DESC:RELATED APPLICATIONS
This application is related to Indian provisional application IN202321086786 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 supplementing of Manganese chloride and methionine and method of using thereof. The present invention further relates to a method of producing protein of interest in a large scale cell culture, comprising supplementing cell culture with Manganese chloride and methionine.
BACKGROUND OF THE INVENTION
Monoclonal antibodies (mAb), such as recombinant immunoglobulins (IgG) are the class of engineered glycoproteins that are recombinantly expressed in animal cell lines and undergo post-translational modification (PTM). Perhaps the most important class of PTM for many biologics is glycosylation, a process that occurs on most eukaryotic secreted and membrane proteins. All IgG antibodies are N-glycosylated at Asn-297 on their Fc region (constant region). Another most widely observed and discussed modification event in IgGs is the oxidation of methionine (Met) to methionine sulfoxide in two positions in the Fc Region.
The constant region of antibodies contains binding sites for immune effector molecules such as the complement system or Fc receptors. These receptors help recruit immune mediators, generally via Fc receptor binding. Antibody Fc receptors mediate the cell-killing effects of mAbs by complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or antibody-mediated phagocytosis by monocytes/macrophages. In ADCC, immune complexes are engaged with Fc?RIIIa (Fc gamma receptor IIIa) on natural killer (NK) cells or tumor cell apoptosis can be induced through the suppression of pro-survival ligands or inhibition of signal receptor dimerization. CDC is activated by the binding of complement component C1q to the mAb Fc region to initiate the complement cascade.
Hence, glycosylation at the Fc region of IgG antibodies has a significant impact on their structure, stability, transport and uptake, half-life, and effector functions. The presence or absence of specific glycan structures, such as core fucose (fucosylation), bisecting GlcNac, and terminal mannose/galactose (galactosylation) /sialic acid residues (Sialyation), can regulate antibody function and affect therapeutic efficacy and immunogenicity. Similarly, 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 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 catalysed 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.
It is known in the art that the culture conditions and production methods can influence the glycosylation profile of a protein produced in a cell culture method. Various factors in cell culture are inextricably linked, such that merely adding for example, galactose to media may on the one hand favourably influence the galactosylation pattern of the protein but may, on the other hand also have other, negative effects on other characteristics of the protein, such as the charge profile, proportion of protein fragments, proportion of aggregates, and titre protein. Oxidation of Met or Trp residues in the Fc was reported for several antibodies. These antibodies were shown to have impaired Fc-mediated activity.
In case of eukaryotic cell chosen for the production of therapeutic recombinant glycoproteins, such as monoclonal antibodies, glycosylation is also highly dependent on the cell culture medium and other production process parameters. Specifically, it has been shown that the composition of growth and feed media, including the concentrations of ammonia, glutamine, glucose, and metal ions, can influence antibody glycosylation. Accordingly, strategies have been developed to control the glycosylation pattern and/or profile of recombinant glycoproteins by supplementation of growth media. For example, WO2017079165 discloses supplementation with a fucose source, e.g. fucose, to cultures of cells engineered to lack GDP-keto-6-deoxyman­ nose-3,5-epimerase, 4-reductase, or GDP-D-mannose-4,6-dehydratase activity to produce glycoproteins with a specific level of fucosylation. WO2017120359 and WO2017120347 discloses supplementation of fucose to reduce afucosylated glycoforms, i.e. increase fucosylation, of antibodies.
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 different antibody in cell culture medium supplemented with a Uridine, Manganese, and Galactose.
WO2020094694 discloses a method for modifying the glycosylation profile of a recombinant glycoprotein produced in cell culture supplemented with fucose, manganese, and taurine.
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.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide a method of producing an antibody, wherein the method comprises: supplementing methionine to a cell culture at a concentration of 0.1g/L to 2.0g/L.
Another object of the present invention is to provide a method of producing Pembrolizumab, wherein the method comprises: culturing a host cell expressing Pembrolizumab in cell culture medium supplemented with methionine at a concentration of 0.1g/L to 2.0g/L.
Another object of the present invention is to provide a method for improving galactosylation profile of an antibody, wherein the method comprises: culturing a host cell expressing said antibody in cell culture medium supplemented MnCl2 at concentration of 0.1 to 2.0 µM.
Another object of the present invention is to provide a method for improving galactosylation profile of Pembrolizumab, wherein the method comprises: culturing a host cell expressing Pembrolizumab in cell culture medium supplemented with MnCl2 at concentration of 0.1 to 2.0 µM.
Another object of the present invention is to provide a method for improving galactosylation profile of Pembrolizumab, wherein the method comprises: culturing a host cell expressing Pembrolizumab in cell culture medium supplemented with MnCl2 at concentration of 0.1 to 2.0 µM and Galactose at concentration of 0 to 4.0 g/L.
Another object of the present invention is to provide a method of producing Pembrolizumab, wherein the method comprises: culturing a host cell expressing Pembrolizumab in cell culture medium supplemented with methionine at a concentration of 0.1g/L to 2.0g/L and MnCl2 at concentration of 0.1 to 2.0 µM.
SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a method of producing an antibody, wherein the method comprises: supplementing methionine to a cell culture at a concentration of 0.1g/L to 2.0g/L.
Another aspect of the present invention is to provide a method of producing Pembrolizumab, wherein the method comprises: culturing a host cell expressing Pembrolizumab in cell culture medium supplemented with methionine at a concentration of 0.1g/L to 2.0g/L.
Another aspect of the present invention is to provide a method for improving galactosylation profile of an antibody, wherein the method comprises: culturing a host cell expressing said antibody in cell culture medium supplemented MnCl2 at concentration of 0.1 to 2.0 µM.
Another aspect of the present invention is to provide a method for improving galactosylation profile of Pembrolizumab, wherein the method comprises: culturing a host cell expressing Pembrolizumab in cell culture medium supplemented with MnCl2 at concentration of 0.1 to 2.0 µM.
Another aspect of the present invention is to provide a method for improving galactosylation profile of Pembrolizumab, wherein the method comprises: culturing a host cell expressing Pembrolizumab in cell culture medium supplemented with MnCl2 at concentration of 0.1 to 2.0 µM and Galactose at concentration of 0 to 4.0 g/L.
Another aspect of the present invention is to provide a method of producing Pembrolizumab, wherein the method comprises: culturing a host cell expressing Pembrolizumab in cell culture medium supplemented with methionine at a concentration of 0.1g/L to 2.0g/L and MnCl2 at concentration of 0.1 to 2.0 µM.
BRIEF DESCRIPTION OF DRAWING
Figure 1: Effect of methionine addition on Oxidation.
Figure 2: Effect of MnCl2 on %Galactosylation.
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 PD-1" binds to PD-1. Pembrolizumab is an example of an antibody that has binding specificity for the PD-1 target.
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 Pembrolizumab.
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 it 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.
A "production medium” is a cell culture medium that favors the production of a recombinant polypeptide, e.g., antibody, of interest, e.g., PD-1 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.
An "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.
"Galactosylation" refers to the type and distribution of galactose residues on polysaccharides and oligosaccharides, for example, N-glycans, O-glycans and glycolipids. The term "galactosylation profile or "galactosylation degree" refers to the quantity of galactose residues on polysaccharides and oligosaccharides, for example N-glycans, O-glycans, and glycolipids."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 (a-gal).
The main embodiment of the present invention is to provide a method of producing an antibody, wherein the method comprises: supplementing methionine to a cell culture at a concentration of 0.1g/L to 2.0g/L.
Another embodiment of the present invention is to provide a method of producing Pembrolizumab, wherein the method comprises: culturing a host cell expressing Pembrolizumab in cell culture medium supplemented with methionine at a concentration of 0.1g/L to 2.0g/L.
Another embodiment of the present invention is to provide a method for improving galactosylation profile of an antibody, wherein the method comprises: culturing a host cell expressing said antibody in cell culture medium supplemented with MnCl2 at concentration of 0.1 to 2.0 µM.
Another embodiment of the present invention is to provide a method for improving galactosylation profile of Pembrolizumab, wherein the method comprises: culturing a host cell expressing Pembrolizumab in cell culture medium supplemented with MnCl2 at concentration of 0.1 to 2.0 µM.
Another embodiment of the present invention is to provide a method for improving galactosylation profile of Pembrolizumab, wherein the method comprises: culturing a host cell expressing Pembrolizumab in cell culture medium supplemented with MnCl2 at concentration of 0.1 to 2.0 µM and Galactose at concentration of 0 to 4.0 g/L.
Another embodiment of the present invention is to provide a method of producing Pembrolizumab, wherein the method comprises: culturing a host cell expressing Pembrolizumab in cell culture medium supplemented with methionine at a concentration of 0.1g/L to 2.0g/L and MnCl2 at concentration of 0.1 to 2.0 µM.
In the present invention, the cell culture contains anti-oxidant at a concentration of about 0.1-10 g/L, 0.2-8 g/L, 0.3-7 g/L or 0.4-5 g/L. Methionine is used in the present invention as anti-oxidant. More preferably the concentration of methionine is 0.3g/L to 3.0g/L.
In the present invention, the cell culture contains Manganese chloride or Manganese at a concentration of about 0.05-5.0 µM, 0.06-4.5 µM, 0.07-4.0 µM, 0.08-3.5µM, 0.09-3.0µM, or 0.1-2.0µM. More preferably the concentration of Manganese chloride is 0.1 to 2.0 µM.
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, 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 is on Day6, Day8, Day10 and Day12.
In the present invention, cell culture is supplemented with Manganese chloride or Manganese on Day1, Day2, Day3, Day4, Day5, Day6, Day7, Day8, Day9, Day10, Day11, Day12, Day13, Day14, Day15 or Day16. More preferably, supplementation of Manganese chloride or Manganese is on Day6, Day8 and Day10.
In the present invention, the cell culture method used is a fed batch culture method and the eukaryotic cell used is CHO cell. Moreover, the cell culture media used for production of recombinant antibody is galactose-free cell culture media.
The present invention is now further described by reference to the following further non-limiting examples. 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: EFFECT OF METHIONINE AND MnCl2 ON OXIDATION AND GALACTOSYLATION
Cell culture comprising Pembrolizumab was treated with different concentration of methionine to analyse the oxidation profile of the cell culture with different concentration. Further, the cell culture was also supplemented with MnCl2 along with varying galactose concentration to understand the role of MnCl2 in galactosylation.
Following table describes the experiment condition:
Batch ID Methionine concentration (g/L) % Oxidized Species % Change
5L BR 0 g/L 6.77
5L BR 0.6 g/L 6.81 0.59084195
5L BR 0.8 g/L 6.32 -6.64697194
5L BR 1.6 g/L 1.34 -80.2067947
50L BR 1.6 g/L 1.61 -76.2186115
Table 1: Effect of methionine addition on Oxidation.
Bioreactor scale Galactose concentration (g/L) MnCl2 concentration(µM) % Galactosylation %Change
5L 0 0 7.66
5L 2 0 11.72 53
250 mL 1 0.1 10.84 42
250 mL 3 0.1 15.72 105
250 mL 1 0.5 27.49 259
250 mL 1 1 31.16 307
250 mL 0 1 29.63 287
250 mL 3 1 31.88 316
5L 0 2 27.98 265
5L 0 1 30.71 301
5L 0 0.3 19.07 149
50L 0 0.3 20.41 166
Table 2: Effect of MnCl2 on %Galactosylation.
Result and Discussion:
From the figure 1 it can be observed that Oxidation has reduced significantly by addition of methionine. Control process where no methionine was added had Oxidized species of 6.77%. However after adding methionine at concentration of 0.4g/L on each Day 6, 8, 10 and 12 the oxidized species were 1.34%. As a result of methionine supplementation the oxidation was reduced by ~80%.
Further, it can also be observed that the same methionine addition strategy was implemented during scale up of the process from 5L to 50L scale, where methionine was added on Day 6, 8, 10 and 12 at a conc. of 0.4g/L. Oxidation profile at 50L scale was similar to that of 5L profile.
From the figure 2 it can be observed that the increase in galactosylation% was only 4% when the galactose concentration was increased from 0 g/L to 2 g/L without MnCl2. Similarly by keeping minimal MnCl2 (0.1 µM) and varying Galactose from 1g/L to 3g/L also yield only about 8% rise in % Galctosylation. However, addition of MnCl2 alone up to 0.3 µM does had a significant impact on % Galactosylation (149-166% increase from control) from 7.66 % to 19-20 %. This indicates the MnCl2 gave significant boost in % Gal levels compared to galactose alone.
Further, it can also be observed that the same MnCl2 addition strategy was implemented during scale up of the process from 5L to 50L scale, where MnCl2 was added at a concentration of 0.3 µM. Galactosylation profile at 50L scale was similar to that of 5L profile. ,CLAIMS:We Claim,
1. A method of producing an antibody, wherein the method comprises supplementing methionine to a cell culture at a concentration of 0.1g/L to 2.0g/L.
2. The method of producing antibody according to claim 1, wherein the method comprises culturing a host cell expressing Pembrolizumab in cell culture medium supplemented with methionine at a concentration of 0.1g/L to 2.0g/L.
3. A method for improving galactosylation profile of an antibody, wherein the method comprises culturing a host cell expressing said antibody in cell culture medium supplemented with MnCl2 at concentration of 0.1 to 2.0 µM.
4. The method according to claim 3, wherein the method comprises culturing a host cell expressing Pembrolizumab in cell culture medium supplemented with MnCl2 at concentration of 0.1 to 2.0 µM.
5. The method of producing antibody according to any of preceding claims, wherein the method comprises culturing a host cell expressing Pembrolizumab in cell culture medium supplemented with methionine at a concentration of 0.1g/L to 2.0g/L and MnCl2 at concentration of 0.1 to 2.0 µM.
6. The method according to any of the preceding claims, wherein the cell culture media used for production of recombinant antibody is galactose-free cell culture media.
7. A method of producing Pembrolizumab, wherein the method comprises supplementing the cell culture medium with methionine.
8. A method for improving galactosylation profile of Pembrolizumab, wherein the method comprises supplementing the cell culture medium with MnCl2.
9. The method according to any of preceding claim, wherein the method comprises supplementation of methionine at a concentration of 0.1g/L to 2.0g/L and MnCl2 at a concentration of 0.1 to 2.0 µM.