WO2024201501A1 - Procédé de production d'une composition pharmaceutique - Google Patents

Procédé de production d'une composition pharmaceutique Download PDF

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Publication number
WO2024201501A1
WO2024201501A1 PCT/IN2024/050297 IN2024050297W WO2024201501A1 WO 2024201501 A1 WO2024201501 A1 WO 2024201501A1 IN 2024050297 W IN2024050297 W IN 2024050297W WO 2024201501 A1 WO2024201501 A1 WO 2024201501A1
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Prior art keywords
temperature
cell culture
culture
antibody
antibody composition
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PCT/IN2024/050297
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English (en)
Inventor
Rama Bhupal Reddy KANDULA
Suman BANDYOPADHYAY
Vikas Chandrawanshi
Deekshith Kumar N
Aravinth SUGANTHAN
Valluri SREEDHARI
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Dr Reddys Laboratories Ltd
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Dr Reddys Laboratories Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation

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 a pharmaceutical composition.
  • the invention provides a mammalian cell culture process to modulate a pharmaceutical composition of a monoclonal antibody comprising culturing the mammalian cells, supplementing the cell culture with manganese or galactose.
  • the invention provides a cell culture process to obtain a monoclonal antibody composition comprising galactosylated glycoform at a target/predetermined range.
  • Nivolumab a human lgG4 monoclonal antibody binds to PD-1 and is approved for treatment of melanoma; non-small cell lung cancer (NSCLC); malignant pleural mesothelioma (MPM); renal cell carcinoma (RCC); and classical Hodgkin lymphoma (cHL). It is produced in mammalian cells, Chinese hamster ovary (CHO) cells, in which the post-translational modifications (PTMs) is an integral part of the mabs produced.
  • NSCLC non-small cell lung cancer
  • MPM malignant pleural mesothelioma
  • RRCC renal cell carcinoma
  • cHL classical Hodgkin lymphoma
  • glycosylation plays a key role in the effector functions of the antibody including its biological activity, efficacy, stability, immunogenicity, clearance rate, antibody-dependent cellular cytoxicity (ADCC), and complementdependent cytoxicity (CDC).
  • ADCC antibody-dependent cellular cytoxicity
  • CDC complementdependent cytoxicity
  • N-linked glycosylation in which glycans are attached to the asparagine of the recognition sequence Asn-X-Thr/Ser, where “X” is any amino acid except proline
  • O-linked glycosylation in which glycans are attached to serine or threonine.
  • the N-linked glycosylation results in heterogeneity in antibody glycoforms which affect the efficacy and safety of the antibody.
  • various studies have been done to understand and characterize the glycoform distributions in antibodies during their production (Radhakrishnan, D., A.S. Robinson, and B.A. Ogunnaike, Controlling the Glycosylation Profile in mAbs Using Time-Dependent Media Supplementation. Antibodies, 2017. 7(1): p. 1)
  • Biosimilars sometimes called ‘similar biological medicinal product’ or ‘follow- on biologic’ or ‘subsequent entry biologic’, include therapeutic mAbs which are similar to the already approved/licensed therapeutic product (the ‘reference product’). Regulatory agencies mandate that biosimilars must demonstrate high similarity to the reference product, in terms of quality characteristics, biological activity, safety and efficacy, in order to have marketing approval (Sullivan, P.M. and LM. DiGrazia, Analytic characterization of biosimilars. Am J Health Syst Pharm, 2017. 74(8): p. 568-579; Guttman et. al. Assessing Glycosimilarity of Biotherapeutics. 2018 https://sciex.com/content/dam/SCIEX/pdf/tech-notes/all/Glycosimilarity.pdf ).
  • glycosylation of mAbs can be influenced by various factors such as pH, temperature, dissolved oxygen, ammonia, and media supplements such as sugar and manganese chloride. These studies focused on individual factors, establishing empirical relationships between the individual factor in question and the specific set of glycoform species it affects (Radhakrishnan, D., AS. Robinson, and B.A. Ogunnaike, Controlling the Glycosylation Profile in mAbs Using Time-Dependent Media Supplementation. Antibodies, 2017. 7(1): p. 1).
  • the present invention relates to a cell culture process for producing an antibody composition, the process comprising culturing mammalian cells at a pH range of 6.7 to 7.4, performing a temperature shift from a first culture temperature to a second culture temperature, supplementing manganese or galactose in the cell culture, thereby obtaining an antibody composition comprising galactosylated glycoform, wherein the percentage of galactosylated glycoform increases with the supplementation of the said supplements.
  • the present invention relates to a cell culture process for producing a pharmaceutical monoclonal antibody composition, the process comprising culturing mammalian cells expressing the monoclonal antibody within a pH range of about 6.7 to about 7.4, performing a temperature shift from a first culture temperature to a second culture temperature, supplementing manganese or galactose in the cell culture, thereby obtaining an antibody composition comprising increased percentage of galactosylated glycoforms. Further, the present invention discloses that the obtained antibody composition comprises galactosylated glycoform wherein the percentage of galactosylated glycoforms is at a target/predetermined range.
  • antibody or “monoclonal antibody” refers to an intact antibody or an antigen binding fragment thereof.
  • antibody composition refers to a population of antibody molecules or fragments thereof that is produced by mammalian cell culture.
  • the population of antibody molecules may have one or several post translational modifications (PTM), imparting the antibody molecules a different molecular weight, charge, solubility or combinations thereof.
  • PTM post translational modifications
  • 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.
  • cell culture process refers to a process of culturing a population of cells that are capable of producing recombinant protein of interest or antibody.
  • galactosylated glycoform or “Gal’ refer to antibodies containing terminal galactose residues such as G1A, G1 B, G1AF, G1 BF, G2, G2F and G2SF.
  • glycoform or “glycovariant” used interchangeably herein refers to different molecular variants of an antibody resulting due to variable glycan structure attached and/or glycan attachment site occupancy on the antibody.
  • glycosyl refers to monosaccharide or polysaccharide moiety attached to another molecule.
  • pH shift refers to the change in pH range during the cell culture process, which includes modification in either or both of the upper and lower limits of the pH range.
  • reference product refers to a currently or previously marketed recombinant protein, also described as the "originator product” or “branded product” serving as a comparator in the studies.
  • supply or “supplementation” as used herein refers to any addition made to cell culture medium/feed to achieve the goals described in this disclosure.
  • target/predetermined levels refers to the glycosylation levels of the ‘reference product’.
  • temperature shift refers to the change in temperature during the cell culture process.
  • the present invention provides a cell culture process for culturing mammalian cells expressing an antibody, the process comprising culturing the cells at a pH range of 6.7 to 7.4 by lowering of temperature of the cell culture from a first temperature to a second temperature, and supplementing manganese or galactose to the cell culture to obtain antibody composition comprising galactosylated variants.
  • 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.
  • Certain glycosylation profile of the pharmaceutical monoclonal antibody (mAb) are desirable based on its mechanism of action as the glycosylation profile effects the stability, safety and efficacy of the antibody.
  • the cell culture process of the present invention may be used to produce an antibody composition comprising galactosylated glycoform at a target/predetermined range.
  • 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 glycosylation profile.
  • the cell culture process envisages culturing the cells within a pH range of about 6.7 to about 7.4, performing a temperature shift and supplementing manganese or galactose.
  • the present invention discloses a cell culture process for producing an antibody composition, wherein the said process comprises a) providing mammalian cells expressing the said antibody b) performing a temperature shift during the cell culture from a first culture temperature to a second culture temperature, wherein the second culture temperature is lower than the first culture temperature c) performing pH shift from a pH range of about 6.7 to about 7.2 to a pH range of about 6.7 to about 7.4 d) supplementing the cell culture with manganese or galactose e) recovering the said recombinant antibody composition wherein the antibody composition obtained comprises of increased percentage of galactosylated glycoforms as compared to the antibody composition obtained from a cell culture process devoid of step (d).
  • the present invention discloses a cell culture process for producing an antibody composition, wherein the said process comprises a) providing mammalian cells expressing the said antibody b) performing a temperature shift during the cell culture from a first culture temperature to a second culture temperature, wherein the second culture temperature is lower than the first culture temperature c) performing pH shift from a pH range of about 6.7 to about 7.2 to a pH range of about 6.7 to about 7.4 d) supplementing the cell culture with manganese or galactose e) recovering the said recombinant antibody composition wherein the antibody composition obtained comprises of increased percentage of galactosylated glycoforms, wherein the increased percentage of galactosylated glycoforms in the antibody composition obtained using the present invention is about 30% to about 42%, as compared to the antibody composition obtained from a cell culture process devoid of step (d).
  • the claimed process comprises supplementing the cell culture with about 0.0625 uM to about 2 uM manganese; or about 1 g/L to about 10 g/L galactose.
  • the present invention discloses a cell culture process for producing a biosimilar monoclonal antibody composition, wherein the said process comprises a) providing mammalian cells expressing the said antibody b) performing a temperature shift during the cell culture from a first culture temperature to a second culture temperature, wherein the second culture temperature is lower than the first culture temperature c) performing pH shift from a pH range of about 6.7 to about 7.2 to a pH range of about 6.7 to about 7.4 d) supplementing the cell culture with manganese or galactose e) recovering the said recombinant antibody composition wherein the biosimilar monoclonal antibody composition obtained comprises of galactosylated glycoforms at a target/predetermined range.
  • the target/predetermined percentage of galactosylated glycoforms for the biosimilar monoclonal antibody composition is about 31 %.
  • the present invention discloses a cell culture process for producing a biosimilar monoclonal antibody composition, wherein the said process comprises a) providing mammalian cells expressing the said antibody b) performing a temperature shift during the cell culture from a first culture temperature to a second culture temperature, wherein the second culture temperature is lower than the first culture temperature c) performing pH shift from a pH range of about 6.7 to about 7.2 to a pH range of about 6.7 to about 7.4 d) supplementing the cell culture with about 0.0625 uM to about 0.125 uM manganese; or about 2 g/L to 4 g/L galactose e) recovering the said recombinant antibody composition wherein the biosimilar monoclonal antibody composition obtained comprises of galactosylated glycoforms at about 31 %.
  • the present embodiment discloses a cell culture process for producing a biosimilar monoclonal antibody composition, wherein the said process comprises a) providing mammalian cells expressing the said antibody b) performing a temperature shift during the cell culture from a first culture temperature to a second culture temperature, wherein the second culture temperature is lower than the first culture temperature c) performing pH shift from a pH range of about 6.7 to about 7.2 to a pH range of about 6.7 to about 7.4 d) supplementing the cell culture with about 2.5 g/L galactose e) recovering the said recombinant antibody composition wherein the biosimilar monoclonal antibody composition obtained comprises of galactosylated glycoforms at about 31 %.
  • the difference between the first culture temperature and the second culture temperature of the cell culture is about 4.5°C.
  • the culture temperature before the temperature shift is about 36.5°C and the culture temperature after the temperature shift is about 32°C.
  • the temperature shift is performed at day 5 of the cell culture.
  • the supplementation of manganese or galactose is performed on the same or different day as that of the temperature shift of the cell culture.
  • the supplementation of manganese or galactose is performed on day 5 or day 7 or day 9 of the cell culture.
  • the manganese is supplemented as manganese chloride.
  • the antibody produced using the present invention is an anti PD-1 antibody.
  • the antibody produced using the present invention is nivolumab.
  • Example I An anti-PD 1 antibody having a heavy chain and light chain as described in US8008449B2, with the sequence set forth in SEQ ID NO: 4 and SEQ ID NO: 11 respectively, was cloned and expressed in a recombinant CHO (rCHO) cell line using techniques described in detail in “Molecular Cloning: A Laboratory Manual (Fourth Edition)”. rCHO cells expressing the antibody were seeded at a density of about 0.9 million cells/mL in basal cell culture medium and cultured at an initial temperature of 36.5°C. The pH range of the cell culture is maintained between pH 6.9 to 7.2. On day 5, a temperature shift was performed, thereby reducing the culture temperature to 32°C and the pH range was maintained at 6.7 to 7.4. Feed medium was added on day 3, 5, 7, 9 and 11 . The culture was harvested on day 12.
  • Example I The cell culture process described in Example I was carried with following modifications. On day 7, the cell culture was supplemented with about 0.0625 pM to 2 pM manganese chloride.
  • Example I The cell culture process described in Example I was carried with following modifications. On day 7, the cell culture was supplemented with about 2 g/L to 10 g/L galactose.
  • Example I The cell culture process described in Example I was carried with following modifications.
  • the cell culture process described in Example I was carried with following modifications.
  • On day 9, the cell culture was supplemented with about 1 g/L to 4 g/L galactose.
  • Example I The cell culture process described in Example I was carried with following modifications. On day 7, the cell culture was supplemented with about 2.5 g/L galactose.
  • the antibody composition comprising % of galactosylated glycoforms obtained in the examples I to IV and V is depicted in Table 1 and 2 respectively.
  • the percentage of galactosylated glycoforms in the reference product (RMP) is about 31 %.
  • Table 1 The composition of galactosylated glycoforms of the anti PD-1 antibody obtained with supplementation of manganese or galactose in different days.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé de culture de cellules de mammifères pour moduler une composition pharmaceutique d'une composition d'anticorps monoclonaux comprenant une glycoforme galactosylée de l'anticorps, le procédé comprenant la culture des cellules de mammifères dans une plage de pH d'environ 6,7 à environ 7,4, la réalisation d'un changement de température d'une première température de culture à une deuxième température de culture, la supplémentation de la culture cellulaire avec du manganèse ou du galactose, l'obtention ainsi d'une composition d'anticorps comprenant un pourcentage accru de glycoformes galactosylées. En outre, le procédé de culture cellulaire (10) divulgué dans la présente invention comprend la culture de cellules de mammifères dans une plage de pH d'environ 6,7 à environ 7,4, le passage d'une première température de culture à une deuxième température de culture, l'ajout de manganèse ou de galactose à la culture cellulaire, ce qui permet d'obtenir une composition d'anticorps monoclonal biosimilaire comprenant des glycoformes galactosylées de l'anticorps monoclonal biosimilaire à une plage cible/prédéfinie par rapport à celle du produit de référence.
PCT/IN2024/050297 2023-03-24 2024-03-22 Procédé de production d'une composition pharmaceutique Ceased WO2024201501A1 (fr)

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IN202341020959 2023-03-24

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017021493A1 (fr) * 2015-08-04 2017-02-09 Richter Gedeon Nyrt. Procédé pour augmenter la teneur en galactose de protéines recombinantes
WO2023021532A1 (fr) * 2021-08-20 2023-02-23 Dr. Reddy’S Laboratories Limited Procédé de production d'une composition pharmaceutique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017021493A1 (fr) * 2015-08-04 2017-02-09 Richter Gedeon Nyrt. Procédé pour augmenter la teneur en galactose de protéines recombinantes
WO2023021532A1 (fr) * 2021-08-20 2023-02-23 Dr. Reddy’S Laboratories Limited Procédé de production d'une composition pharmaceutique

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