Cell culture method for improving galactosylation level of antibody
1. A cell culture method for improving the galactosylation level of antibodies is characterized in that CHO cells are used as an expression system, and galactose, uridine and manganese chloride tetrahydrate are added in the feeding process.
2. The method of claim 1, the CHO cell is selected from the group consisting of CHO-S, CHO-K1, CHO-GS, or CHO-DG44 cell; preferably, the CHO cells are CHO-K1 cells.
3. The method according to claim 2, wherein the total amount of galactose is added in the range of 3.30 to 9.40g/kg of the initial culture solution, the total amount of uridine is added in the range of 0.92 to 2.60g/kg of the initial culture solution, and the total amount of manganese chloride tetrahydrate is added in the range of 1.48 to 4.20mg/kg of the initial culture solution;
preferably, the total amount of galactose is 3.45-9.20g/kg of the initial culture solution, the total amount of uridine is 0.94-2.51g/kg of the initial culture solution, and the total amount of manganese chloride tetrahydrate is 1.51-4.03mg/kg of the initial culture solution;
preferably, the total amount of galactose is 5.18-9.20g/kg of the initial culture solution, the total amount of uridine is 1.41-2.51g/kg of the initial culture solution, and the total amount of manganese chloride tetrahydrate is 2.27-4.03mg/kg of the initial culture solution;
preferably, the total amount of galactose is 3.45g/kg of the initial culture solution, the total amount of uridine is 0.94g/kg of the initial culture solution, and the total amount of manganese chloride tetrahydrate is 1.51mg/kg of the initial culture solution;
preferably, the total amount of galactose is 5.18g/kg of the initial culture solution, the total amount of uridine is 1.41g/kg of the initial culture solution, and the total amount of manganese chloride tetrahydrate is 2.27mg/kg of the initial culture solution;
preferably, the total amount of galactose is 6.90g/kg of the initial culture solution, the total amount of uridine is 1.88g/kg of the initial culture solution, and the total amount of manganese chloride tetrahydrate is 3.02mg/kg of the initial culture solution;
preferably, the total amount of galactose is 9.20g/kg of the initial culture solution, the total amount of uridine is 2.51g/kg of the initial culture solution, and the total amount of manganese chloride tetrahydrate is 4.03mg/kg of the initial culture solution.
4. The method according to any one of claims 1 to 3, wherein the cell culture method comprises the steps of:
a) adding a basal culture medium into a shake flask or a cell reactor, and inoculating cells for culture;
b) equally dividing the galactose, the uridine and the manganese chloride tetrahydrate into five equal parts, respectively adding the five equal parts on the 3 rd, 5th, 7 th, 9 th and 11 th days of culture, and adding a supplementary culture medium;
c) cooling to 33.0 ℃ after the culture is carried out for 5 days, maintaining the temperature, and continuing the culture until 13-15 days are finished;
the method can increase the glycosylation level of the harvested antibody by at least 199%;
preferably, the method is capable of increasing the level of glycosylation of the harvested antibody by 218%;
preferably, the method is capable of increasing the glycosylation level of the harvested antibody by 227%;
preferably, the method is capable of increasing the level of glycosylation of the harvested antibody by 232%.
5. The method of claim 4, wherein the feed medium is in one or more combinations.
6. The method of claim 4, wherein the antibody is a monospecific antibody or a bispecific antibody or a multispecific antibody.
7. The method of claim 4, wherein the antibody is a monoclonal antibody.
8. The method of claim 7, wherein the monoclonal antibody is an anti-CD 20 monoclonal antibody.
9. The method of claim 8, wherein said anti-CD 20 monoclonal antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein said VH comprises the complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein said HCDR1 comprises the amino acid sequence set forth in SEQ ID NO 5, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO 6 and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO 7; wherein the VL comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO. 8, LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 9 and LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 10.
10. The method of claim 9, wherein the anti-CD 20 monoclonal antibody is rituximab.
Background
The CHO cell (Chinese Hamster ovary cell) is one of the most extensive host cells for producing antibody drugs, compared with other cell expression systems, the CHO cell expression system can be stably integrated with exogenous genes, and meanwhile, the interior of the cell contains a complicated post-translational modification system of eukaryotic cells, so that the expressed antibody drugs are similar to human monoclonal antibodies in antibody structure, immunogenicity, glycosylation type and mode, and the like, therefore, the drug property and the drug effect of the antibodies can be ensured, meanwhile, the CHO cell per se secretes less endogenous protein, and the subsequent separation and purification of the antibodies are facilitated, and common CHO cells comprise CHO-S, CHO-K1, CHO-GS, CHO-DG44 cells and the like.
There are two main important action modes (effector functions) of antibody (including Fc fusion protein) drugs for targeting and killing target cells (such as tumor cells): ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complementary dependent cytotoxicity). Wherein CDC activates the classical pathway of complement by binding a specific antibody to a corresponding antigen on the surface of a cell membrane to form a complex, and the formed attack membrane complex exerts a lytic effect on a target cell.
Therefore, the mechanism of action of CDC is an important index for in vitro research, evaluation, and screening of antibody drug development. In addition, the effect is directly related to the level and type of glycosylation modification. Currently, antibody engineering against CDC antibody effector functions is mainly focused on the engineering of Fc to enhance the interaction of the antibody with Fc γ R, ultimately increasing effector function.
However, methods for modifying the glycoform or glycosylation level of an antibody by engineering Fc through genetic means have a long period of time, and require long-term verification of the safety, stability and effectiveness of the expressed antibody. In addition to altering CDC effects through genetic engineering of antibody Fc, improvements in upstream or downstream production processes may be relied upon to increase CDC activity of an antibody. Development time for improving upstream or downstream production processes is shorter than that of genetic approaches, and overall development costs can be reduced.
Therefore, there is still a need to develop an improved antibody production process to change glycosylation profile, increase CDC activity of antibody, and meet the actual production requirement.
Disclosure of Invention
The invention provides an improved process method, which adopts a cell culture process of a CHO expression system, can improve the galactosylation level of an antibody and the CDC activity of the antibody by adding galactose, manganese chloride tetrahydrate and uridine in the feeding process of cell culture, and is simple and easy to operate.
To achieve the above objects, in some embodiments, the present invention uses CHO cells as an expression system, and galactose, manganese chloride tetrahydrate, and uridine are added during feeding;
further, in some embodiments, the present invention uses CHO-S, CHO-K1, CHO-GS or CHO-DG44 cells as expression systems, and galactose, manganese chloride tetrahydrate and uridine are added during feeding;
further, in some embodiments, the present invention uses CHO-K1 cell as an expression system, and galactose, manganese chloride tetrahydrate and uridine are added during feeding.
In some embodiments, galactose is added in a total amount of 3.30 to 9.40g/kg of the initial culture solution during feeding, uridine is added in a total amount of 0.92 to 2.60g/kg of the initial culture solution during feeding, and manganese chloride tetrahydrate is added in a total amount of 1.48 to 4.20mg/kg of the initial culture solution during feeding;
further, the total amount of galactose added in the feeding process is 3.45-9.20g/kg of initial culture solution, the total amount of uridine added in the feeding process is 0.94-2.51g/kg of initial culture solution, and the total amount of manganese chloride tetrahydrate added in the feeding process is 1.51-4.03mg/kg of initial culture solution;
further, the total amount of galactose added during feeding is 5.18-9.20g/kg of the initial culture solution, the total amount of uridine added during feeding is 1.41-2.51g/kg of the initial culture solution, and the total amount of manganese chloride tetrahydrate added during feeding is 2.27-4.03mg/kg of the initial culture solution;
preferably, in some embodiments, galactose is added in a total amount of 3.45g/kg of the initial culture solution during the feeding, uridine is added in a total amount of 0.94g/kg of the initial culture solution during the feeding, and manganese chloride tetrahydrate is added in a total amount of 1.51mg/kg of the initial culture solution during the feeding.
Preferably, in some embodiments, galactose is added in a total amount of 5.18g/kg of the initial culture solution during the feeding, uridine is added in a total amount of 1.41g/kg of the initial culture solution during the feeding, and manganese chloride tetrahydrate is added in a total amount of 2.27mg/kg of the initial culture solution during the feeding.
Preferably, in some embodiments, galactose is added in a total amount of 6.90g/kg of the initial culture solution during the feeding, uridine is added in a total amount of 1.88g/kg of the initial culture solution during the feeding, and manganese chloride tetrahydrate is added in a total amount of 3.02mg/kg of the initial culture solution during the feeding.
Preferably, in some embodiments, galactose is added in a total amount of 9.20g/kg of the initial culture solution during the feeding, uridine is added in a total amount of 2.51g/kg of the initial culture solution during the feeding, and manganese chloride tetrahydrate is added in a total amount of 4.03mg/kg of the initial culture solution during the feeding.
In some embodiments, the cell culture method comprises the steps of:
a) adding a basal culture medium into a shake flask or a cell reactor, and inoculating any one of the cells for culture;
b) dividing the galactose, the uridine and the manganese chloride tetrahydrate into five equal parts, adding the five equal parts on the 3 rd, 5th, 7 th, 9 th and 11 th days of culture respectively, and adding a supplementary culture medium;
c) cooling to 33.0 ℃ after the culture is carried out for 5 days, maintaining the temperature, and continuing the culture until 13-15 days are finished;
the method can increase the glycosylation level of the harvested antibody by at least 199%;
further, the method can increase the glycosylation level of the harvested antibody by 218%;
still further, the method can increase the glycosylation level of the harvested antibody by 227%;
further, the method can increase the glycosylation level of the harvested antibody by 232%.
In some embodiments, the feed medium of the invention is a combination of one or more.
In some embodiments, the feed media of the invention are feed media a and feed media B.
In some embodiments, the cell culture method comprises the steps of:
a) adding a basal culture medium into a shake flask, and inoculating cells for culture;
b) adding a feed medium A, a feed medium B, 3.30-9.40g/kg of galactose of the initial culture solution, 0.92-2.60g/kg of uridine of the initial culture solution and 1.48-4.20mg/kg of manganese chloride tetrahydrate of the initial culture solution; on days 3, 5, 7, 9 and 11, dividing the substances into 5 equal parts each day for supplementing;
c) cooling to 33.0 ℃ after the culture is carried out for 5 days, maintaining the temperature, and continuing to culture until the culture is finished for 14 days;
the method can increase the glycosylation level of the harvested antibody by at least 199%;
further, the method can increase the glycosylation level of the harvested antibody by 218%;
still further, the method can increase the glycosylation level of the harvested antibody by 227%;
further, the method can increase the glycosylation level of the harvested antibody by 232%.
In some embodiments, the cell culture method comprises the steps of:
a) adding a basal culture medium into a shake flask, and inoculating cells for culture;
b) adding 25.0% (w/w) of feed medium A by weight of the initial culture solution, 1.5% (w/w) of feed medium B by weight of the initial culture solution, and 3.30-9.40g/kg of galactose of the initial culture solution, 0.92-2.60g/kg of uridine of the initial culture solution, 1.48-4.20mg/kg of manganese chloride tetrahydrate of the initial culture solution; on days 3, 5, 7, 9 and 11, dividing the substances into 5 equal parts each day for supplementing;
c) cooling to 33.0 ℃ after the culture is carried out for 5 days, maintaining the temperature, and continuing to culture until the culture is finished for 14 days;
the method can increase the glycosylation level of the harvested antibody by at least 199%;
further, the method can increase the glycosylation level of the harvested antibody by 218%;
still further, the method can increase the glycosylation level of the harvested antibody by 227%;
further, the method can increase the glycosylation level of the harvested antibody by 232%.
In some embodiments, the cell culture method comprises the steps of:
a) adding a basal culture medium into a shake flask, and inoculating cells for culture;
b) adding 25.0% (w/w) of feed medium A by weight of the initial culture solution, 1.5% (w/w) of feed medium B by weight of the initial culture solution, and 3.45-9.20g/kg of galactose of the initial culture solution, 0.94-2.51g/kg of uridine of the initial culture solution, 1.51-4.03mg/kg of manganese chloride tetrahydrate of the initial culture solution; on days 3, 5, 7, 9 and 11, dividing the substances into 5 equal parts each day for supplementing;
c) cooling to 33.0 ℃ after the culture is carried out for 5 days, maintaining the temperature, and continuing to culture until the culture is finished for 14 days;
the method can increase the glycosylation level of the harvested antibody by at least 199%;
further, the method can increase the glycosylation level of the harvested antibody by 218%;
still further, the method can increase the glycosylation level of the harvested antibody by 227%;
further, the method can increase the glycosylation level of the harvested antibody by 232%.
In some embodiments, the cell culture method comprises the steps of:
a) adding a basal culture medium into a shake flask, and inoculating cells for culture;
b) adding 25.0% (w/w) of feed medium A by weight of the initial culture solution, 1.5% (w/w) of feed medium B by weight of the initial culture solution, and 5.18-9.20g/kg of galactose of the initial culture solution, 1.41-2.51g/kg of uridine of the initial culture solution, 2.27-4.03mg/kg of manganese chloride tetrahydrate of the initial culture solution; on days 3, 5, 7, 9 and 11, dividing the substances into 5 equal parts each day for supplementing;
c) cooling to 33.0 ℃ after the culture is carried out for 5 days, maintaining the temperature, and continuing to culture until the culture is finished for 14 days;
the method can increase the glycosylation level of the harvested antibody by 218%;
still further, the method can increase the glycosylation level of the harvested antibody by 227%;
further, the method can increase the glycosylation level of the harvested antibody by 232%.
In some embodiments, the cell culture method comprises the steps of:
a) adding a basal culture medium into a shake flask, and inoculating cells for culture;
b) adding 25.0% (w/w) of feed medium A by weight of the initial culture solution, 1.5% (w/w) of feed medium B by weight of the initial culture solution, and 3.45g/kg of galactose of the initial culture solution, 0.94g/kg of uridine of the initial culture solution, 1.51mg/kg of manganese chloride tetrahydrate of the initial culture solution; on days 3, 5, 7, 9 and 11, dividing the substances into 5 equal parts each day for supplementing;
c) cooling to 33.0 ℃ after the culture is carried out for 5 days, maintaining the temperature, and continuing to culture until the culture is finished for 14 days;
the method can increase the glycosylation level of the harvested antibody by 199%.
In some embodiments, the cell culture method comprises the steps of:
a) adding a basal culture medium into a shake flask, and inoculating cells for culture;
b) adding 25.0% (w/w) of feed medium A by weight of the initial culture solution, 1.5% (w/w) of feed medium B by weight of the initial culture solution, and 5.18g/kg of galactose of the initial culture solution, 1.41g/kg of uridine of the initial culture solution, 2.27mg/kg of manganese chloride tetrahydrate of the initial culture solution; on days 3, 5, 7, 9 and 11, dividing the substances into 5 equal parts each day for supplementing;
c) cooling to 33.0 ℃ after the culture is carried out for 5 days, maintaining the temperature, and continuing to culture until the culture is finished for 14 days;
the method can increase the glycosylation level of the harvested antibody by 218 percent.
In some embodiments, the cell culture method comprises the steps of:
a) adding a basal culture medium into a shake flask, and inoculating cells for culture;
b) adding 25.0% (w/w) of feed medium A by weight of the initial culture solution, 1.5% (w/w) of feed medium B by weight of the initial culture solution, and 6.90g/kg of galactose of the initial culture solution, 1.88g/kg of uridine of the initial culture solution, 3.02mg/kg of manganese chloride tetrahydrate of the initial culture solution; on days 3, 5, 7, 9 and 11, dividing the substances into 5 equal parts each day for supplementing;
c) cooling to 33.0 ℃ after the culture is carried out for 5 days, maintaining the temperature, and continuing to culture until the culture is finished for 14 days;
the method can improve the glycosylation level of the harvested antibody by 227%.
In some embodiments, the cell culture method comprises the steps of:
a) adding a basal culture medium into a shake flask, and inoculating cells for culture;
b) adding 25.0% (w/w) of feed medium A by weight of the initial culture solution, 1.5% (w/w) of feed medium B by weight of the initial culture solution, and 9.20g/kg of galactose of the initial culture solution, 2.51g/kg of uridine of the initial culture solution, 4.03mg/kg of manganese chloride tetrahydrate of the initial culture solution; on days 3, 5, 7, 9 and 11, dividing the substances into 5 equal parts each day for supplementing;
c) cooling to 33.0 ℃ after the culture is carried out for 5 days, maintaining the temperature, and continuing to culture until the culture is finished for 14 days;
the method can increase the glycosylation level of the harvested antibody by 232 percent.
In some embodiments, the cell culture method comprises the steps of:
a) adding a basic culture medium into a cell reactor, and inoculating cells for culture;
b) adding 25.0% (w/w) of feed medium A by weight of the initial culture solution, 1.5% (w/w) of feed medium B by weight of the initial culture solution, and 6.90g/kg of galactose of the initial culture solution, 1.88g/kg of uridine of the initial culture solution, 3.02mg/kg of manganese chloride tetrahydrate of the initial culture solution; on days 3, 5, 7, 9 and 11, dividing the substances into 5 equal parts each day for supplementing;
c) the temperature is reduced to 33.0 ℃ by the 5th day of culture, and the temperature is maintained to continue the culture until the 14 th day is finished.
In some embodiments, the basal Medium is selected from Dynamis AGT Medium or ActiPro.
In some embodiments, feed medium a is selected from Cell Boost 7a, and feed medium B is selected from Cell Boost 7B.
In one embodiment, the antibody is a monospecific antibody or a bispecific antibody or a multispecific antibody.
In one embodiment, the antibody is a monoclonal antibody, further the monoclonal antibody is an anti-CD 20 monoclonal antibody.
In one embodiment, the anti-CD 20 monoclonal antibody of the invention comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises the complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID No. 5, HCDR2 comprises the amino acid sequence set forth in SEQ ID No. 6 and HCDR3 comprises the amino acid sequence set forth in SEQ ID No. 7; wherein the VL comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO. 8, LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 9 and LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 10.
In one embodiment, the anti-CD 20 monoclonal antibody of the present invention comprises HCDR1 comprising the amino acid sequence shown by SYNMH (SEQ ID NO:5), HCDR2 comprising the amino acid sequence shown by AIYPGNGDTSYNQKFKG (SEQ ID NO:6) and HCDR3 comprising the amino acid sequence shown by STYYGGDWYFNV (SEQ ID NO: 7); wherein the LCDR1 comprises the amino acid sequence shown as RASSSVSYIH (SEQ ID NO:8), LCDR2 comprises the amino acid sequence shown as ATSNLAS (SEQ ID NO:9) and LCDR3 comprises the amino acid sequence shown as QQWTSNPPT (SEQ ID NO: 10).
In one embodiment, the anti-CD 20 monoclonal antibody of the invention is rituximab.
Heavy chain of an exemplary anti-CD 20 monoclonal antibody of the invention:
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:1)
light chain of an exemplary anti-CD 20 monoclonal antibody of the invention:
QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:2)
heavy chain variable region of an exemplary anti-CD 20 monoclonal antibody of the invention:
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRG LEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAV YYCARSTYYGGDWYFNVWGAGTTVTVSA(SEQ ID NO:3)。
light chain variable region of an exemplary anti-CD 20 monoclonal antibody of the invention:
QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIY ATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGG GTKLEIK(SEQ ID NO:4)
Detailed Description
Before the present invention is described in detail, it is to be understood that this invention is not limited to the particular methodology and experimental conditions set forth herein as such may vary. In addition, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
Definition of
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.
The term "initial medium" refers to a medium at the time of first addition of a medium and completion of cell inoculation, wherein the cell inoculation density may be generally 0.8 to 1.2X 106Cells/ml.
The term "monoclonal antibody" refers to a preparation of antibody molecules having a single amino acid composition, and not to the method of production thereof. Monoclonal antibodies or antigen-binding fragments thereof can be produced, for example, by hybridoma techniques, recombinant techniques, phage display techniques, synthetic techniques such as CDR grafting, or a combination of such or other techniques known in the art.
The term "monospecific" antibody refers to an antibody having one or more binding sites each binding to the same epitope of the same antigen.
The term "bispecific" means that an antibody is capable of specifically binding to at least two different antigenic determinants, e.g., two binding sites each formed by a pair of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) bind to different antigens or different epitopes on the same antigen. Such bispecific antibodies are in the 1+1 format. Other bispecific antibody formats are the 2+1 format (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or the 2+2 format (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). Typically, bispecific antibodies comprise two antigen binding sites, each antigen binding site being specific for a different antigenic determinant.
The term "multispecific" antibody refers to an antibody having at least two antigen binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. Multispecific antibodies are antibodies that have binding specificities for at least two different epitopes. The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or the domain of a light chain involved in binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies typically have similar structures, with each variable domain comprising four conserved Framework Regions (FRs) and three complementarity determining regions. (see, e.g., Kindt et al Kuby Immunology, 6th ed., page W.H.Freeman and Co.91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains from antibodies that bind to a particular antigen can be used to isolate antibodies that bind the antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al, j.immunol.150: 880- & ltwbr & gt 887 & gt (1993); clarkson et al, Nature 352: 624-628(1991).
"complementarity determining regions" or "CDR" or "hypervariable region" (used interchangeably herein with hypervariable region "HVR") are regions in an antibody variable domain which are mutated in sequence and form structurally defined loops ("hypervariable loops") and/or which contain antigen-contacting residues ("antigen-contacting points"). The CDRs are primarily responsible for binding to an epitope of the antigen. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR 3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including, for example: chothia (Chothia et Al (1989) Nature 342:877- & 883, Al-Lazikani et Al, "Standard constraints for the structural organization of immunology", Journal of Molecular Biology,273,927- & 948(1997)), based on antibody sequence variations Kabat (Kabat et Al, Sequences of Proteins of Immunological Interest, 4 th edition, U.S. Depatm of Health and Human Services, National Institutes of Health (1987)), AbM (fundamental of Molecular), activity university Collection (London), Muinnocent tissue (IMGT) (international patent publication).
Unless otherwise indicated, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), reference is made to the numbering positions according to the Kabat numbering system (Kabat et al, Sequences of Proteins of Immunological Interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
Drawings
FIG. 1 is the cell viability of the cell feed batch experiment in example 1.
FIG. 2 is a graph of glycosylation content of the cell feed batch experiment in example 1.
FIG. 3 is a schematic diagram of the glycosyl structure of the antibody of the example.
FIG. 4 cell viability of the cell feed batch experiment in example 2.
FIG. 5 is the viable cell density of the cell feed batch experiment in example 2.
FIG. 6 is a graph of antibody expression levels for the cell feed batch experiment in example 2.
FIG. 7 is a graph of glycosylation content of the cell feed batch experiment in example 2.
Detailed Description
A full disclosure and description of how to make and use the methods and compositions of the present invention is provided to those of ordinary skill in the art by the following examples, which are not intended to limit the scope of what is encompassed by the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
The Chinese hamster ovary cell line CHO-K1 in the examples of the present invention was purchased from ATCC under the accession number: CCL-61TM。
The basal Medium used in the present invention, Dynamis AGT Medium, was purchased from Gibco under the accession number A26175-03; ActiPro is available from HyClone, cat # SH 31037.04;
feed medium used in the present invention: cell Boost 7a was purchased from HyClone, cat #: SH 31026.05; cell Boost 7b was purchased from HyClone, cat #: SH31027.04CN, respectively;
galactose used in the present invention was purchased from Spectrum, cat # usa: G5924C; manganese chloride was purchased from spectra, usa, cat # s: m1106; uridine was purchased from Ameresco, usa under the cat number: 0975;
the anti-CD 20 antibody used in the examples of the invention is rituximab.
Example 1: CHO-K1 cell shake flask culture for expressing rituximab
In the cell culture experiment, 80mL of basal Medium (Dynamis AGT Medium) was added to 250mL of shake flasks on day 0, and CHO-K1 cells were cultured at 1.0X 106cells/mL were inoculated at 36.5 ℃. + -. 0.5 ℃ with 6%. + -. 1% CO2130 + -10 r/min, the temperature is reduced and controlled at 33 ℃ on the 5th day of culture, and the temperature is maintained until the 14 th day of cell culture, and then the culture is finished and the harvest is performed. Feeding was carried out on days 3, 5, 7, 9 and 11, respectively, and 25.0% (w/w) of feed medium A (Cell Boost 7a) by weight of the initial culture broth, 1.5% (w/w) of feed medium B (Cell Boost 7B) by weight of the initial culture broth, and 3.45g/kg of galactose of the initial culture broth, 0.94g/kg of manganese chloride of the initial culture broth, 1.51mg/kg of uridine of the initial culture broth (group A) were added for 5 days in total; or 5.18g/kg of galactose of the initial culture solution, 1.41g/kg of manganese chloride of the initial culture solution, 2.27mg/kg of uridine of the initial culture solution (group B); or 6.90mg/kg of galactose, 1.88g/kg of manganese chloride, 3.02mg/kg of uridine (group C) in the initial culture solution; or 9.20g/kg of galactose, 2.51g/kg of manganese chloride, and 4.03mg/kg of uridine (group D) in the initial culture broth. On days 3, 5, 7, 9 and 11, dividing the above materials into 5 parts every day, etcAdding the components. And (3) detecting the glucose content according to a biochemical analyzer on days 3, 5, 7, 9 and 11, and adding the glucose concentrated solution to increase the glucose concentration in the cell sap to 4.0g/L when the glucose content is lower than 3.0 g/L. Cell viability was measured on days 3, 5, 7, 9, and 11, and levels of glycosylation (G0F, G1F, G2F) and CDC activity were measured on day 14.
The CELL viability was measured using a CELL viability analyzer (manufacturer: BECKMAN, U.S.A., Cat. No.: Vi-CELL XR); the antibody glycosyl is subjected to enzymolysis by PNGase enzyme according to the instruction of a reagent kit (manufacturer NEB, product number: P0704L), and then the glycosylation (G0F, G1F and G2F) levels are detected by HPLC-MS (manufacturer Agilent, product number: Agilent 1260); the CDC activity of the antibody was measured using a microplate reader (manufacturer: U.S. Thermo, cat # MULTISKAN FC), and the structural diagrams of G0F, G1F and G2F are shown in FIG. 3. The glycosylation level in the invention is mainly examined on the content of G0F, G1F and G2F, wherein G0F indicates that the antibody Fc has no galactosylation, G1F indicates that the glycosylation end of the Fc segment has 1 galactosylation modification, and G2F indicates that the glycosylation end of the Fc segment has 2 galactosylation modifications. It has been shown that increasing galactosylation of antibodies (i.e., increasing the content of G1F and G2F and decreasing the content of G0F) can increase CDC activity, probably because the glycosylation-modified sugar chain structure can maintain the steric idea of the antibody, stabilize the structure of the antibody, and protect the antibody from hydrolysis by certain enzymes. (Jason Hodoniczky, Yuan Zhi Zheng and David C. James, Control of Recombinant Monoclonal Antibody effects by Fc N-Glycan modification in Vitro, Biotechnol. prog.2005 and Wangchong, modern immunology, development of Monoclonal Antibody glycosylation modification, vol. 37, No. 3, 2017).
This experiment shows that, compared with the control (no galactose, manganese chloride and uridine are added during feeding), the content of G2F and G1F gradually increases, the content of G0F gradually decreases and the relative activity of antibody CDC gradually increases as the addition amount of galactose, manganese chloride and uridine increases during feeding (see fig. 2 and table 1). In addition, as the addition amount of galactose, manganese chloride and uridine was increased, there was no significant difference in cell viability rate compared to the control, and the cell viability rate was maintained at 90% or more at 14 days after the end of the culture (see fig. 1). In conclusion, the addition of galactose, manganese chloride and uridine during the feeding process can significantly increase the content of G1F and G2F, reduce the content of G0F, increase the galactosylation level in group A by 199%, increase in group B by 218%, increase in group C by 227%, increase in group D by 232%, and increase the CDC activity of the antibody (see Table 1); in addition, galactose, manganese chloride and uridine are added during feeding, so that the activity of cells is not influenced.
Table 1: antibody glycosylation level and CDC relative activity
Note: the allowable range of the content error of each substance in the table is +/-2 percent; the initial weight of the culture solution in the shake flask culture was 0.08 kg.
Example 2: CHO-K1 cell reactor culture for expressing rituximab
This experiment was selected from group C of example 1 for scale-up testing.
In the cell culture test, day 0 in the cell culture test, at a density of 1.0X 106The cells were inoculated at one/ml with CHO-K1 cells into a 2L cell reactor (manufacturer: Sartorious, Germany, Cat.: BIOSTATR A) with DO set at 40%, pH controlled at 6.90. + -. 0.25, culture temperature at 36.5 deg.C, cooled to 33.0 deg.C by day 5, maintained at that temperature until day 14 of cell culture, and then terminated and harvested. The culture was carried out until the feed was performed on days 3, 5, 7, 9 and 11, respectively, and 25.0% (w/w) of feed medium A (Cell Boost 7a) by weight of the initial culture broth, 1.5% (w/w) of feed medium B (Cell Boost 7B) by weight of the initial culture broth, and 6.90g/kg of galactose of the initial culture broth, 1.88g/kg of manganese chloride of the initial culture broth, and 3.02mg/kg of uridine of the initial culture broth were added for 5 days. On days 3, 5, 7, 9 and 11, the above substances were divided into 5 equal parts each day for addition. Detecting the glucose content by a biochemical analyzer every day, and adding the glucose concentrated solution to increase the glucose concentration in the cell sap to 4.0g/L when the glucose is lower than 3.0 g/L. According to the condition of foam generated by aeration, manually adding when the foam is 5cm higher than the liquid levelThe total amount of 10% ADCF Antifoam Solution (manufacturer: Hyclone, cat # SH30897.01, USA) cannot exceed 50 ppm. The cell viability and cell density were measured every day, and the antibody expression level was measured from day 6 and the glycosylation (G0F, G1F, G2F) level was measured at day 14.
CELL viability and CELL density were determined using a CELL viability analyzer (manufacturer: U.S. BECKMAN, cat # Vi-CELL XR), antibody concentration was determined using a biochemical analyzer (manufacturer: Switzerland Roche, cat # CEDEX BIO HT), antibody glycosylation was enzymatically hydrolyzed using PNGase enzyme, enzymatic hydrolysis was performed according to the kit instructions (manufacturer NEB, cat # P0704L), and then levels of glycosylation (G0F, G1F, G2F) were determined using HPLC-MS (manufacturer: U.S. Agilent, cat # Agilent 1260). The experiment was repeated three times, corresponding to experiment one, experiment two and experiment three in FIGS. 4-7, with the initial broth weight of experiment one, experiment two and experiment three being 1.2 kg.
TABLE 2 content of three experiments G0F, G1F and G2F
Experiments show that the growth conditions of three experimental cells in the reactor are basically consistent, and the highest viable cell density is 29.0 multiplied by 106Around one/ml, no significant difference exists (see figure 4), the change trend of the cell viability rate is basically consistent, the cell viability rate is maintained to be more than 90% until the culture is finished, and no significant difference exists in three experiments (see figure 5). The final expression level of the antibody can reach more than 4.0mg/mL, and the antibody expression level between batches is not obviously different (see figure 6). In addition, after a certain proportion of galactose, manganese chloride and uridine is added, the content ratio of G1F + G2F to G0F is the same as that of the experiment before amplification, no difference exists among three amplification experiments (see figure 7 and table 2), and the cell density, the activity rate, the antibody yield and the like can be maintained without being influenced. And the method is verified to have good stability and can meet the requirement of actual amplification production.
SEQUENCE LISTING
<110> Xinda biopharmaceuticals (Suzhou) Limited
<120> a cell culture method for increasing galactosylation level of antibody
<130> P21012834C
<150> 202010184316.6
<151> 2020-03-16
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 451
<212> PRT
<213> Artificial Sequence
<220>
<223> heavy chain of anti-CD 20 antibody
<400> 1
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly
100 105 110
Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Lys
450
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<212> PRT
<213> Artificial Sequence
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<223> light chain of anti-CD 20 antibody
<400> 2
Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys
210
<210> 3
<211> 121
<212> PRT
<213> Artificial Sequence
<220>
<223> heavy chain variable region of anti-CD 20 antibody
<400> 3
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly
100 105 110
Ala Gly Thr Thr Val Thr Val Ser Ala
115 120
<210> 4
<211> 106
<212> PRT
<213> Artificial Sequence
<220>
<223> light chain variable region of anti-CD 20 antibody
<400> 4
Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
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<212> PRT
<213> Artificial Sequence
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<223> HCDR1 of anti-CD 20 antibody
<400> 5
Ser Tyr Asn Met His
1 5
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<212> PRT
<213> Artificial Sequence
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<223> HCDR2 of anti-CD 20 antibody
<400> 6
Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys
1 5 10 15
Gly
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<213> Artificial Sequence
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<223> HCDR3 of anti-CD 20 antibody
<400> 7
Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val
1 5 10
<210> 8
<211> 10
<212> PRT
<213> Artificial Sequence
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<223> LCDR1 of anti-CD 20 antibody
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Arg Ala Ser Ser Ser Val Ser Tyr Ile His
1 5 10
<210> 9
<211> 7
<212> PRT
<213> Artificial Sequence
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<223> LCDR2 of anti-CD 20 antibody
<400> 9
Ala Thr Ser Asn Leu Ala Ser
1 5
<210> 10
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> LCDR3 of anti-CD 20 antibody
<400> 10
Gln Gln Trp Thr Ser Asn Pro Pro Thr
1 5
