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WO2023067626A1 - Procédé de culture cellulaire pour produire une composition d'anticorps - Google Patents

Procédé de culture cellulaire pour produire une composition d'anticorps Download PDF

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Publication number
WO2023067626A1
WO2023067626A1 PCT/IN2022/050935 IN2022050935W WO2023067626A1 WO 2023067626 A1 WO2023067626 A1 WO 2023067626A1 IN 2022050935 W IN2022050935 W IN 2022050935W WO 2023067626 A1 WO2023067626 A1 WO 2023067626A1
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WO
WIPO (PCT)
Prior art keywords
antibody
cell culture
antibody composition
variants
composition
Prior art date
Application number
PCT/IN2022/050935
Other languages
English (en)
Inventor
Kannayakana Halli Maheshwarappa DAYANANDA
Shailja DWIVEDI
Original Assignee
Dr. Reddy's Laboratories Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dr. Reddy's Laboratories Limited filed Critical Dr. Reddy's Laboratories Limited
Publication of WO2023067626A1 publication Critical patent/WO2023067626A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39516—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum from serum, plasma
    • A61K39/39525—Purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/91—Cell lines ; Processes using cell lines

Definitions

  • the Biological material used in the invention was not obtained from India.
  • the present invention relates to a cell culture process for culturing mammalian cells producing an antibody composition.
  • the present invention discloses a method of reducing the % of acidic variants in an antibody composition, the method comprising culturing the mammalian cells in cell culture medium having low cystine content, wherein the % of acidic variant is reduced as compared to a composition produced using culture medium having high cystine content.
  • the present invention provides a method comprises reducing the cysteinylation and/or glutathionylation in a cell culture process producing an antibody composition by using cell culture medium having low cystine content as compared to a cell culture process using culture medium having high cystine content.
  • Recombinant monoclonal antibodies have emerged as an important class of biopharmaceutical drugs.
  • Recombinant mAbs are inherently complex and heterogenous molecules which is due to various factors like post-translational modifications that occur within the cells they are expressed and chemical modifications that may occur during different steps of manufacturing.
  • the resultant mAb variants often lead unwanted effect on stability and activity of these drugs. Therefore, regulatory agencies warrant that these recombinant mAb variants are well managed to ensure their safety and intended efficacy.
  • Most of the recombinant mAbs are of IgG isotype and they share the evolutionary conserved disulfide bonds between the cysteine residues described for naturally human IgG.
  • the free cysteines are susceptible to chemical modification such as cysteinylation and glutathionylation. Such chemical modification results to acidic variants of the antibodies.
  • the increased level of acidic variants can affect the biophysical and biochemical properties in some cases the bioactivity of the protein (Prade et.al. Cysteine in cell culture media induces acidic lgG1 species by disrupting the disulfide bond network. Biotech and Bioeng. 2021;118:1091-1104).
  • Secukinumab is a recombinant fully humanized monoclonal immunoglobulin G1 (lgG1 )/K antibody that selectively targets IL-17A and blocks its interaction with the IL-17 receptor. It has a non-canonical cysteine present at 97th position in the light chain (CysL97) of secukinumab (WO2016103146). This cysteine is in the CDR3 region of the light chain of the antibody. Any chemical modification of these non- canonical cysteines resulting to high levels of acidic variants that may negatively affect the activity and stability of the antibody.
  • the present invention discloses a method to modulate these chemical modifications of the non-canonical free cysteine.
  • the method comprises reduction of cysteinylation and/or glutathionylation in a cell culture process expressing an antibody composition using cell culture medium having low cystine content.
  • the present invention relates to an antibody composition comprising antibody variants resulted due to chemical modifications, which are the critical quality attributes (CQA) of the antibody.
  • CQA critical quality attributes
  • the invention also discloses a method to reduce the % of acidic variants in an antibody composition by culturing mammalian cells in cell culture medium having low cystine content.
  • the present invention provides a method to reduce cysteinylated and/or glutathionylated variants in an antibody composition by using a cell culture process comprising culturing mammalian cells producing the said antibody in cell culture medium having low cystine content.
  • antibody encompasses whole antibodies and any antigen binding fragment (i.e., “antigen-binding portion”) or single chains or fusion protein thereof.
  • An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen binding portion thereof.
  • biosimilar refers to a recombinant pharmaceutical protein, commonly with identical amino acid sequence to a reference product that contains, similar, very similar to or same post-translational modifications as the reference product yielding no clinically meaningful difference in terms of safety, purity and potency.
  • cysteine refers to a cysteine residue in an antibody sequence which are involved in evolutionary conserved disulfide linkages described in immunoglobulins.
  • cell culture process refers to a process of culturing a population of cells that are capable of producing recombinant protein of interest or antibody.
  • charge variants i.e. variant species having acidic or basic charge due to various post translational modification can be detected by Ion exchange chromatography (IEX) or Isoelectric focusing (IEF).
  • main peak refers to the peak that elutes in abundance (major peak) during ion exchange chromatography.
  • ACV acidic variant peak
  • BCV basic variant peak
  • cystemylated variant refers to the thiol variant comprising such bonded cysteine in their amino acid chain.
  • free cysteine refers to a cysteine that is not involved in disulfide bonding. These include free cysteine residues resulting due to reduction of the cysteines involved in conserved disulfide linkages in immunoglobulins and native free cysteine residues that are not involved in conserved disulfide linkages described in immunoglobulins.
  • glycosylation refers to modification of protein wherein formation of disulfide bond between protein cysteines and glutathione (GSH) cysteine takes place.
  • GSH glutathione
  • glutathionylated variant refers to the variant comprising such bonded cysteine with glutathione.
  • target/predetermined value refers to the quantifiable value for antibody characteristic that is indicative of its biosimilarity of a biosimilar antibody to the ‘reference product’.
  • thiol variant refers an antibody variant that may result due to chemical modification of sulfhydryl group present in free cysteine in the antibody.
  • the exemplary chemical modifications include cysteinylation, cystinylation and glutathionylation.
  • the present invention discloses a method of reducing the % of acidic variants in an antibody composition. Further, the present invention discloses a method of reducing cysteinylation and/or glutathionylation in a cell culture process producing the antibody composition by using cell culture medium having low cystine content.
  • mammalian cell or cell type which is suitable for expression of recombinant proteins in a cell culture medium may be used for the present invention.
  • mammalian cells that may be used with the present invention include Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK21 ) cells and murine myeloma cells (NSO and Sp2/0) human retinoblasts (PER.C6 cell line), human embryonic kidney cell line (HEK-293 cell line) (Dumont, J., et al., Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives. Crit Rev Biotechnol, 2016. 36(6): p. 1110-1122).
  • CHO cell lines expressing recombinant proteins may be used in accordance with the present invention.
  • Cell culture medium is understood by those skilled in the art to refer to a nutrient solution in which cells, such as animal or mammalian cells, are grown.
  • a cell culture medium generally includes one or more of the following components: an energy source (e.g., a carbohydrate such as glucose); amino acids; vitamins; lipids or free fatty acids; and trace elements, e.g., inorganic compounds or naturally occurring elements in the micromolar range.
  • Cell culture medium can also contain additional components, such as hormones and other growth factors (e.g., insulin, transferrin, epidermal growth factor, serum, and the like); salts (e.g., calcium, magnesium and phosphate); sugars (e.g.
  • Mannose, galactose, fucose amino acids
  • amino acids glutamine
  • buffers e.g., HEPES
  • nucleosides and bases e.g., adenosine, thymidine, hypoxanthine
  • antibiotics e.g., gentamycin
  • cell protective agents e.g., a Pluronic polyol (Pluronic F68).
  • DMEM Dulbecco's Modified Eagles Medium
  • RPMI-1640 Medium Sigma-Aldrich
  • EX-CELL® Advanced CHO Fed-batch Medium Sigma-Aldrich
  • Cell BoostTM 7a and 7b GE Healthcare Bio-Sciences AB.
  • DMEM Dulbecco's Modified Eagles Medium
  • RPMI-1640 Medium Sigma-Aldrich
  • EX-CELL® Advanced CHO Fed-batch Medium Sigma-Aldrich
  • Cell BoostTM 7a and 7b GE Healthcare Bio-Sciences AB
  • the methods described in the present invention are in recognition of the fact that various parameters of the cell culture process may be used to obtain antibody composition of desired antibody composition.
  • the method of the present invention envisages a cell culture process to reduce cysteinylation and/or glutathionylation, the method comprising culturing mammalian cells in a cell culture medium having low cystine content.
  • the present invention provides a method of reducing acidic variant content in an antibody composition, the method comprising culturing the cells producing the said antibody in a lower cystine containing cell culture medium and the said reduction in acidic variant is in comparison with an antibody composition produced in cell culture medium having higher cystine content.
  • the acidic variants in the said antibody composition comprises of thiol variants.
  • the present invention discloses a method of producing an antibody composition, the method comprises culturing the mammalian cells expressing the antibody in cell culture medium having low cystine content, wherein the % of acidic variant of the antibody is about 18.0% and/or the % of main species of the antibody is about 74%.
  • the invention of the present application provides a method of producing a homogenous antibody composition comprising of a thiol variant, wherein the said method comprises culturing of cells producing the said antibody in a low cystine containing cell culture medium.
  • the present invention discloses a method to reduce cysteinylation and/or glutathionylation in an antibody composition, the method comprising culturing mammalian cells in a cell culture medium having low cystine content, wherein the obtained antibody composition comprises reduced cysteinylated and/or glutathionylated variants of the antibody as compared to the antibody composition obtained using cell culture medium having high cystine content.
  • the present invention discloses a method to improve an anti-IL-17A antibody composition, the method comprises culturing the mammalian cell in cell culture medium having low cystine content, wherein the improvement in the antibody composition is in terms of increased % of free cysteine variant and decreased % of cysteinylated variant, and decreased % of glutathionylated variant, when compared with the antibody composition produced using cell culture medium having high cystine content.
  • a method of reducing thiol variant heterogeneity in an antibody composition comprising culturing the cells producing the said antibody in a low cystine containing cell culture medium and the said reduction in thiol variant heterogeneity is in comparison with an antibody composition produced in cell culture medium having higher cystine content.
  • the antibody produced using the present invention is a biosimilar monoclonal antibody.
  • the present invention discloses a method to produce a biosimilar antibody composition, wherein the method comprises culturing the mammalian cells expressing the biosimilar antibody in a cell culture medium having low cystine content, thereby the obtained biosimilar antibody composition comprises acidic variant, wherein the % of acidic variant is in a target/predetermined value.
  • the present invention discloses a method to produce a biosimilar antibody composition, wherein the method comprises culturing the mammalian cells expressing the biosimilar antibody in a cell culture medium having low cystine content, thereby the obtained biosimilar antibody composition comprises cysteinylated and/or glutathionylated variants of the antibody, wherein the % of cysteinylated and/or glutathionylated variants are in target/predetermined value.
  • the method of the present invention is applicable to antibody composition comprising thiol variants wherein the thiol variants arise from presence of free cysteine or chemical modification of free sulfhydryl present on free cysteines derived from other wise disulfide bonded cysteines and/or non-canonical free cysteines.
  • the method of the present invention is applicable to antibody composition of antibodies having a non-canonical cysteine residue in the light chain.
  • the said non-canonical residue is in the CDR region of the light chain, preferably in the CDR3 region of the light chain.
  • the method of the present invention is applicable to antibody composition of antibodies having a non-canonical cysteine at 97th position of light chain, the position being designated according to Kabat numbering scheme.
  • the method of present invention is applicable to antibody molecules which have more than one cysteine residues that can give rise to thiol variants, and wherein these multiple cysteine residues are homogenously modified or have mix of possible chemical modification.
  • an antibody molecule having a free cysteine in each of its light chain, each of the cysteine residue has same modification e.g. cysteinylation, or has different modification on each light chain e.g. cysteinylation in one light chain while glutathionylation in other light chain.
  • the thiol variants in said antibody composition comprises from the variant/variants selected from the group comprising free cysteine variant, cysteinylated variant, glutathionylated variant, having intramolecular or intermolecular disulfide bond scrambling, antibody dimer variants.
  • the method of the present invention results in a homogenous antibody composition wherein the free cysteine variant is the most abundant species present in the composition.
  • the method of the present invention reduces the thiol variants heterogeneity arising due to modification of the free cysteine in the said antibody composition and the resultant composition is essentially free of any modification of the free cysteines.
  • the cell culture process disclosed in the present invention further comprises of a temperature shift from 37°C to 32°C. Further, the temperature shift in the present invention is performed on day 5.
  • the cell culture process disclosed in the present invention further comprises of supplementation of glucose.
  • the cell culture disclosed further comprises of addition of feed medium on day 3, 5, 7, 9, 11 and 13.
  • the mammalian cell used to produce the antibody composition of the present invention is Chinese hamster ovary (CHO) cells.
  • the antibody produced using the cell culture process of the present invention is an anti-IL-17A antibody.
  • the antibody produced using the cell culture method of the present invention has the following light and heavy chain amino acid sequences:
  • rCHO recombinant CHO
  • rCHO cells expressing the antibody were seeded at a density of about 0.5 million cells/mL in basal cell culture medium I and cultured at a temperature of about 37°C.
  • the cell culture medium was supplemented with glucose.
  • a temperature shift was applied on day 5, thereby reducing the culture temperature to about 32°C.
  • the feed medium I was added on day 3, 5, 7, 9, 11 and 13.
  • the culture was harvested on day 13.
  • the cystine content in basal cell culture medium and feed medium is given in table 1 .
  • the titer of the antibody produced is given in table 2.
  • the charge variant content and the thiol variant content in the antibody composition produced is given in table 2 and 3 respectively.
  • Example I The cell culture process described in Example I was carried with following modifications. Basal cell culture medium II and the feed medium II was used for culturing cells. The culture was harvested on day 14. The titer of the antibody produced is given in table 2. The charge variant content and the thiol variant content in the antibody composition produced is given in table 2 and 3 respectively.

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  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
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  • Wood Science & Technology (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
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  • Mycology (AREA)
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  • Veterinary Medicine (AREA)
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  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne une composition d'anticorps comprenant des variants d'anticorps résultant de modifications chimiques, constituant les attributs de qualité critiques (AQC) de l'anticorps. L'invention concerne également un procédé permettant de réduire le pourcentage de variants acides dans une composition d'anticorps en cultivant des cellules de mammifères dans un milieu de culture cellulaire possédant une faible teneur en cystine. La présente invention concerne également un procédé permettant de réduire la variante cystéinylée et/ou glutathionylée dans une composition d'anticorps à l'aide d'un procédé de culture cellulaire consistant à cultiver des cellules de mammifères produisant ledit anticorps dans un milieu de culture cellulaire possédant une faible teneur en cystine.
PCT/IN2022/050935 2021-10-20 2022-10-20 Procédé de culture cellulaire pour produire une composition d'anticorps WO2023067626A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN202141047524 2021-10-20
IN202141047524 2021-10-20

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WO2023067626A1 true WO2023067626A1 (fr) 2023-04-27

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016103146A1 (fr) * 2014-12-22 2016-06-30 Novartis Ag Réduction sélective de résidus de cystéine dans des anticorps anti-il-17
WO2017025897A2 (fr) * 2015-08-12 2017-02-16 Pfizer Inc. Cystéines d'anticorps coiffés et non coiffés, et leur utilisation dans les conjugaisons anticorps-médicament
EP3492582A1 (fr) * 2017-12-01 2019-06-05 UCB Biopharma SPRL Procédés de culture cellulaire

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016103146A1 (fr) * 2014-12-22 2016-06-30 Novartis Ag Réduction sélective de résidus de cystéine dans des anticorps anti-il-17
WO2017025897A2 (fr) * 2015-08-12 2017-02-16 Pfizer Inc. Cystéines d'anticorps coiffés et non coiffés, et leur utilisation dans les conjugaisons anticorps-médicament
EP3492582A1 (fr) * 2017-12-01 2019-06-05 UCB Biopharma SPRL Procédés de culture cellulaire

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NATARAJAN VIJAYASANKARAN ET AL.: "Effect of Cell Culture Medium Additives on Color and Acidic Charge Variants of a Monoclonal Antibody", BIOTECHNOLOGY PROGRESS, vol. 34, September 2018 (2018-09-01), pages 1298 - 1307, XP055875309, DOI: 10.1002/btpr.2668 *

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