DESC:RELATED APPLICATIONS
This application is related to Indian provisional application IN202321089627 filed on 29th Dec. 2023 and is incorporated herein in its entirety.
FIELD OF THE INVENTION
The present invention relates to modulation of afucosylation in cell culture containing Pertuzumab by pH shift. The present invention further relates to a method of producing antibody in large-scale cell culture, with modulation of afucosylation using pH shift.
BACKGROUND OF THE INVENTION
Antibody efficacies critically depend on the structure of the oligosaccharides attached to the polypeptide as well as the amino acid sequences of the heavy and light chains. IgG1 has two N-linked oligosaccharide chains bound to the Fc region. The oligosaccharides are of the complex biantennary type, composed of a trimannosyl core structure with or without core fucose, bisecting N-acetylglucosamine (GlcNAc), galactose, and terminal sialic acid, all of which give rise to structural heterogeneities. These heterogeneities make comparability control difficult because of differences in pharmacokinetics, pharmacodynamics, and immunogenicity. Antibody-dependent cellular cytotoxicity (ADCC) is triggered upon binding of the antibody Fc region to lymphocyte receptors (Fc?Rs), and is dependent on the content of the fucose attached to the innermost GlcNAc of the N-linked Fc oligosaccharide.
Antibody-dependent cellular cytotoxicity (ADCC) is dependent on the fucose content of oligosaccharides bound to monoclonal antibodies (MAbs).
N-glycosylation of antibody Fc regions is essential for binding to Fc?R, which engages antibody effector functions. Fucosylation is one of the most common modifications involving oligosaccharides on glycoproteins or glycolipids. Fucosylation comprises the attachment of a fucose residue to N-glycans, O-glycans and glycolipids. Fucosylation is an important post translational modification which can affect the binding of IgG to Fc?RIIIa receptor leading to ADCC activity.
WO2015011660 discloses a method or process for controlling, inhibiting or reducing protein fucosylation in a eukaryote and/or a eukaryotic protein expression system. More specifically method comprises carrying out the protein expression and/or post-translational modification in the presence of an elevated total concentration of manganese or manganese ions.
WO2021066772 discloses a cell culture medium for the production of antibodies having reduced fucosylation, comprising an effective amount of a fucose analog and ZN+2 obtained by adding the fucose analog to the cell culture medium not before the 5th day.
WO2020042022 discloses a method for producing an afucosylated protein in a host cell comprising introducing a nucleic acid encoding at least one modified enzyme of the fucosylation pathway to the host cell to produce the afucosylated protein.
Antibody afucosylation is crucial for ADCC activity. Therefore, it is essential to create an enhanced, low-cost technique that can modulate the antibody's overall efficacy and ADCC activity.
Present invention address this critical issue by proposing a unique method involve pH shifting for modulation of afucosylation level in cell culture containing monoclonal antibody.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide a method for modulation of afucosylation level of antibody comprising a pH shift of cell culture.
Another object of the present invention is to provide a method for modulation of afucosylation level of anti-HER2 antibody comprising a pH shift of cell culture.
Another object of the present invention is to provide a method for modulation of afucosylation level of Pertuzumab comprising a pH shift of cell culture.
Another object of the present invention is to provide a method for modulation of afucosylation level of antibody comprising a pH shift of cell culture between 6.0 and 8.0 when cell culture achieve viable cell count of 18-22 x 106 cells/mL.
Another object of the present invention is to provide a method for modulation of afucosylation level of anti-HER2 antibody comprising a pH shift of cell culture between 6.0 and 8.0 when cell culture achieve viable cell count of 18-22 x 106 cells/mL.
Another object of the present invention is to provide a method for modulation of afucosylation level of Pertuzumab comprising a pH shift of cell culture between 6.0 and 8.0 when cell culture achieve viable cell count of 18-22 x 106 cells/mL.
Another object of the present invention is to provide a method for modulation of afucosylation level of Pertuzumab comprising temperature shift between 40oC and 30oC when cell culture achieve viable cell count of 18-22 x 106 cells/mL.
Another object of the present invention is to provide a method for modulation of afucosylation level of Pertuzumab comprising performing a pH shift or temperature shift of cell culture.
Another object of the present invention is to provide a method for modulation of afucosylation level of Pertuzumab comprising performing a pH shift of cell culture between 6.0 and 8.0 and temperature shift between 40oC and 30oC when cell culture achieve viable cell count of 18-22 x 106 cells/mL.
SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a method for modulation of afucosylation level of antibody comprising a pH shift of cell culture.
Another aspect of the present invention is to provide a method for modulation of afucosylation level of anti-HER2 antibody comprising a pH shift of cell culture.
Another aspect of the present invention is to provide a method for modulation of afucosylation level of Pertuzumab comprising a pH shift of cell culture.
Another aspect of the present invention is to provide a method for modulation of afucosylation level of antibody comprising a pH shift of cell culture between 6.0 and 8.0 when cell culture achieve viable cell count of 18-22 x 106 cells/mL.
Another aspect of the present invention is to provide a method for modulation of afucosylation level of anti-HER2 antibody comprising a pH shift of cell culture between 6.0 and 8.0 when cell culture achieve viable cell count of 18-22 x 106 cells/mL.
Another aspect of the present invention is to provide a method for modulation of afucosylation level of Pertuzumab comprising a pH shift of cell culture between 6.0 and 8.0 when cell culture achieve viable cell count of 18-22 x 106 cells/mL.
Another aspect of the present invention is to provide a method for modulation of afucosylation level of Pertuzumab comprising temperature shift between 40oC and 30oC when cell culture achieve viable cell count of 18-22 x 106 cells/mL.
Another aspect of the present invention is to provide a method for modulation of afucosylation level of Pertuzumab comprising performing a pH shift or temperature shift of cell culture.
Another aspect of the present invention is to provide a method for modulation of afucosylation level of Pertuzumab comprising performing a pH shift of cell culture between 6.0 and 8.0 and temperature shift between 40oC and 30oC when cell culture achieve viable cell count of 18-22 x 106 cells/mL.
BRIEF DESCRIPTION OF DRAWING
Figure 1: Effect of pH on afucosylation at Ambr 250.
Figure 2: Effect of pH on galactosylation at Ambr 250.
Figure 3: Effect of pH on high mannose at Ambr 250.
Figure 4: Effect of pH on galactosylation at 2000L bioreactor.
Figure 5: Effect of pH on high mannose at 2000L bioreactor.
Figure 6: Effect of pH on afucosylation at 2000L bioreactor.

DETAILED DESCRIPTION OF THE INVENTION
The following is a detailed description of embodiments of the invention. The embodiments are in such details as to clearly communicate the invention. However, the amount of details offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents of embodiments, and alternative falling within the spirit and scope of the present invention.
DEFINITION
The following definitions are provided to facilitate understanding of certain terms used throughout the specification.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred embodiments of compositions, methods and materials are described herein. For the purposes of the present disclosure, the following terms are defined below.
The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.
The words "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively. The words "consist", "consisting", and its variants, are to be interpreted exclusively, rather than inclusively. While various embodiments in the specification are presented using “comprising” language, under other circumstances, a related embodiment is also intended to be interpreted and described using “consisting of’ or “consisting essentially of’ language.
The term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), nanobodies, modified antibodies, subunits of antibodies, antibody derivatives, artificial antibodies, combinations of antibodies with proteins and antibody fragments sufficiently long to display the desired biological activity, the monoclonal antibodies as used herein may be human antibodies. As used herein, an antibody, or antigen-binding fragment thereof, that has "binding specificity for the HER2" binds to HER2. Pertuzumab is an example of an antibody that has binding specificity for the HER2 complex.
In the present invention antibody is selected from Ranibizumab, Denosumab, Pembrolizumab, Vedolizumab, Aflibercept, Daratumumab, Trastuzumab, Secukinumab, Pertuzumab, Nivolumab, Golimumab, Dupilumab, Etanercept, Atezolizumab, Risankizumab, Bevacizumab, Dulaglutide or Rituximab. More preferably, antibody selected is Pertuzumab.
The terms "culture" and "cell culture" as used herein refer to a eukaryotic cell population that is suspended in a medium (see definition of "medium" below) under conditions suitable to survival and/or growth of the cell population. As will be clear to those of ordinary skill in the art, these terms as used herein can refer to the combination comprising the mammalian cell population and the medium in which the population is suspended.
The term "Galactosylation" herein refers to the type and distribution of galactose residues on polysaccharides and oligosaccharides. Galactose refers to a group of monosaccharides, which include open chain and cyclic forms. An important disaccharide form of galactose is galactose-alpha-1, 3-galactose (-gal).
The term “Fucosylation” herein refers to the process of adding fucose sugar units to a molecule.
The term “Afucosylation” herein refers to biochemical phenomenon that involves the alteration of glycoproteins by the removal of fucose residues from their carbohydrate structures.
The term “High – mannose” herein refers to the glycans contain unsubstituted terminal mannose sugars. These glycans typically contain between five and nine mannose residues attached to the chitobiose (GlcNAc2) core.
The term “ADCC” herein refers to a powerful immune response mechanism employed by the body to combat infected or abnormal cells, such as cancer cells. It relies on a well-coordinated interplay between antibodies, immune cells, and target cells.
The main embodiment of the present invention is to provide a method for modulation of afucosylation level of antibody comprising a pH shift of cell culture.
Another embodiment of the present invention is to provide a method for modulation of afucosylation level of anti-HER2 antibody comprising a pH shift of cell culture.
Another embodiment of the present invention is to provide a method for modulation of afucosylation level of Pertuzumab comprising a pH shift of cell culture.
Another embodiment of the present invention is to provide a method for modulation of afucosylation level of antibody comprising a pH shift of cell culture between 6.0 and 8.0 when cell culture achieve viable cell count of 18-22 x 106 cells/mL.
Another embodiment of the present invention is to provide a method for modulation of afucosylation level of anti-HER2 antibody comprising a pH shift of cell culture between 6.0 and 8.0 when cell culture achieve viable cell count of 18-22 x 106 cells/mL.
Another embodiment of the present invention is to provide a method for modulation of afucosylation level of Pertuzumab comprising a pH shift of cell culture between 6.0 and 8.0 when cell culture achieve viable cell count of 18-22 x 106 cells/mL.
Another embodiment of the present invention is to provide a method for modulation of afucosylation level of Pertuzumab comprising temperature shift between 40oC and 30oC when cell culture achieve viable cell count of 18-22 x 106 cells/mL.
Another embodiment of the present invention is to provide a method for modulation of afucosylation level of Pertuzumab comprising performing a pH shift or temperature shift of cell culture.
Another embodiment of the present invention is to provide a method for modulation of afucosylation level of Pertuzumab comprising performing a pH shift of cell culture between 6.0 and 8.0 and temperature shift between 40oC and 30oC when cell culture achieve viable cell count of 18-22 x 106 cells/mL.
In the present invention pH shift of cell culture is of about 5.0-9.0, 5.2-8.8, 5.4-8.6, 5.6-8.4, 5.8-8.2, 6.0-8.0, 6.2-7.8, 6.4-7.6, 6.6-7.4, or 6.8-7.2. More preferably, pH shift of cell culture is 6.8-7.2.
In the present invention galactosylation profile of antibody is not affected due to pH shift of cell culture.
In the present invention high mannose level of antibody is not affected due to pH shift of cell culture.
In the present invention afucosylation profile of antibody is not affected due to manganese chloride.
In the present invention, the cell culture media is supplemented with glutamine, anti-clumping agent, kolliphor, manganese chloride, glucose, or EDTA.
In the present invention, the concentration of glutamine is maintained in cell culture media at of about 0.1-20 mM, 0.5-19.5 mM, 1-18 mM, 1.5-17.5 mM, 2-17 mM, 2.5-16.5 mM, 3-16 mM, 3.5-15.5 mM, 415 mM, 4.5-14.5 mM, 5-14 mM, 5.5-13.5 mM, 6-13 mM, 6.5-12.5 mM, 7-12 mM, 7.5-11.5 mM, 8-11mM, 8.5-10.5 mM, 9-10 mM, or 9.5-10.5 mM. More preferably, the concentration of glutamine after supplementation is maintained around 4mM to 15 mM.
In the present invention, the concentration of anti-clumping agent is maintained in cell culture media at of about 0.1-20%, 0.5-19.5%, 1-18%, 1.5-17.5%, 2-17%, 2.5-16.5%, 3-16%, 3.5-15.5%, 4-15%, 4.514.5%, 5-14%, 5.5-13.5%, 6-13%, 6.5-12.5%, 7-12%, 7.5-11.5%, 8-11%, 8.5-10.5%, 9-10%, or 9.5-10.5%. More preferably, the concentration of anti-clumping agent after supplementation is maintained around 0.2% to 1.5%.
In the present invention, the concentration of kolliphor is maintained in cell culture media at of about 0.01-2%, 0.02-0.19%, 0.03-0.18%, 0.04-0.17%, 0.05-0.16%, 0.06-0.15%, 0.07-0.14%, 0.08-0.13%, 0.070.12%, 0.08-0.11%, or 0.09-0.1%. More preferably, the concentration of kolliphor after supplementation is maintained around 0.05% to 0.16%.
In the present invention, the concentration of manganese chloride or manganese is maintained in cell culture media at of about 1-30 µM, 2-29 µM, 3-28 µM, 4-27 µM, 5-26 µM, 6-25 µM, 7-24 µM, 8-23 µM, 9-22 µM, 10-21 µM, 11-20 µM, 12-19 µM, 13-18 µM, 14-17 µM, or 15-18 µM. More preferably, the concentration of manganese chloride or manganese after supplementation is maintained around 1µM to 15 µM.
In the present invention, the concentration of glucose is maintained in cell culture media at of about 1-15 g/L, 2-14 g/L, 3-13 g/L, 4-12 g/L, 5-11 g/L, 6-10 g/L, 7-9 g/L, or 8-9 g/L. More preferably, the concentration of glucose after supplementation is maintained around 3g/L to 6 g/L.
In the present invention, EDTA is added to the cell culture media before harvesting.
In the present invention, the cell culture media contains EDTA at a concentration of about 0-8 mM, 0.5-7.5 mM, 1-7 mM, 1.5-6.5 mM, 2-6 mM, 2.5-5.5 mM, 3-5 mM, 3.5-4.5 mM, or 4-5 mM. More preferably, the concentration of EDTA after supplementation is maintained around 0.5mM to 2mM.
In the present invention, the temperature of cell culture media is maintained at about 40°C to 30°C, when cell culture achieve viable cell count of 18-22 x 106 cells/mL the temperature is shifted from 37oC to 34oC.
The embodiments of the present invention are further described using specific examples herein after. The examples are provided for better understanding of certain embodiments of the invention and not, in any manner, to limit the scope thereof. Possible modifications and equivalents apparent to those skilled in the art using the teachings of the present description and the general art in the field of the invention shall also from the part of this specification and are intended to be included within the scope of it.
EXAMPLE: MODULATION OF AFUCOSYLATION LEVEL OF PERTUZUMAB
Experiment was conducted at Ambr250 scale for modulation of afucosylation for Pertuzumab. Experiment containing various combination of various concentration of MnCl2 and pH are described below in the table.
During the experiment, temperature was shifted from 37oC to 34oC when cell count had reached (18-22) × 106 cells/mL. At the same time pH was also shifted as per experimental design.
pH MnCl2
7.00±0.20 1
7.00±0.20 0.3
7.00±0.20 1
7.00±0.20 0.65
7.00±0.20 0.3
7.00±0.05 0.3
7.00±0.05 1
6.85±0.05 0.65
6.85±0.05 1
6.85±0.05 0.3
6.85±0.05 0.3
6.85±0.05 1
Table 1: Range of pH and MnCl2
Result and Discussion:
MnCl2 concentration had no effect on % afucosylation. However decreasing the pH lead to decrease in % afucosylation of Pertuzumab. When pH was set at 7.00±0.20, % afucosylation was observed to be in the range of (3.30-3.66). When the pH was maintained at 7.05±0.05, % afucosylation was within the range of (2.65-2.75) %. However further reducing the pH to 6.85±0.05, reduced the % afucosylated species to (2.18-2.35) %. Reducing the pH had no significant effect on % galactosylated species or % high mannose of Pertuzumab (Fig.1 to 3).
The same process was scaled up to 2000L scale for Pertuzumab. Similar results were obtained. No significant difference was observed in % galactosylation and % high mannose. However % afucosylation has decreased when pH was decreased from 7.00 ± 0.20 (3.93%) to 6.85 ± 0.05 (1.60%) (Fig.4 to 6). ,CLAIMS:We Claim,
1. A method for modulation of afucosylation level of antibody comprising a pH shift of cell culture.
2. The method according to claim 1, wherein the method for modulation of afucosylation level of anti-HER2 antibody comprises pH shift of cell culture between 6.0 and 8.0 when cell culture achieves a viable cell count of 18-22 x 106 cells/mL.
3. A method for modulation of afucosylation level of Pertuzumab comprising a pH shift of cell culture.
4. The method according to claim 3, wherein the method for modulation of afucosylation level of Pertuzumab comprises pH shift of cell culture between 6.0 and 8.0 when cell culture achieves a viable cell count of 18-22 x 106 cells/mL.
5. A method for modulation of afucosylation level of Pertuzumab comprising performing a pH shift or temperature shift of cell culture.
6. The method according to claim 5, wherein the method comprises temperature shift from 40oC to 30oC when cell culture achieve viable cell count of 18-22 x 106 cells/mL.
7. A method for modulation of afucosylation level of Pertuzumab comprising pH shift of cell culture between 6.8 and 7.2 when cell culture achieves a viable cell count of 18-22x 106 cells/mL.
8. The method according to any of preceding claims, wherein culture media is supplemented with glutamine, anti-clumping agent, kolliphor, manganese chloride, or EDTA.