Cl and 10 mM GlcNAc into the production medium in combination, and the specific steps refer to Embodiment 1.

[0079] Embodiment 10

[0080] The method for regulating the glycoform of the antibody provided in the embodiment regulates the ratio of G0F, G1F and G2F in the glycoform of the antibody by adding 100 μM 2FG, 10 mM NH4Cl and 20 mM Fucose into the production medium in combination, and the specific steps refer to Embodiment 1.

[0081] Embodiment 11

[0082] The method for regulating the glycoform of the antibody provided in the embodiment regulates the ratio of G0F, G1F and G2F in the glycoform of the antibody by adding 100 μM 2FG, 10 mM GlcNAc and 20 mM Fucose into the production medium in combination, and the specific steps refer to Embodiment 1.

[0083] Embodiment 12

[0084] The method for regulating the glycoform of the antibody provided in the embodiment regulates the ratio of G0F, G1F and G2F in the glycoform of the antibody by adding 100 μM 2FG, 10 mM GlcNAc and 10 mM NH4Cl into the production medium in combination, and the specific steps refer to Embodiment 1.

[0085] Example 13

[0086] The method for regulating the glycoform of the antibody provided in this example regulates the ratio of G0F, G1F and G2F in the glycoform of the antibody by adding 100 μM 2FG, 10 mM GlcNAc, 10 mM NH4Cl and 20 mM Fucose to the production medium, and the specific steps are referred to Example 1.

[0087] Example 14

[0088] The method for regulating the glycoform of the antibody provided in this example regulates the ratio of G0F, G1F and G2F in the glycoform of the antibody by adding 150 μM 2FG to the production medium, and the specific steps are referred to Example 1.

[0089] Comparative Example 1

[0090] The method for regulating the glycoform of the antibody provided in this example is different from that of Example 1 only in that no glycoform regulator is added to the production medium.

[0091] Table 1 Ratio of the glycoform of the antibody

[0092]

[0093]

[0094] The mass of the glycoform of the antibody protein in each example and comparative example was measured, and the results are shown in Table 1.

[0095] As shown in Table 1, the production medium containing the glycoform regulator was used to culture the cells in each example of the present application, which can significantly increase the ratio of G0F and decrease the ratio of G1F and G2F, indicating that the technical solution of the present application can achieve the effect of significantly increasing the ratio of G0F and decreasing the ratio of G1F and G2F by adding the glycoform regulator to the medium used in the culture process of the cells expressing the protein.

[0096] Although the present application has been illustrated and described with reference to specific embodiments, it is to be understood that the above embodiments are merely used to illustrate the technical solutions of the present application, and are not intended to limit the present application; it should be understood by those skilled in the art that the technical solutions described in the above embodiments can be modified, or some or all of the technical features can be replaced equivalently without departing from the spirit and scope of the present application; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application; therefore, this means that all these replacements and modifications within the scope of the present application are included in the appended claims.

Claims

1. A method of modulating the glycoform of an antibody, characterized in that, The method comprises the following steps: adding a glycoform regulator to a culture process of antibody-expressing cells to regulate the ratio of G0F, G1F and G2F in the glycoform of the antibody, increase the ratio of G0F and decrease the ratio of G1F and G2F; the glycoform regulator comprises at least one of fucose, ammonium chloride, N-acetylglucosamine and 2-deoxy-2-fluoro-D-galactose; the glycoform regulator is a combination of 2-deoxy-2-fluoro-D-galactose and N-acetylglucosamine; or a combination of 2-deoxy-2-fluoro-D-galactose, N-acetylglucosamine and fucose; or a combination of 2-deoxy-2-fluoro-D-galactose, N-acetylglucosamine and ammonium chloride; or a combination of 2-deoxy-2-fluoro-D-galactose, fucose and ammonium chloride; or a combination of 2-deoxy-2-fluoro-D-galactose, N-acetylglucosamine, ammonium chloride and fucose; the addition concentration of the fucose is 20 mM; the addition concentration of the ammonium chloride is 10 mM; the addition concentration of the N-acetylglucosamine is 10 mM; the addition concentration of the 2-deoxy-2-fluoro-D-galactose is 100 µM.

2. The method of modulating antibody glycoform according to claim 1, wherein, The culture process of the antibody-expressing cells comprises: The antibody-expressing cells were cultured in growth medium to a density of 4 × 10⁶ cells / year. 6 ~8×10 6 When the cell density reaches 8 × 10⁶ cells / mL, the first centrifugation and medium change should be performed using growth medium; the cells should be cultured until the density is greater than 8 × 10⁶. 6 When the concentration is cells / mL, a second centrifugation and medium exchange is performed using production medium containing the glycoform regulator.

3. The method of modulating the glycoform of an antibody according to claim 1 or 2, characterized in that, the culture time of the antibody-expressing cells is 14-16 days.

4. The method of modulating antibody glycoform according to claim 1, wherein, The culture mode of the antibody-expressing cells comprises any one of batch culture, fed-batch culture or perfusion culture.

5. The method of modulating antibody glycoform according to claim 1, wherein, The type of the antibody-expressing cells comprises CHO cells.

6. A method of preparing an antibody drug, characterized by, The method for regulating the glycoform of the antibody comprises any one of claims 1-5.

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