DESC:RELATED APPLICATIONS
This application is related to Indian provisional application IN202321086787 filed 19th Dec. 2023 and is incorporated herein in its entirety
FIELD OF THE INVENTION
The present invention relates to a cell culture medium comprising supplementation of Methionine and method of using thereof. The present invention further relates to a method of producing antibody in a large scale cell culture, comprising supplementation cell culture with Methionine.
BACKGROUND OF THE INVENTION
Monoclonal antibodies play a pivotal role in therapeutic applications, but their production is hindered by susceptibility to oxidation during cell culture. Oxidation can compromise the structural integrity and efficacy of antibodies, leading to reduced therapeutic potential. Existing methods have limitations in effectively preventing oxidation.
The FcRn binding region in the CH2-CH3 interface of the Fc contains two methionine residues located at positions 252 in the CH2 domain and 428 in the CH3 domain, which are highly conserved among IgG isotypes. Both residues are surface exposed, structurally close to the FcRn binding interface and susceptible to oxidation. It has been described that Met252 and Met428 oxidation is accompanied by conformational changes in the backbone.
Oxidation of Met252 and Met428 was shown to impair affinity to FcRn and PK properties of monoclonal antibodies where IgG preparations with high oxidation levels exhibited significantly faster clearance compared with nondegraded batches.
Galactosylation is also critical for complement-dependent cytotoxicity (CDC) activity, while the role of terminal galactose in antibody-dependent cellular cytotoxicity (ADCC) is less clear. Variations in galactosylation can lead to differences in protein structural diversity and glycan heterogeneity, which can impact the biophysical and pharmacological properties of glycoproteins. Therefore, modulation of galactosylation in IgG antibodies is crucial for optimizing their therapeutic potential and modulation of oxidation levels also paramount importance for maintaining the chemical and structural integrity of antibody therapeutics.
Galactosylation step is catalyzed by galactosyltransferase-mediated reaction that employs UDP-galactose as the sugar Substrate and Mn+2 as a cofactor for galactosyltransferase. Percentage galactosylation can be modulated by fine tuning the amount of the substrate (UDP-galactose), the cofactor for galactosyltransferase (Mn+2) or both.
WO2020252082 discloses a cell culture supplemented with Uridine, Manganese, and Galactose. It further discloses method of culturing host cell in production medium comprising zinc. More particularly, it discloses a method for production of antibody in cell culture medium supplemented with a Uridine, Manganese, and Galactose.
WO2016196621 discloses a method for achieving a glycosylation profile by addition of manganese to a cell culture. More particularly, it discloses method for production of antibody in cell culture supplemented with Manganese.
WO2020094694 discloses a method for modifying the glycosylation profile of a recombinant glycoprotein produced in cell culture supplemented with fucose, manganese, and taurine.
Oxidation of Met or Trp residues in the Fc was reported for several antibodies. These antibodies were shown to have impaired Fc-mediated activity. Hence, it is necessary to develop an improved method which can reduce the oxidation profile of antibody containing cell culture.
Thus, there is a need in the art for identification of methods that can predictably modify the galactosylation profile and reduce oxidation species of recombinant glycoproteins of interest to better resemble that of a reference recombinant glycoprotein that meets all safety, efficacy, and regulatory standards.
This invention addresses critical issue by proposing a unique approach involving methionine supplementation during cell culture, presenting a substantial advancement in the field of biotechnology.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide a method for producing antibody comprising: supplementing cell culture medium with a methionine to reduce oxidation of cell culture.
Another object of the present invention is to provide a method for producing Vedolizumab comprising: supplementing cell culture medium with a methionine to reduce oxidation of monoclonal antibody during cell culture process.
Another object of the present invention is to provide a method for improving galactosylation profile of antibody comprising: supplementing cell culture medium with a galactose and MnCl2.
Another object of the present invention is to provide a method for improving galactosylation profile of Vedolizumab comprising: supplementing cell culture medium with a galactose and MnCl2.
Another object of the present invention is to provide a method for improving seed generation profile of antibody comprising: addition of anti-clumping agent and glutamine to the seed media.
Another object of the present invention is to provide a method for improving seed generation profile of antibody comprising: addition of anti-clumping agent, kolliphor, and glutamine to the production media.
Another object of the present invention is to provide a method for improving Galactosylation profile and reducing oxidation level of Vedolizumab comprising: supplementing cell culture medium with a galactose, MnCl2, and methionine.
Another object of the present invention is to provide a method of producing Vedolizumab, wherein the method comprises: culturing a host cell expressing Vedolizumab in cell culture medium supplemented with methionine at a concentration of 0.1g/L to 2.0g/L, Galactose 0g/L to 8g/L and MnCl2 at concentration of 1-15 µM.
Another object of the present invention is to provide a method for improving seed generation profile of antibody comprising: addition of anti-clumping agent at concentration of 0.2 to 1.5%, kolliphor at a concentration of 0.05% to 0.16%, and glutamine at a concentration of 4mM to 15mM to the production media.
SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a method for producing antibody comprising: supplementing cell culture medium with a methionine to reduce oxidation of cell culture.
Another aspect of the present invention is to provide a method for producing Vedolizumab comprising: supplementing cell culture medium with a methionine to reduce oxidation of monoclonal antibody during cell culture process.
Another aspect of the present invention is to provide a method for improving galactosylation profile of antibody comprising: supplementing cell culture medium with a galactose and MnCl2.
Another aspect of the present invention is to provide a method for improving galactosylation profile of Vedolizumab comprising: supplementing cell culture medium with a galactose and MnCl2.
Another aspect of the present invention is to provide a method for improving seed generation profile of antibody comprising: addition of anti-clumping agent and glutamine to the seed media.
Another aspect of the present invention is to provide a method for improving seed generation profile of antibody comprising: addition of anti-clumping agent, kolliphor, and glutamine to the production media.
Another aspect of the present invention is to provide a method for improving galactosylation profile and reducing oxidation level of Vedolizumab comprising: supplementing cell culture medium with a galactose, MnCl2, and methionine.
Another aspect of the present invention is to provide a method of producing Vedolizumab, wherein the method comprises: culturing a host cell expressing Vedolizumab in cell culture medium supplemented with methionine at a concentration of 0.1g/L to 2.0g/L, Galactose 0g/L to 8g/L and MnCl2 at concentration of 1-15 µM.
Another aspect of the present invention is to provide a method for improving seed generation profile of antibody comprising: addition of anti-clumping agent at concentration of 0.2 to 1.5%, kolliphor at a concentration of 0.05% to 0.16%, and glutamine at a concentration of 4mM to 15mM to the production media.
BRIEF DESCRIPTION OF DRAWING
Figure 1: Effect of methionine addition on oxidation at 5L scale.
Figure 2: Oxidation profile during scale up.
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 a4ß7 complex" binds to a4ß7. Vedolizumab is an example of an antibody that has binding specificity for the a4ß7 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 Vedolizumab.
The terms "culture", "cell culture" and "eukaryotic 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 terms “medium” and “cell culture medium” (plural, “media”) refer to a nutrient source used for growing or maintaining cells. As is understood by a person of skill in the art, the nutrient source may contain components required by the cell for growth and/or survival or may contain components that aid in cell growth and/or survival. Vitamins, essential or non-essential amino acids (e.g., cysteine and cystine), and trace elements (e.g., copper) are examples of medium components. Examples of cell culture media include growth medium and production medium.
The term "growth medium" refers to a cell culture medium that favors the growth, i.e., increase in number, of cultured cells and is used during the growth or expansion phase of the cell culturing process.
The term "production medium” refers to a cell culture medium that favors the production of a recombinant polypeptide, e.g., antibody, of interest, e.g., an anti-a4ß7 antibody.
The term “supplementation” refers to the addition of specific components or compounds to the cell culture medium to support optimal cell growth, productivity, and desired metabolic activity. These supplements can enhance cellular functions, increase yields, and maintain stable conditions throughout the cultivation process.
The term "antioxidant" herein refers to an agent that inhibits the oxidation of other molecules. Examples of antioxidants herein include L-methionine, citrate, lipoic acid, uric acid, glutathione, tocopherol, carotene, lycopene, cysteine, phosphonate compounds, e.g., etidronic acid, desferoxamine and malate.
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 galactosealpha-1,3-galactose (-gal).
This invention addresses the persistent challenge of antibody oxidation during cell culture processes. By introducing methionine supplementation, the method significantly mitigates oxidative stress, ensuring enhanced stability and quality of monoclonal antibodies.
The main embodiment of the present invention is to provide a method for producing antibody comprising: supplementing cell culture medium with a methionine to reduce oxidation of cell culture.
Another embodiment of the present invention is to provide a method for producing Vedolizumab comprising: supplementing cell culture medium with a methionine to reduce oxidation of monoclonal antibody during cell culture process.
Another embodiment of the present invention is to provide a method for improving galactosylation profile of antibody comprising: supplementing cell culture medium with a galactose and MnCl2.
Another embodiment of the present invention is to provide a method for improving galactosylation profile of Vedolizumab comprising: supplementing cell culture medium with a galactose and MnCl2.
Another embodiment of the present invention is to provide a method for improving seed generation profile of antibody comprising: addition of anti-clumping agent and glutamine to the seed media.
Another embodiment of the present invention is to provide a method for improving seed generation profile of antibody comprising: addition of anti-clumping agent, kolliphor, and glutamine to the production media.
Another embodiment of the present invention is to provide a method for improving galactosylation profile and reducing oxidation level of Vedolizumab comprising: supplementing cell culture medium with a galactose, MnCl2, and methionine.
Another embodiment of the present invention is to provide a method of producing Vedolizumab, wherein the method comprises: culturing a host cell expressing Vedolizumab in cell culture medium supplemented with methionine at a concentration of 0.1g/L to 2.0g/L, Galactose 0g/L to 8g/L and MnCl2 at concentration of 1-15 µM.
Another embodiment of the present invention is to provide a method for improving seed generation profile of antibody comprising: addition of anti-clumping agent at concentration of 0.2 to 1.5%, kolliphor at a concentration of 0.05% to 0.16%, and glutamine at a concentration of 4mM to 15mM to the production media.
In the present invention, the cell culture contains anti-oxidant at a concentration of about 0.1-2g/L, 0.5-19.5 g/L, 1-19 g/L, 1.5-18.5 g/L, 2-18 g/L, 2.5-17.5 g/L, 3-17 g/L, 3.5-16.5 g/L, 4-16 g/L, 4.5- 15.5 g/L, 5-15 g/L, 5.5-14.5 g/L, 6-14 g/L, or 6.5-13.5 g/L. More preferably the concentration of anti-oxidant is 0.1-1 g/L.
In the present invention, the cell culture contains galactose at a concentration of about 0-20 g/L, 1-19 g/L,2-18 g/L, 3-17 g/L, 4-16 g/L, 5-15 g/L, 6-14 g/L, 7-13 g/L, 8-12 g/L, 9-11 g/L, or 10-12 g/L. More preferably the concentration of galactose is 0-8 g/L.
In the present invention, the cell culture contains manganese chloride or manganese at a concentration 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 is 1-15 µM.
In the present invention, the cell culture contains manganese chloride or manganese at a stock concentration of about 150-250 g/L.
In the present invention, the cell culture contains anti-clumping agent at a concentration 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%, 4.5- 14.5%, 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 is 0.2-1.5%.
In the present invention, the cell culture contains glutamine at a concentration 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, 4- 15 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, 8-11 mM, 8.5-10.5 mM, 9-10 mM, or 9.5-10.5 mM. More preferably the concentration of glutamine is 4-15 mM.
In the present invention, the cell culture contains kolliphor at a concentration 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%, 0.07- 0.12%, 0.08-0.11%, or 0.09-0.1%. More preferably the concentration of kolliphor is 0.05-0.16%.
In the present invention cell culture is supplemented with methionine on Day1, Day2, Day3, Day4, Day5, Day6, Day7, Day8, Day9, Day10, Day11, Day12, Day13, Day14, Day15 or Day16. More preferably supplementation of methionine on Day4, Day6, Day8, Day10, or Day12.
In the present invention, a supplementation of methionine to the cell culture medium for producing composition comprising anti- a4ß7 antibody such as Vedolizumab, where the composition has decreased level of oxidation.
In the present invention the level of oxidation is reduced to about ~60% or less, ~55%, ~50%, ~45%, ~40%, ~35%, ~30%, ~25%, or ~20% or less compared to the cell culture without any methionine supplementation.
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 1: METHOD OF REDUCTION OF OXIDATION OF CELL CULTURE
In the present invention, cell culture comprising Vedolizumab is treated with different concentration of Methionine to analyze the oxidation profile of the cell culture with different concentration.
Following table describes the experiment condition:
Sr. No. Experiment condition
1 No Methionine
2 0.2g/L Methionine on Day 6
3 0.4g/L Methionine on Day 6
4 0.2g/L Methionine on Day 6 and Day 10
5 0.2g/L Methionine on Day 6, 10 and 12
Table 1: Experiment condition for Oxidation profiling of cell culture
Result and Discussion:
From the figure 1 it can be observed that percentage Oxidation has reduced significantly by addition of Methionine. Control process where no methionine was added had oxidation of 7.54 %. However after adding methionine on Day 6, oxidation decreased by 29% from control. Increasing the concentration of methionine on Day 6 from 0.2g/L to 0.4g/L had same reduction in oxidation.
Furthermore, oxidation was significantly reduced by adding methionine at different time interval. Specifically, maximum oxidation reduction by ~51% were observed by adding methionine at a concentration of 0.2g/L on Day6, Day10, and Day12.
From figure 2 it can be observed that the same methionine addition strategy was implemented during scale up of the process from 5L to 50L and 2000L scale, where methionine was added on Day 6, 10 and 12 at a conc. of 0.2g/L.
Oxidation profile at 50L and 2000L was similar to that of 5L profile. ,CLAIMS:We Claim,
1. A method for producing antibody comprising supplementing cell culture medium with methionine to reduce oxidation of cell culture.
2. The method according to claim 1, wherein the method comprises supplementation of methionine at a concentration of 0.1 g/L to 2.0 g/L.
3. A method for producing Vedolizumab comprising supplementing cell culture medium with methionine to reduce oxidation of cell culture.
4. The method according to claim 3, wherein the method comprises supplementation of methionine at a concentration of 0.1g/L to 2.0 g/L.
5. A method for improving galactosylation profile of antibody comprising supplementing cell culture medium with a galactose and MnCl2.
6. The method for producing antibody according to any of preceding claims, wherein method for improving galactosylation profile and reducing oxidation level of Vedolizumab comprises supplementing cell culture medium with a galactose, MnCl2, and methionine.
7. The method of producing antibody according to claim 1, wherein the method comprises culturing a host cell expressing Vedolizumab in cell culture medium supplemented with methionine at a concentration of 0.1g/L to 2.0g/L, Galactose 0g/L to 8g/L and MnCl2 at concentration of 1-15 µM.
8. The method according to any of preceding claims, wherein the method comprises improving seed generation profile of antibody comprises; addition of anti-clumping agent at concentration of 0.2 to 1.5%, kolliphor at a concentration of 0.05% to 0.16%, and glutamine at a concentration of 4mM to 15mM to the production media.