A method for regulating antibody glycosylation

Abstract

The present invention belongs to the field of biomedicine and specifically relates to a method for regulating antibody glycosylation, comprising adding Mn at a specific time during the culture cycle of antibody-producing cells. 2+ , N‑acetylglucosamine, and fucose.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine and specifically to a method for regulating antibody glycosylation. Background Art

[0002] Glycosylation is an important post-translational modification of therapeutic monoclonal antibodies. The two most common forms are O-glycosylation (oligosaccharides are linked to hydroxyl-containing amino acids such as Ser, Thr or Tyr) and N-glycosylation (oligosaccharides are linked to Asn-X-Ser / Thr, where X is any amino acid except Pro). N-glycosylation can affect the physicochemical properties of monoclonal antibodies, including protein folding, solubility, binding, stability, immunogenicity and pharmacokinetics. The efficacy of therapeutic monoclonal antibodies may depend on complement-dependent cytotoxicity (CDC) activity and / or antibody-dependent cell-mediated cytotoxicity (ADCC) activity. Increasing the galactosylation level of antibodies can increase the CDC activity of antibodies; increasing the defucosylation of antibodies can increase the ADCC activity of antibodies; high mannose forms will shorten the half-life of antibodies, affect the pharmacokinetics of antibodies, and high mannose forms have significant immunogenicity and may cause severe immune responses. Therefore, in the development of antibody drugs, glycoform distribution is usually regarded as one of the critical quality attributes (CQA) of the product, and the distribution of glycoforms needs to be strictly controlled.

[0003] The upstream control strategies for antibody glycosylation are mainly focused on genetically engineered cells, culture medium adjustment, and bioprocess control, including process parameters during the culture process. The use of genetic engineering methods to adjust glycoforms can often accurately obtain the target glycoforms, but genetic engineering methods require genetic modification of cells, which has the disadvantages of high cost and long cycle for antibody drugs. Culture medium adjustment and process control are usually accompanied by changes in other quality attributes of antibodies and reduced yields. It is necessary to provide a low-cost, short-cycle method for controlling antibody glycosylation modification to achieve flexible adjustment of glycoform distribution, enhance process controllability, and improve antibody quality. Summary of the invention

[0004] In one aspect, the present application provides a method for regulating antibody glycosylation, the method comprising adding Mn at a specific time during the culture cycle of antibody-producing cells. 2+ , and N-acetylglucosamine and fucose were added at another specific time.

[0005] In some embodiments, the Mn 2+ The amount of Mn added is 0.1-2.0 μmol / L. 2+The amount of Mn added is 0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.5 μmol / L, 0.6 μmol / L, 0.7 μmol / L, 0.8 μmol / L, 0.9 μmol / L, 1.0 μmol / L, 1.1 μmol / L, 1.2 μmol / L, 1.3 μmol / L, 1.4 μmol / L, 1.5 μmol / L, 1.6 μmol / L, 1.7 μmol / L, 1.8 μmol / L, 1.9 μmol / L, 2.0 μmol / L or a range formed by any of the foregoing values. In some embodiments, the Mn 2+ The amount of Mn added is 0.4-2.0 μmol / L or 0.7-2.0 μmol / L. 2+ The amount of Mn added is 0.7-2.0 μmol / L. 2+ The amount of Mn added is 0.7 μmol / L, 1.0 μmol / L or 2.0 μmol / L. 2+ The amount added was 2.0 μmol / L.

[0006] In some embodiments, the Mn 2+ The Mn is added on the start day, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or any of the foregoing days of the culture cycle. 2+ Added on the start day or 8th day of the culture cycle. In some specific embodiments, the Mn 2+ Added on the start day of the culture cycle. In some specific embodiments, the Mn 2+ Add on day 8 of the culture cycle.

[0007] In some embodiments, the amount of N-acetylglucosamine added is 1-10mmol / L. In some embodiments, the amount of N-acetylglucosamine added is 1mmol / L, 2mmol / L, 3mmol / L, 4mmol / L, 5mmol / L, 6mmol / L, 7mmol / L, 8mmol / L, 9mmol / L, 10mmol / L or the range formed by any of the foregoing values. In some embodiments, the amount of N-acetylglucosamine added is 2-10mmol / L, 2-8mmol / L or 2-5mmol / L. In some embodiments, the amount of N-acetylglucosamine added is 2-10mmol / L. In some embodiments, the amount of N-acetylglucosamine added is 2mmol / L, 5mmol / L or 10mmol / L. In some specific embodiments, the amount of N-acetylglucosamine added is 5mmol / L.

[0008] In some embodiments, the N-acetylglucosamine is added on the start day, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or any of the foregoing days of the culture period. In some specific embodiments, the N-acetylglucosamine is added on the 5th day of the culture period.

[0009] In some embodiments, the amount of fucose added is 10-100mmol / L. In some embodiments, the amount of fucose added is 10mmol / L, 20mmol / L, 30mmol / L, 40mmol / L, 50mmol / L, 60mmol / L, 70mmol / L, 80mmol / L, 90mmol / L, 100mmol / L or the range formed by any of the foregoing values. In some embodiments, the amount of fucose added is 10-80mmol / L, 10-60mmol / L or 20-50mmol / L. In some embodiments, the amount of fucose added is 20-50mmol / L. In some specific embodiments, the amount of fucose added is 20mmol / L or 50mmol / L.

[0010] In some embodiments, the fucose is added on the start day, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or any of the foregoing days of the culture cycle. In some specific embodiments, the fucose is added on day 5 of the culture cycle.

[0011] In some embodiments, the Mn 2+The amount of Mn added is 0.1-2.0 μmol / L, the amount of N-acetylglucosamine added is 1-10 mmol / L, and the amount of fucose added is 10-100 mmol / L. 2+ The addition amounts are 0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.5 μmol / L, 0.6 μmol / L, 0.7 μmol / L, 0.8 μmol / L, 0.9 μmol / L, 1.0 μmol / L, 1.1 μmol / L, 1.2 μmol / L, 1.3 μmol / L, 1.4 μmol / L, 1.5 μmol / L, 1.6 μmol / L, 1.7 μmol / L, 1.8 μmol / L, 1.9 μmol / L, 2.0 μmol / L or the range formed by any of the above values, the amount of N-acetylglucosamine added is 1mmol / L, 2mmol / L, 3mmol / L, 4mmol / L, 5mmol / L, 6mmol / L, 7mmol / L, 8mmol / L, 9mmol / L, 10mmol / L or the range formed by any of the above values, and the amount of fucose added is 10mmol / L, 20mmol / L, 30mmol / L, 40mmol / L, 50mmol / L, 60mmol / L, 70mmol / L, 80mmol / L, 90mmol / L, 100mmol / L or the range formed by any of the above values. In some embodiments, the Mn 2+ The amount of Mn added is 0.4-2.0 μmol / L or 0.7-2.0 μmol / L, the amount of N-acetylglucosamine added is 2-10mmol / L, 2-8mmol / L or 2-5mmol / L, and the amount of fucose added is 10-80mmol / L, 10-60mmol / L or 20-50mmol / L. In some embodiments, the Mn 2+ The amount of Mn added is 0.7-2.0 μmol / L, the amount of N-acetylglucosamine added is 2-10mmol / L, and the amount of fucose added is 20-50mmol / L. 2+ The amount of Mn added is 0.7 μmol / L, 1.0 μmol / L or 2.0 μmol / L, the amount of N-acetylglucosamine added is 2 mmol / L, 5 mmol / L or 10 mmol / L, and the amount of fucose added is 20 mmol / L or 50 mmol / L. In some specific embodiments, the Mn 2+The added amount of is 2.0 μmol / L, the added amount of N-acetylglucosamine is 5 mmol / L, and the added amount of fucose is 20 mmol / L or 50 mmol / L.

[0012] In some embodiments, the Mn 2+ The N-acetylglucosamine is added on the starting day, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or any of the foregoing days of the culture period; the fucose is added on the starting day, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or any of the foregoing days of the culture period. In some specific embodiments, the Mn 2+ The N-acetylglucosamine and fucose are added on the starting day or the 8th day of the culture cycle, and the N-acetylglucosamine and fucose are added on the 5th day of the culture cycle.

[0013] In some embodiments, 0.1-2.0 μmol / L Mn is added on the first day or on day 8 of the culture cycle. 2+ , 2-10 mmol / L N-acetylglucosamine and 10-100 mmol / L fucose are added on the 5th day of the culture cycle. In some embodiments, 0.7-2.0 μmol / L Mn is added on the starting day or the 8th day of the culture cycle. 2+ , 2-10 mmol / L N-acetylglucosamine and 20-50 mmol / L fucose are added on the 5th day of the culture cycle. In some specific embodiments, 2 μmol / L Mn 2+ , 5 mmol / L N-acetylglucosamine and 20 mmol / L or 50 mmol / L fucose were added on the 5th day of the culture cycle.

[0014] In some embodiments, the Mn 2+ Provided by manganese chloride.

[0015] On the other hand, the present application provides a method for producing an antibody, wherein the culture conditions of the antibody-producing cells are: 36.5°C, 8% CO2, 130 rpm, the culture period is 14 days, and 2 μmol / L Mn is added on the 8th day of the culture period. 2+ , 5 mmol / L N-acetylglucosamine and 20 mmol / L or 50 mmol / L fucose were added on the 5th day of the culture cycle.

[0016] In some embodiments, the Mn 2+ Provided by manganese chloride.

[0017] In some embodiments, the antibody is penampalimab, bemosubinib, daratumumab, mepolizumab, ramucirumab, pertuzumab, guselkumab, rituximab, trastuzumab, adalimumab, bevacizumab, epezetuzumab, obinutuzumab, pembrolizumab, lomosozumab, alirocumab, bimegizumab, denosumab, elotuzumab, nivolumab, secukinumab, ustekinumab, belimumab, nemolizumab, lencanezumab, rozelixizumab, faricizumab, or atezolizumab.

[0018] In some embodiments, the antibody is an anti-TIM-3 antibody or an anti-LAG-3 antibody.

[0019] Table S1. CDR sequences of anti-TIM-3 antibodies

[0020]

[0021] In some embodiments, the anti-TIM-3 monoclonal antibody comprises HCDR1 shown in the amino acid sequence of SEQ ID NO: 1, HCDR2 shown in the amino acid sequence of SEQ ID NO: 2, HCDR3 shown in the amino acid sequence of SEQ ID NO: 3, LCDR1 shown in the amino acid sequence of SEQ ID NO: 4, LCDR2 shown in the amino acid sequence of SEQ ID NO: 5, and LCDR3 shown in the amino acid sequence of SEQ ID NO: 6.

[0022] In some embodiments, the anti-TIM-3 monoclonal antibody comprises HCDR1 shown in the amino acid sequence of SEQ ID NO: 7, HCDR2 shown in the amino acid sequence of SEQ ID NO: 8, HCDR3 shown in the amino acid sequence of SEQ ID NO: 9, LCDR1 shown in the amino acid sequence of SEQ ID NO: 10, LCDR2 shown in the amino acid sequence of SEQ ID NO: 11, and LCDR3 shown in the amino acid sequence of SEQ ID NO: 12.

[0023] In some embodiments, the anti-TIM-3 monoclonal antibody comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 13 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 14.

[0024] In some embodiments, the anti-TIM-3 monoclonal antibody comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 15 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 16.

[0025] In some embodiments, the anti-TIM-3 monoclonal antibody comprises a heavy chain shown in the amino acid sequence of SEQ ID NO: 17 and a light chain shown in the amino acid sequence of SEQ ID NO: 18.

[0026] In some embodiments, the anti-TIM-3 monoclonal antibody comprises a heavy chain shown in the amino acid sequence of SEQ ID NO: 19 and a light chain shown in the amino acid sequence of SEQ ID NO: 20.

[0027] Table S2. CDR sequences of anti-LAG-3 antibodies

[0028]

[0029] In some embodiments, the anti-LAG-3 monoclonal antibody comprises HCDR1 shown in the amino acid sequence of SEQ ID NO: 21, HCDR2 shown in the amino acid sequence of SEQ ID NO: 22, HCDR3 shown in the amino acid sequence of SEQ ID NO: 23, LCDR1 shown in the amino acid sequence of SEQ ID NO: 24, LCDR2 shown in the amino acid sequence of SEQ ID NO: 25, and LCDR3 shown in the amino acid sequence of SEQ ID NO: 26.

[0030] In some embodiments, the anti-LAG-3 monoclonal antibody comprises HCDR1 shown in the amino acid sequence of SEQ ID NO: 27, HCDR2 shown in the amino acid sequence of SEQ ID NO: 28, HCDR3 shown in the amino acid sequence of SEQ ID NO: 29, LCDR1 shown in the amino acid sequence of SEQ ID NO: 30, LCDR2 shown in the amino acid sequence of SEQ ID NO: 31, and LCDR3 shown in the amino acid sequence of SEQ ID NO: 32.

[0031] In some embodiments, the anti-LAG-3 monoclonal antibody comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 33 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 34.

[0032] In some embodiments, the anti-LAG-3 monoclonal antibody comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 35 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 36.

[0033] In some embodiments, the anti-LAG-3 monoclonal antibody comprises a heavy chain shown in the amino acid sequence of SEQ ID NO: 37 and a light chain shown in the amino acid sequence of SEQ ID NO: 38.

[0034] In some embodiments, the anti-LAG-3 monoclonal antibody comprises a heavy chain shown in the amino acid sequence of SEQ ID NO: 39 and a light chain shown in the amino acid sequence of SEQ ID NO: 40.

[0035] It will be understood by those skilled in the art that, unless otherwise specified, the term "CDR" or "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions defined by any known scheme. Although Tables S1-S2 have shown CDR sequences, however, when referring to antibodies defined by specific CDR sequences disclosed herein, the scope of the antibodies also encompasses antibodies defined by CDR sequences converted to other arbitrary numbering system definitions (e.g., a combination of one or more of the definitions of Kabat, Chothia, CCG, IMGT or Contact, etc., which are well known in the art).

[0036] The method of the present application can be used to cultivate a variety of cells. In some embodiments, the cultured cells are eukaryotic cells, such as plant and / or animal cells. The animal cells can be mammalian cells, fish cells, insect cells, amphibian cells or bird cells. A variety of mammalian cell lines suitable for cultivation can be obtained from depository institutions and commercial suppliers. Cells that can be used in the method of the present application include but are not limited to Chinese hamster ovary cells (CHO), CHO-S cells, CHO-DG44 cells, or any other cell type known to those skilled in the art.

[0037] In some embodiments, the basal medium for cell culture may include serum-free and / or animal-free products or components. As will be appreciated by those skilled in the art, animal or mammalian cells may be cultured in a defined medium without undue experimentation, provided that it is suitable for the specific cells being cultured. Commercial culture media may be used, including but not limited to: CD OptiCHO, Hycell, CD CHO, GrowthA, Dynamis, Iscov's Modified Dulbecco's Medium, RPMI 1640, Minimum Essential Medium-α (MEM-α), Dulbecco's Modified Eagle's Medium (DMEM), DME / F12, Earle's Basal Medium with Earle's BSS, High Glucose DMEM with Glutamine, High Glucose DMEM without Glutamine, Low Glucose DMEM without Glutamine, Glasgow's MEM, GMEM with Glutamine, Ham's F-10 with Glutamine, Ham's with Glutamine F-12, IMDM with HEPES and glutamine, IMDM with HEPES and without glutamine, McCoy's Modified Medium, Medium 199, Eagle's MEM-Earle's BSS with glutamine, Eagle's MEM-Earle's BSS without glutamine, Eagle's MEM-Hanks BSS without glutamine, NCTC-109 with glutamine, Richter's CM with glutamine, RPMI 1640 with HEPES, glutamine and / or penicillin-streptomycin, RPMI 1640 with glutamine, RPMI 1640 without glutamine, or any other medium known to those of skill in the art formulated for a particular cell type. Supplemental components or ingredients, including optional components, may be added to the above exemplary media at appropriate concentrations or amounts as needed or as desired and as known and practiced by those of skill in the art using routine skills.

[0038] In some embodiments, the feed medium of the specific nutrient that is difficult to prepare or consumes rapidly in the cell culture can also be supplemented in the cell culture. Such nutrients can be amino acids, such as tyrosine, cysteine ​​and / or cystine. For example, concentrated tyrosine solution can be fed independently to the cell culture grown in the cell culture medium containing tyrosine. Concentrated solutions of tyrosine and cystine can also be fed independently to the cell culture grown in the cell culture medium lacking tyrosine, cystine and / or cysteine.

[0039] The method for regulating antibody glycosylation or producing antibodies provided in the present application has low cost and short cycle, can realize flexible regulation of glycoform distribution, enhances process controllability and reproducibility, ensures consistency between antibody batches, and improves the quality of antibodies at the same time. DETAILED DESCRIPTION

[0040] the term

[0041] Unless otherwise specified, the following terms used in this application have the following meanings. A particular term should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to the common meaning in the art. When a trade name appears in this application, it is intended to refer to the corresponding commodity or its active ingredient.

[0042] The term "antibody" is used in the broadest sense to cover natural antibodies and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and single-chain antibodies, as long as they show the desired antigen-binding activity.

[0043] The term "variable domain" or "variable region" refers to the domain of an antibody that is involved in binding the antibody to an antigen. For example, a natural four-chain antibody (e.g., derived from humans, mice, etc.) has a heavy chain variable region (also referred to as a heavy chain variable domain, VH or VH domain) and a light chain variable region (also referred to as a light chain variable domain, VL or VL domain). In most cases, each variable domain of a natural antibody is essentially composed of four "framework regions (FR)" and three "complementarity determining regions (CDR)". The four framework regions are respectively referred to as framework region 1 (or FR1), framework region 2 (or FR2), framework region 3 (or FR3), and framework region 4 (or FR4); the framework region is separated by three complementary determining regions, which are respectively referred to as complementary determining region 1 (or CDR1), complementary determining region 2 (or CDR2), and complementary determining region 3 (or CDR3) in the art and hereinafter. Therefore, the general structure of the variable domain can be expressed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable domain confers specificity to the antibody for the antigen by providing the antigen binding site.

[0044] The term "complementarity determining region" (CDR), also known as "hypervariable region" (HVR). A natural four-chain antibody usually contains six CDRs, three in the heavy chain variable region, namely heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3); three in the light chain variable region, namely light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3).

[0045] There are many methods to divide and define CDR at present. Among them, Kabat definition divides CDR based on sequence variability, and is the most commonly used (Elvin A.Kabat, et al, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and Chothia definition is based on the position of structural loop (Cyrus Chothia, et al, Canonical Structures for the Hypervariable Regions of Immunoglobulins, J.Mol. Biol. 196:901-917 (1987)). AbM definition is a compromise between Kabat definition and Chothia definition, and is used by AbM antibody modeling software of Oxford Molecular. The basis of "contact" definition CDR is the analysis of available complex crystal structure. In addition, there are definitions such as IMGT and CCG. However, it should be noted that the boundaries of the CDRs of the same antibody variable region obtained by different division definitions may be different, that is, the CDR sequences of the same antibody variable region divided and defined by different methods may be different. Therefore, when it comes to defining antibodies by specific CDR sequences, the scope of the antibodies also covers antibodies defined by CDR sequences of any other definitions (e.g., a combination of one or more of the definitions of Kabat, IMGT, Chothia, Contact, AbM, CCG, etc.).

[0046] The term "culture cycle" refers to the cycle from cell inoculation to shake flask culture, "D0" refers to the starting day of the culture cycle, which is day 0 of the culture cycle, "D5" refers to day 5 of the culture cycle, and so on.

[0047] The terms "include" or "comprising" should be construed in an open, non-exclusive sense, ie, "including, but not limited to."

[0048] As used herein, "about" means within the acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation according to the practice in the art. Alternatively, "about" may mean a range of up to ±5%, such as fluctuations within ±2%, within ±1%, or within ±0.5% of a given specific numerical range. When a specific value is given in the application or claims, unless otherwise stated, the meaning of "about" should be considered to be within the acceptable error range for that specific value. In this article, unless otherwise stated, all values ​​involving dosages, times, step parameters or conditions are modified by "about" by default.

[0049] Example

[0050] Although the foregoing disclosure has been described in detail by way of illustration and example for purposes of clear understanding, it will be apparent to one of ordinary skill in the art, based on the teachings of the present disclosure, that certain changes and modifications may be made to the disclosure without departing from the spirit and scope of the appended claims. The following examples are provided by way of illustration only and are not intended to be limiting. One of ordinary skill in the art will readily recognize a variety of non-critical parameters that may be changed or modified to produce substantially similar results.

[0051] Unless otherwise specified, all reagents used in the examples can be purchased from commercial sources.

[0052] The present application also provides the following specific implementation schemes, but is not limited thereto:

[0053] Embodiment 1. A method for regulating antibody glycosylation, characterized in that Mn is added at a specific time during the culture cycle of antibody-producing cells. 2+ , and N-acetylglucosamine and fucose were added at another specific time.

[0054] Embodiment 2. The method according to embodiment 1, characterized in that the Mn 2+ The amount added is 0.1-2.0 μmol / L.

[0055] Embodiment 3. The method according to any one of embodiments 1-2, characterized in that the Mn 2+ The amount added is 0.7-2.0 μmol / L.

[0056] Embodiment 4. The method according to any one of embodiments 1-3, characterized in that the Mn 2+ Add on the start day or 8th day of the culture cycle.

[0057] Embodiment 5. The method according to any one of embodiments 1-4 is characterized in that the amount of N-acetylglucosamine added is 2-10 mmol / L.

[0058] Embodiment 6. The method according to any one of embodiments 1-5 is characterized in that the N-acetylglucosamine is added on the 5th day of the culture cycle.

[0059] Embodiment 7. The method according to any one of Embodiments 1-6 is characterized in that the amount of fucose added is 10-100 mmol / L.

[0060] Embodiment 8. The method according to any one of Embodiments 1-7 is characterized in that the amount of fucose added is 20-50 mmol / L.

[0061] Embodiment 9. The method according to any one of embodiments 1-8 is characterized in that the fucose is added on the 5th day of the culture cycle.

[0062] Embodiment 10. The method according to any one of embodiments 1-9, characterized in that the Mn 2+ The added amount of is 0.1-2.0 μmol / L, the added amount of N-acetylglucosamine is 2-10 mmol / L, and the added amount of fucose is 10-100 mmol / L.

[0063] Embodiment 11. The method according to any one of embodiments 1-10, characterized in that the Mn 2+ The added amount of is 0.7-2.0 μmol / L, the added amount of N-acetylglucosamine is 2-10 mmol / L, and the added amount of fucose is 20-50 mmol / L.

[0064] Embodiment 12. The method according to any one of embodiments 1-10, characterized in that the Mn 2+ The added amount of is 2 μmol / L, the added amount of N-acetylglucosamine is 5 mmol / L, and the added amount of fucose is 20 mmol / L or 50 mmol / L.

[0065] Embodiment 13. The method according to any one of embodiments 1-12, characterized in that the Mn 2+ The N-acetylglucosamine and fucose are added on the starting day or the 8th day of the culture cycle, and the N-acetylglucosamine and fucose are added on the 5th day of the culture cycle.

[0066] Embodiment 14. The method according to any one of embodiments 1-13, characterized in that 0.1-2.0 μmol / L Mn is added on the starting day or the 8th day of the culture cycle. 2+ , 1-10 mmol / L N-acetylglucosamine and 10-100 mmol / L fucose were added on the 5th day of the culture cycle.

[0067] Embodiment 15. The method according to any one of embodiments 1 to 14, characterized in that 0.7-2.0 μmol / L Mn is added on the starting day or the 8th day of the culture cycle. 2+ , 2-10 mmol / L N-acetylglucosamine and 20-50 mmol / L fucose were added on the 5th day of the culture cycle.

[0068] Embodiment 16. The method according to any one of embodiments 1-15, characterized in that 2 μmol / L Mn 2+ , 5 mmol / L N-acetylglucosamine and 20 mmol / L or 50 mmol / L fucose were added on the 5th day of the culture cycle.

[0069] Embodiment 17. The method according to any one of Embodiments 1-16, characterized in that the Mn 2+ Provided by manganese chloride.

[0070] Embodiment 18. A method for producing an antibody, characterized in that the culture conditions of the antibody-producing cells are: 36.5°C, 8% CO2, 130 rpm, the culture period is 14 days, and 2 μmol / L Mn is added on the 8th day of the culture period. 2+ , 5 mmol / L N-acetylglucosamine and 20 mmol / L or 50 mmol / L fucose were added on the 5th day of the culture cycle.

[0071] Embodiment 19. The method according to any one of embodiments 1-18 is characterized in that the antibody is penampalimab, bemosubinumab, daratumumab, mepolizumab, ramucirumab, pertuzumab, guselkumab, rituximab, trastuzumab, adalimumab, bevacizumab, epezetuzumab, obinutuzumab, pembrolizumab, lomosozumab, alirocumab, bimegizumab, denosumab, elotuzumab, nivolumab, secukinumab, ustekinumab, belimumab, nemolizumab, lencanezumab, rozelixizumab, faricizumab or atezolizumab.

[0072] Embodiment 20. The method according to any one of Embodiments 1-19, characterized in that the antibody is an anti-TIM-3 antibody or an anti-LAG-3 antibody.

[0073] Embodiment 21. The method according to Embodiment 20, characterized in that the anti-TIM-3 monoclonal antibody comprises HCDR1 shown in the amino acid sequence of SEQ ID NO: 1, HCDR2 shown in the amino acid sequence of SEQ ID NO: 2, HCDR3 shown in the amino acid sequence of SEQ ID NO: 3, LCDR1 shown in the amino acid sequence of SEQ ID NO: 4, LCDR2 shown in the amino acid sequence of SEQ ID NO: 5, and LCDR3 shown in the amino acid sequence of SEQ ID NO: 6; or

[0074] The anti-TIM-3 monoclonal antibody comprises HCDR1 shown in the amino acid sequence of SEQ ID NO: 7, HCDR2 shown in the amino acid sequence of SEQ ID NO: 8, HCDR3 shown in the amino acid sequence of SEQ ID NO: 9, LCDR1 shown in the amino acid sequence of SEQ ID NO: 10, LCDR2 shown in the amino acid sequence of SEQ ID NO: 11, and LCDR3 shown in the amino acid sequence of SEQ ID NO: 12.

[0075] Embodiment 22. The method according to Embodiment 20 or 21, characterized in that the anti-TIM-3 monoclonal antibody comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 13 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 14; or

[0076] The anti-TIM-3 monoclonal antibody comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 15 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 16.

[0077] Embodiment 23. The method according to any one of Embodiments 20-22, characterized in that the anti-TIM-3 monoclonal antibody comprises a heavy chain shown in the amino acid sequence of SEQ ID NO: 17 and a light chain shown in the amino acid sequence of SEQ ID NO: 18; or

[0078] The anti-TIM-3 monoclonal antibody comprises a heavy chain shown in the amino acid sequence of SEQ ID NO: 19 and a light chain shown in the amino acid sequence of SEQ ID NO: 20.

[0079] Embodiment 24. The method according to Embodiment 20 is characterized in that the anti-LAG-3 monoclonal antibody comprises HCDR1 shown in the amino acid sequence of SEQ ID NO: 21, HCDR2 shown in the amino acid sequence of SEQ ID NO: 22, HCDR3 shown in the amino acid sequence of SEQ ID NO: 23, LCDR1 shown in the amino acid sequence of SEQ ID NO: 24, LCDR2 shown in the amino acid sequence of SEQ ID NO: 25 and LCDR3 shown in the amino acid sequence of SEQ ID NO: 26; or

[0080] The anti-LAG-3 monoclonal antibody comprises HCDR1 shown in the amino acid sequence of SEQ ID NO: 27, HCDR2 shown in the amino acid sequence of SEQ ID NO: 28, HCDR3 shown in the amino acid sequence of SEQ ID NO: 29, LCDR1 shown in the amino acid sequence of SEQ ID NO: 30, LCDR2 shown in the amino acid sequence of SEQ ID NO: 31 and LCDR3 shown in the amino acid sequence of SEQ ID NO: 32.

[0081] Embodiment 25. The method according to Embodiment 20 or 24, characterized in that the anti-LAG-3 monoclonal antibody comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 33 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 34; or

[0082] The anti-LAG-3 monoclonal antibody comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 35 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 36.

[0083] Embodiment 26. The method according to any one of Embodiments 20, 24-25, characterized in that the anti-LAG-3 monoclonal antibody comprises a heavy chain shown in the amino acid sequence of SEQ ID NO: 37 and a light chain shown in the amino acid sequence of SEQ ID NO: 38; or

[0084] The anti-LAG-3 monoclonal antibody comprises a heavy chain represented by the amino acid sequence of SEQ ID NO: 39 and a light chain represented by the amino acid sequence of SEQ ID NO: 40.

[0085] Example 1 Seed solution preparation and cultivation

[0086] Take one daratumumab cryopreserved working cell bank cell (1 mL) from the liquid nitrogen tank, thaw it in a 37°C water bath, transfer it to a cell culture shake flask containing basal culture medium, and culture it in a carbon dioxide constant temperature incubator. The cell density is about 1.0-6.0×10 6 cells / mL, and the subculture density is about 0.2-1.0×10 6 cells / ml, the subculture medium was a basal medium containing 25 μmol / L MSX (Sigma), and the shaking flask culture conditions were: 36.5°C, 8% CO2, 130 rpm. The culture medium in the logarithmic growth phase and with good cell status was used as the seed medium.

[0087] The seed solution was inoculated into a cell culture shake flask containing a basal medium and placed in a carbon dioxide constant temperature incubator for 14 days. The culture conditions were: 36.5°C, 8% CO2, 130 rpm. 2mmol / kg sodium valproate solution was added to D5, and feed medium was added to D3, D5, D7, D9, D11, and D13. The cells were harvested after 14 days of culture, and the antibodies were purified after centrifugation to determine the antibody expression level and glycoform.

[0088] The antibody expression was calculated by high performance liquid chromatography at a wavelength of 280 nm.

[0089] The samples were digested with PNGase F to remove N-glycosides, labeled with 2-AB (Sigma), separated by hydrophilic interaction chromatography column, and detected by fluorescence detector to calculate the content of each glycoform.

[0090] Example 2 Mn 2+ Effect of concentration on glycoform distribution

[0091] According to the method of Example 1, manganese chloride (0.4 μmol / L, 0.6 μmol / L, 0.7 μmol / L, 1 μmol / L) was added on the first day of the culture cycle to culture cells, and the antibody expression level and glycoform were measured. The results are shown in Table 1. 2+ Significantly increased the galactosylation level of antibodies, but after adding to a certain concentration, the galactosylation modification reached a plateau and there was no significant increase; Mn 2+ The regulation of Man5 (high mannose glycoform) and defucosylation was weak, and the effect on antibody expression was small.

[0092] Table 1 Mn 2+ Effect of concentration on glycoform distribution

[0093] <![CDATA[Mn 2+ (μmol / L)]]> Expression level (g / L) Man5 (%) Defucosylation (%) Galactosylation (%) 0 4.58 2.37 4.76 11.55 0.4 4.62 2.59 4.77 26.48 0.6 4.67 2.42 4.66 27.58 0.7 4.65 2.33 4.71 28.19 1.0 4.65 2.24 4.65 28.33

[0094] Example 3 Effect of fucose concentration on glycoform distribution

[0095] According to the method of Example 1, fucose (20 mmol / L, 100 mmol / L) was added to culture cells until D5, and the antibody expression and glycoform were measured. The results are shown in Table 2. The addition of fucose significantly increased the galactosylation level of the antibody, reduced the Man5 level and the defucosylation level, and a higher concentration of fucose would affect the antibody expression.

[0096] Table 2 Effect of fucose concentration on glycoform distribution

[0097] Fucose (mmol / L) Expression (g / L) Man5 (%) Defucosylation (%) Galactosylation (%) 0 4.58 2.37 4.76 11.55 20 4.28 2.19 2.20 12.43 100 2.52 1.73 1.27 20.71

[0098] Example 4 Mn 2+ Effects of combined addition of β-glucose and fucose on glycoform distribution

[0099] According to the method of Example 1, 0.7 μmol / L manganese chloride was added on the first day of the culture cycle, and fucose (20 mmol / L, 100 mmol / L) was added to culture cells on D5 to measure the antibody expression and glycoform. The results are shown in Table 3. Lower concentrations of fucose can break through Mn 2+ Single regulation of the plateau phase of galactosylation modification can significantly and continuously increase the galactosylation level, Man5 level and defucosylation level.

[0100] Table 3 Mn 2+ Effects of different fucose concentrations on glycoform distribution

[0101] <![CDATA[Mn 2+ (μmol / L)]]> Fucose (mmol / L) Expression level (g / L) Man5 (%) Defucosylation (%) Galactosylation (%) 0.7 0 4.66 2.32 4.05 28.61 0.7 20 4.23 1.47 2.8 33.45 0.7 100 2.76 1.24 1.71 46.71

[0102] Example 5 Mn 2+ Effect of combined addition of 1,2-dihydropyruvate and N-acetylglucosamine on the distribution of glycoforms

[0103] According to the method of Example 1, 0.7 μmol / L manganese chloride was added on the first day of the culture cycle, and N-acetylglucosamine (2 mmol / L, 5 mmol / L, 10 mmol / L) was added to culture cells until D5, and the antibody expression and glycoform were measured. The results are shown in Table 4. The addition of N-acetylglucosamine can reduce the galactosylation level of the antibody and the Man5 level, and has little effect on the antibody expression.

[0104] Table 4 Mn 2+ Effects of different concentrations of N-acetylglucosamine on the distribution of glycoforms

[0105] <![CDATA[Mn 2+ (μmol / L)]]> N-acetylglucosamine (mmol / L) Expression level (g / L) Man5 (%) Defucosylation (%) Galactosylation (%) 0.7 0 4.13 2.95 3.89 27.92 0.7 2 4.39 2.16 3.93 17.63 0.7 5 4.35 1.91 3.67 15.09 0.7 10 4.20 1.68 3.55 13.17

[0106] Example 6 Mn 2+ Effects of combined addition of N-acetylglucosamine and fucose on glycoform distribution

[0107] According to the method of Example 1, N-acetylglucosamine (5 mmol / L) and fucose (20 mmol / L, 50 mmol / L) were added to culture until D5, and 2 μmol / L manganese chloride was added to culture cells on D8 to measure the antibody expression and glycoform. The results are shown in Table 5. During the culture process, only 2 μmol / L Mn 2+ The levels of galactosylation, Man5 and defucosylation were 1.83%, 2.76% and 25.85%, respectively. 2+ Adding 20 mmol / L fucose to the system increased galactosylation and reduced Man5 and defucosylation levels. The galactosylation level increased to 30.80%, while the Man5 and defucosylation levels decreased to 1.35% and 1.98%, respectively. 2+ Adding 5 mmol / L N-acetylglucosamine to the system can simultaneously reduce the levels of Man5, defucosylation, and galactosylation to 1.53%, 2.24%, and 16.80%, respectively. 2+ Gradually increasing the concentration of fucose in the system of 5 mmol / L N-acetylglucosamine can gradually increase the levels of galactosylation and defucosylation, and continuously reduce the level of Man5. When the fucose concentration increased from 20 mmol / L to 50 mmol / L, the galactosylation level increased from 19.49% to 22.80%, the defucosylation level increased from 1.61% to 1.86%, and Man5 decreased from 1.12% to 0.80%.

[0108] Table 5 Mn 2+ Effects of combined addition of N-acetylglucosamine and fucose on glycoform distribution

[0109] <![CDATA[Mn 2+ (μmol / L)]]> N-acetylglucosamine (mmol / L) Fucose (mmol / L) Expression level (g / L) Man5 (%) Defucosylation (%) Galactosylation (%) 2 0 0 3.58 1.83 2.76 25.85 2 0 20 3.51 1.35 1.98 30.80 2 5 0 3.54 1.53 2.24 16.80 2 5 20 3.56 1.12 1.61 19.49 2 5 50 3.28 0.80 1.86 22.80

[0110] Mn 2+, N-acetylglucosamine and fucose can be added in combination to flexibly adjust the anti-TIM-3 antibody (the heavy chain is shown in SEQ ID NO: 17 and the light chain is shown in SEQ ID NO: 18, the heavy chain is shown in SEQ ID NO: 19 and the light chain is shown in SEQ ID NO: 20), the anti-LAG-3 antibody (the heavy chain is shown in SEQ ID NO: 37 and the light chain is shown in SEQ ID NO: 38, the heavy chain is shown in SEQ ID NO: 39 and the light chain is shown in SEQ ID NO: 40), penamplimab, bemosubishizumab, daratumumab, mepolizumab, ramucirumab, pertuzumab, guselkumab, rituximab, trastuzumab, adalimumab, bevacizumab, epezetuzumab, obinutuzumab, pembrolizumab, lomosozumab, alirocumab, bimegizumab, denosumab, elotuzumab, nivolumab, secukinumab, ustekinumab, belimumab, nemolizumab, lencanezumab, rozelixizumab, faricizumab, atezolizumab and other antibodies Man5, defucosylation and galactosylation levels to obtain antibodies that meet the drug quality standards.

Claims

1. A method for regulating antibody glycosylation, characterized in that: Add 0.7-2.0 μmol / L Mn on the first day or on the 8th day of the culture cycle of antibody-producing cells. 2+ , 2-10 mmol / L N-acetylglucosamine and 20-50 mmol / L fucose were added on the 5th day of the culture cycle.

2. The method according to claim 1, characterized in that On the 8th day of the culture cycle, 2 μmol / L Mn 2+ , 5 mmol / L N-acetylglucosamine and 20 mmol / L or 50 mmol / L fucose were added on the 5th day of the culture cycle.

3. The method according to claim 1, characterized in that The Mn 2+ Provided by manganese chloride.

4. A method for producing an antibody, characterized in that: The culture conditions for antibody-producing cells were: 36.5°C, 8% CO2, 130 rpm, and a culture period of 14 days. On the 8th day of the culture period, 2 μmol / L Mn 2+ , 5 mmol / L N-acetylglucosamine and 20 mmol / L or 50 mmol / L fucose were added on the 5th day of the culture cycle.

5. The method according to any one of claims 1 to 4, characterized in that: The antibody is an anti-TIM-3 antibody, an anti-LAG-3 antibody, penamprimab, bemosubizumab, daratumumab, mepolizumab, ramucirumab, pertuzumab, guselkumab, rituximab, trastuzumab, adalimumab, bevacizumab, epezetuzumab, obinutuzumab, pembrolizumab, lomosozumab, alirocumab, bimegizumab, denosumab, elotuzumab, nivolumab, secukinumab, ustekinumab, belimumab, nemolizumab, lencanezumab, rozelixizumab, faricizumab or atezolizumab.

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