Method for modulation of daratumumab glycosylation profile

Abstract

The present invention provides a method for modulation of galactosylation and mannosylation level of Daratumumab by supplementation of manganese chloride and modulation of afucosylation level of Daratumumab by maintaining pCO2 with pH of cell culture.

Description

[0001] METHOD FOR MODULATION OF DARATUMUMAB GLYCOSYLATION PROFILE

[0002] RELATED APPLICATIONS

[0003] This application is related to Indian provisional application IN202421003607 filed 18thJan. 2024 and is incorporated herein in its entirety.

[0004] FIELD OF THE INVENTION

[0005] The present inventions relates to modification of glycosylation by modulating galactosylation, mannosylation and afucosylation using manganese chloride (MnCh), pH and pCO2 concentration in culture vessel.

[0006] BACKGROUND OF THE INVENTION

[0007] Monoclonal antibodies (mAbs) are immunoglobulins, made by homogeneous hybrid cells (B cells) that are derived from the same parent cell. Monoclonal antibodies are attached to the specific protein of the pathogens or abnormal cells and inactivate their ability to bind or invade new cells. They are used to treat cancer, infectious viral and bacterial diseases with more of a targeted therapy with less side effects and more specificity.

[0008] Most monoclonal antibodies are typically of the immunoglobulin G (IgG) subclass, and they are glycosylated at the conserved asparagine position 297 (Asn-297) in the CH2 domain of the Fc region. During the synthesis of N-glycans, multiple sugar moieties can be added to form different glycoforms, e.g., GO, Gl, G2, afucosylated complex. Glycosylation plays an important role for complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) functions through modulating the binding to the Fey receptor. Particular glycoforms may be necessary to achieve therapeutic efficacy. These glycoforms may be targeted by glycosylation engineering, but may also be affected by cell culture conditions.

[0009] Post-translational modifications impact the stability, pharmacokinetics (PK), efficacy, and immunogenicity of mAbs. These modifications are also critical for the Fc receptor binding, and consequently, key antibody effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Therefore ADCC / CDC mechanisms are highly dependent on the glycosylation profile of the Fc portion and the polymorphism of the Fey receptor. Variations in glycosylation can lead to differences in protein structural diversity and glycan heterogeneity, which can impact the biophysical and pharmacological properties of glycoproteins. Therefore, modulation of glycosylation in IgG antibodies is crucial for optimizing their therapeutic potential for maintaining the chemical and structural integrity of antibody therapeutics. In this invention, we have addressed to modulate galactosylation, mannosylation, afucosylation for monoclonal antibody.

[0010] Amongst cell culture process parameters, pCCh in the culture fluid is of substantial interest because pCO2levels can vary across bioreactor scales and it indirectly influence afucosylation in monoclonal antibody production through their effects on cell metabolism, cell growth and culture conditions.

[0011] WO2022261021 discloses a method for obtaining recombinant glycosylated protein with increased level of afucosylated glycoforms with use of buffer suitable for fucosidase activity.

[0012] WO2016061424 discloses a method for achieving a predetermined glycosylation profile of an anti a4- integrin antibody by adjusting copper concentration in cell culture.

[0013] W02020094694 discloses a method for modifying a glycosylation profile of a recombinant glycoprotein by supplementing cell culture with fucose, manganese, and taurine.

[0014] During upstream production, the glycan profile on mAbs can be varied by varying of the cell line, process conditions, media and feed formulations and genetic engineering in earlier development stages. The effect of these variables in modulating the glycosylation levels is complicated and difficult to implement considering the complications involved during the cell’s uptake, growth and harvest.

[0015] There is a need in industry for simple and efficient methods to manipulate glycosylation during recombinant production of therapeutic antibodies. Thus, the present invention provide a method to modulate glycosylation by modulation of galactosylation and mannosylation by supplementing manganese chloride, and afucosylation by maintaining pCCh with pH of cell culture.

[0016] OBJECTS OF THE INVENTION

[0017] The main object of the present invention is to provide a method for modulation of galactosylation and mannosylation level of antibody by supplementing manganese chloride to the cell culture. Another object of the present invention is to provide a method for modulation of galactosylation and mannosylation level of Daratumumab by supplementing manganese chloride to the cell culture.

[0018] Another object of the present invention is to provide a method for modulation of afucosylation level of antibody by maintaining pCCh with pH of cell culture.

[0019] Another object of the present invention is to provide a method for modulation of afucosylation level of Daratumumab by maintaining pCCh with pH of cell culture.

[0020] Another object of the present invention is to provide a method for modulation of glycosylation level of Daratumumab by supplementing manganese chloride and maintaining pCCh with pH of cell culture.

[0021] Another object of the present invention is to provide a method for modulation of glycosylation level of Daratumumab by modulating galactosylation and mannosylation through supplementation of manganese chloride and modulating afucosylation through maintaining pCCh with pH of cell culture.

[0022] Another object of the present invention is to provide a method for modulation of glycosylation level of Daratumumab by supplementation of manganese chloride at a concentration of about 0 to 5 pM and maintaining pCCh with pH of about 30 to 150 mmHg.

[0023] SUMMARY OF THE INVENTION

[0024] The main aspect of the present invention is to provide a method for modulation of galactosylation and mannosylation level of antibody by supplementing manganese chloride to the cell culture.

[0025] Another aspect of the present invention is to provide a method for modulation of galactosylation and mannosylation level of Daratumumab by supplementing manganese chloride to the cell culture.

[0026] Another aspect of the present invention is to provide a method for modulation of afucosylation level of antibody by maintaining pCCh with pH of cell culture. Another aspect of the present invention is to provide a method for modulation of afucosylation level of Daratumumab by maintaining pCCh with pH of cell culture.

[0027] Another aspect of the present invention is to provide a method for modulation of glycosylation level of Daratumumab by supplementing manganese chloride and maintaining pCCh with pH of cell culture.

[0028] Another aspect of the present invention is to provide a method for modulation of glycosylation level of Daratumumab by modulating galactosylation and mannosylation through supplementation of manganese chloride and modulating afucosylation through maintaining pCCh with pH of cell culture.

[0029] Another aspect of the present invention is to provide a method for modulation of glycosylation level of Daratumumab by supplementation of manganese chloride at a concentration of about 0 to 5 pM and maintaining pCCh with pH of about 30 to 150 mmHg.

[0030] BRIEF DESCRIPTION OF DRAWING

[0031] Figure 1: Upstream process flow for Daratumumab.

[0032] Figure 2: Effect of MnCh concentration on galactosylation

[0033] Figure 3: Effect of MnCh concentration on mannosylation

[0034] Figure 4: Effect of pCO2 concentration on afucosylation

[0035] Figure 5: Effect of pCO2 concentration on afucosylation (Higher scale)

[0036] DETAILED DESCRIPTION OF THE INVENTION

[0037] DEFINITION

[0038] The following definitions are provided to facilitate understanding of certain terms used throughout the specification.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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 Daratumumab.

[0044] 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.

[0045] 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, and trace elements are examples of medium components. Examples of cell culture media include growth medium and production medium.

[0046] 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.

[0047] The term "production medium” refers to a cell culture medium that favors the production of a recombinant polypeptide, e.g., antibody, of interest.

[0048] 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).

[0049] The term "Mannosylation" herein refers to a biochemical process that involves the addition of mannose sugar residues to proteins.

[0050] The term “Afucosylation” herein refers to biochemical phenomenon that involves the alteration of glycoproteins by the removal of fucose residues from their carbohydrate structures.

[0051] 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.

[0052] 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.

[0053] The term “pCCh” herein refers to a partial pressure of carbon dioxide in cell culture.

[0054] The main embodiment of the present invention is to provide a method for modulation of galactosylation and mannosylation level of antibody by supplementing manganese chloride to the cell culture. Another embodiment of the present invention is to provide a method for modulation of galactosylation and mannosylation level of Daratumumab by supplementing manganese chloride to the cell culture.

[0055] Another embodiment of the present invention is to provide a method for modulation of afucosylation level of antibody by maintaining pCCh with pH of cell culture.

[0056] Another embodiment of the present invention is to provide a method for modulation of afucosylation level of Daratumumab by maintaining pCCh with pH of cell culture.

[0057] Another embodiment of the present invention is to provide a method for modulation of glycosylation level of Daratumumab by supplementing manganese chloride and maintaining pCCh with pH of cell culture.

[0058] Another embodiment of the present invention is to provide a method for modulation of glycosylation level of Daratumumab by modulating galactosylation and mannosylation through supplementation of manganese chloride and modulating afucosylation through maintaining pCCh with pH of cell culture.

[0059] Another embodiment of the present invention is to provide a method for modulation of glycosylation level of Daratumumab by supplementation of manganese chloride at a concentration of about 0 to 5 pM and maintaining pCCh with pH of about 30 to 150 mmHg.

[0060] In this present invention, the concentration of manganese chloride or manganese is maintained in cell culture media at of about 1-30 pM, 2-29 pM, 3-28 pM, 4-27 pM, 5-26 pM, 6-25 pM, 7-24 pM, 8-23 pM, 9-22 pM, 10-21 pM, 11-20 pM, 12-19 pM, 13-18 pM, 14-17 pM, or 15-18 pM. More preferably, the concentration of manganese chloride or manganese after supplementation is maintained around 1 mM to 15mM.

[0061] In the present invention the concentration of pCCh is of about 0-400 mmHg, 20-380 mmHg, 40- 360 mmHg, 40-340 mmHg, 60-320 mmHg, 80-300 mmHg, 100-280 mmHg, 120-260 mmHg, 140- 240 mmHg, 160-220 mmHg, or 180-200 mmHg. More preferably, the concentration of pCCh is maintained around 30mmhGto 150 mmHg. In the present invention concentration of pCCh is modulated using overlay and sparging strategy.

[0062] In the present invention the pH of cell culture is about 4-10, 4.5-9.5, 5-9, 5.5-8.5, 6-8, 6.5-7.5, or 7-7.5. More preferably, the pH range suitable for Afucosylation is 6.0 - 8.0.

[0063] In the present invention, the cell culture media is supplemented with glutamine, anti-clumping agent or glucose.

[0064] In the present invention, the cell culture media is supplemented with 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,

[0065] 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,

[0066] 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 after supplementation is maintained around 4mM to 15mM.

[0067] In the present invention, the cell culture media is supplemented with 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%, 4.514.5%, 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%, or9.5-10.5%. More preferably, the concentration of anti-clumping agent after supplementation is maintained around 0.2% to 1.5%.

[0068] In the present invention, the cell culture media is supplemented with glucose at a concentration of about 1-15 gm / L, 1.5-14.5 gm / L, 2-14 gm / L, 2.5-13.5 gm / L, 3-13 gm / L, 3.5-12.5 gm / L, 4-12 gm / L, 4.5-11.5 gm / L, 5-11 gm / L, 5.5-10.5 gm / L, 6-10 gm / L, 6.5-9.5 gm / L, 7-9 gm / L, or 7.5-8.5 gm / L. More preferably, the concentration of glucose after supplementation is maintained around 2 gm / L to 8gm / L.

[0069] 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: MODULATION OF GALACTOSYLATION BY MnCh AND pCOi

[0070] The upstream cell culture process is broadly divided into two stages i.e. seed expansion and production stage. During the seed stage, a sufficient volume of culture is generated in order to start the production stage. Production scale bioreactor is supplemented with feed (Excell advance feed 1 and Cell vento 4 feed) and supplement (MnCh) at predefined interval that help in desired protein quality. Upstream process flow is presented in the Fig. 1.

[0071] Results & Discussion:

[0072] In Fig. 2, it was observed that there is rise in % galactosylation when MnCh concentration increased from 0 pM / L to 2 pM / L. There is ~2.6 to 2.8 times rise in % galactosylation when MnCh concentration increased from 0 pM / L to 1 pM / L. With 2 pM MnCh concentration there is ~2.5 times rise in % galactosylation compared to 0 pM / L MnCh concentration.

[0073] It was observed that there was significant increase in % galactosylation with MnCh concentration from 0 pM / L to 1 pM / L. But, there was no remarkable rise in % galactosylation from 1 pM to 2pM / L MnCh concentration.

[0074] Increasing MnCh concentration beyond 1 pM also has a kind of saturation effect and shows negligible impact on the rise in % galactosylation.

[0075] Table 1: Experimental plan deciphering the impact of MnCh on % galactosylation and % mannosylation

[0076] In Fig. 3, it was observed that there is reduction in % mannosylation when MnCh concentration increased from 0 pM / L to 2 pM / L. There is -20-30 % reduction in % mannosylation when MnCh concentration increased from 0 pM / L to 2 pM / L. It was observed that there was a significant decrease of 23% in % mannosylation with MnCh concentration from 0 pM / L to 1 pM / L. With MnCh concentration from 1 pM / L to 2 pM / L reduction in % mannosylation has been reduced to 9 %.

[0077] In the above study, higher pCCh was maintained at low pH (6.85 ± 0.1) and lower pCCh was maintained at high pH (7.25 ± 0.1) in comparison to control process (7.0 ± 0.2).

[0078] Table 2: Experimental Plan deciphering the impact of pCCh on %AF species

[0079] In Fig. 4, it was observed that there is 6% reduction in % afucosylation at higher pCCh concentration and 6% increase in % afucosylation at lower pCCh concentration compared to control process. As per observed in Fig.4, Fig. 5 also shows that there was significant decrease in % afucosylation with increase in pCCh concentration at higher scale (5L and 50L) also.

[0080] Table 3: Experimental Plan deciphering the impact of pCCh on %AF species at5L and 50L scale

[0081] The studies described herein have also established that the changes in the galactosylation, mannosylation and afucosylation profiles obtained by implementation of the methods of the present invention are scale-independent. There is no significant impact on culture growth and productivity with addition of the supplement at recommended concentration (MnCh) and change in physical parameter (pCCh). Therefore, current finding suggest that optimizing MnCh concentration, can improve percentage galactosylated species and mannosylated species whereas optimizing pH and pCCh concentration can improve percentage afucosylated species in Daratumumab production from CHO cells.

Claims

We Claim,1. A method for modulation of galactosylation and mannosylation level of antibody by supplementing manganese chloride to the cell culture.

2. The method of claim 1, wherein the method for modulation of galactosylation and mannosylation level of Daratumumab by supplementing manganese chloride to the cell culture.

3. A method for modulation of afucosylation level of antibody by maintaining pCO2 with pH of cell culture.

4. The method of claim 3, wherein the method for modulation of afucosylation level of Daratumumab by maintaining pCO2 with pH of cell culture.

5. A method for modulation of glycosylation level of Daratumumab by modulating galactosylation and mannosylation through supplementation of manganese chloride and modulating afucosylation through maintaining pCO2 with pH of cell culture.

6. The method according to any of preceding claims, wherein the method for modulation of glycosylation level of Daratumumab by supplementation of manganese chloride at a concentration of about 0 to 5 pM and maintaining pCO2 with pH of about 30 to 150 mmHg.

7. The method according to any of preceding claims, wherein the cell culture media is supplemented with glutamine, anti-clumping agent or glucose.

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