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JP-7855505-B2 - Anti-VEGF protein composition and method for producing the same

JP7855505B2JP 7855505 B2JP7855505 B2JP 7855505B2JP-7855505-B2

Inventors

  • ローレンス ショーン
  • ジョンソン エイミー
  • キャシー ミーガン
  • マストロジャコモ ジェイミー
  • ワン シュンハイ
  • リ ニン

Assignees

  • リジェネロン・ファーマシューティカルズ・インコーポレイテッド

Dates

Publication Date
20260508
Application Date
20200818
Priority Date
20191206

Claims (20)

  1. A method for producing aflibercept recovered from host cells cultured in synthetic medium (CDM), (a) A step of providing a host cell that has been genetically modified to express aflibercept, (b) A step of culturing the host cells in the CDM under conditions suitable for the host cells to express the aflibercept, wherein the cumulative concentration of nickel in the CDM is 0.4 μM or less or 0.4 μM and less: i. The cumulative concentration of iron in the CDM is 55.0 μM or less; ii. The cumulative concentration of copper in the CDM is 0.8 μM or less; iii. The cumulative concentration of zinc in the CDM is 56.0 μM or less; iv . The cumulative concentration of cysteine in the CDM is 10.0 mM or less; and v . A step in which the CDM contains an antioxidant, and the cumulative concentration of the antioxidant is one or more of 0.001 mM to 10.0 mM for any single antioxidant, (c) A step of recovering aflibercept produced by the host cells, wherein the recovered material has a yellowish-brown color equal to or lower than the yellowish-brown color of the European color standard BY7, and the concentration of aflibercept is 5.0 g/L. A method that includes [this].
  2. The method according to claim 1 , wherein the antioxidant is taurine, hypotaurine, glycine, thioctic acid, glutathione, choline, hydrocortisone, vitamin C, vitamin E, or a combination thereof.
  3. The method according to claim 1 , wherein the cumulative concentration of antioxidants in the CDM is 0.001 mM to 10.0 mM for any single antioxidant, and the cumulative concentration of all antioxidants is 30.0 mM or less than 30.0 mM.
  4. The method according to claim 1, wherein the titer of aflibercept, viable cell concentration, viability, ammonia, or osmotic pressure remains substantially unchanged.
  5. The method according to claim 1, wherein the host cells are selected from the group consisting of CHO, NS0, Sp2/0, fetal kidney cells, and BHK.
  6. The method according to claim 1, wherein the recovered material contains one or more aflibercept variants, and the variant has at least one oxidized amino acid residue.
  7. The method according to claim 6 , wherein the oxidized amino acid residue is selected from the group consisting of methionine, tryptophan, histidine, phenylalanine, tyrosine, and combinations thereof.
  8. The method according to claim 7 , wherein the oxidized amino acid residue is histidine.
  9. The method according to claim 7 , wherein the oxidized amino acid residue is tryptophan.
  10. The method according to claim 6, wherein the aflibercept variant comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , and combinations thereof.
  11. The method according to claim 1, wherein the antioxidant is taurine, hypotaurine, glycine, thioctic acid, glutathione, choline, hydrocortisone, vitamin C, vitamin E, or a combination thereof.
  12. A method for producing aflibercept recovered from host cells cultured in synthetic medium (CDM), (a) A step of culturing the host cells in the CDM under conditions suitable for the host cells to express the aflibercept, wherein the cumulative concentration of nickel in the CDM is 0.4 μM or less or 0.4 μM and less: i. The cumulative concentration of iron in the CDM is 55.0 μM or less; ii. The cumulative concentration of copper in the CDM is 0.8 μM or less; iii. The cumulative concentration of zinc in the CDM is 56.0 μM or less; iv . The cumulative concentration of cysteine in the CDM is 10.0 mM or less; and v . A step in which the CDM contains an antioxidant, and the cumulative concentration of the antioxidant is one or more of 0.001 mM to 10.0 mM for any single antioxidant, (c) A step of recovering aflibercept produced by the host cells, wherein the recovered material has a yellowish-brown color equal to or lower than the yellowish-brown color of the European color standard BY7, and the concentration of aflibercept is 5.0 g/L . method.
  13. A method for producing aflibercept recovered from mammalian host cells cultured in a cell culture medium supplemented with synthetic medium (CDM), (a) A step of providing a mammalian host cell that has been genetically modified to express aflibercept, (b) A step of culturing the mammalian host cells under conditions suitable for the expression of the aflibercept, wherein the cumulative concentration of nickel in the CDM is 0.25 μM to 0.4 μM, and is as follows: i. The cumulative concentration of iron in the CDM is 34 μM to 41.5 μM; ii. The cumulative concentration of copper in the CDM is 0.05 μM to 0.1 μM; iii. The cumulative concentration of zinc in the CDM is 3 μM to 3.5 μM; iv. The cumulative concentration of cysteine in the CDM is 6 mM to 7 mM; and v. The CDM contains an antioxidant, the antioxidant comprises hypotaurine, taurine, and glycine, the cumulative concentration of hypotaurine is 1.0 mM to 7.5 mM, the cumulative concentration of taurine is 1.0 mM to 7.5 mM, and the cumulative concentration of glycine is 2.0 mM to 9.5 mM. Process and, (c) A step of recovering aflibercept produced by the mammalian host cells. and Methods that include...
  14. A method for producing aflibercept, (a) A step of expressing aflibercept in mammalian host cells in cell culture medium supplemented with synthetic medium (CDM), wherein the cumulative concentration of nickel in the CDM is 0.25 μM to 0.4 μM, and is hereafter: i. The cumulative concentration of iron in the CDM is 34 μM to 41.5 μM; ii. The cumulative concentration of copper in the CDM is 0.05 μM to 0.1 μM; iii. The cumulative concentration of zinc in the CDM is 3 μM to 3.5 μM; iv. The cumulative concentration of cysteine in the CDM is 6 mM to 7 mM; and v. The CDM contains an antioxidant, the antioxidant comprises hypotaurine, taurine, and glycine, the cumulative concentration of hypotaurine is 1.0 mM to 7.5 mM, the cumulative concentration of taurine is 1.0 mM to 7.5 mM, and the cumulative concentration of glycine is 2.0 mM to 9.5 mM. Process and, (b) A step of recovering aflibercept produced by the mammalian host cells. and Methods that include...
  15. The method according to claim 13 or 14, wherein the recovered material has a yellowish-brown color equal to or less than that of the European color standard BY2, and the concentration of aflibercept is 5.0 g/L.
  16. The method according to claim 15, wherein the antioxidant further comprises thioctic acid, glutathione, choline, hydrocortisone, vitamin C, vitamin E, or a combination thereof.
  17. The method according to claim 15, wherein the cumulative concentration of all antioxidants is 30.0 mM or less than 30.0 mM.
  18. The method according to claim 13 or 14, wherein the titer of aflibercept, viable cell concentration, viability, ammonia, or osmotic pressure remains unchanged.
  19. The method according to claim 13 or 14, wherein the mammalian host cell is selected from the group consisting of CHO, NS0, Sp2/0, fetal kidney cells, and BHK.
  20. The method according to claim 13 or 14, wherein the recovered material comprises one or more aflibercept variants, and the variant has at least one oxidized amino acid residue.

Description

Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, the entirety of which is incorporated herein by reference. The ASCII copy was created on 13 August 2020, is denoted as 070816-02251_SL.txt, and has a size of 134,385 bytes. Cross-reference of related applications This application claims priority and benefits of U.S. Provisional Patent Application No. 63/065,012, filed on 13 August 2020, the contents of which are incorporated herein by reference in their entirety. Field: The present invention generally relates to anti-VEGF compositions and methods for producing the same. Background: Protein-based biopharmaceutical compositions have emerged as important research products for the treatment of ophthalmic diseases, cancer, autoimmune diseases, infectious diseases, and other diseases and disorders. Biopharmaceuticals are one of the rapidly growing product segments of the pharmaceutical industry. The class of cell-derived dimeric mitogens that exhibit selectivity for vascular endothelial cells has been identified as vascular endothelial growth factor (VEGF) and is referred to by this name. Persistent angiogenesis can cause or exacerbate certain diseases, including psoriasis, rheumatoid arthritis, hemangiomas, angiofibromas, diabetic retinopathy, and neovascular glaucoma. VEGF activity inhibitors are useful for treating these diseases, as well as other VEGF-induced pathological angiogenesis and vascular permeability conditions, such as tumor angiogenesis. Angiopoietin and members of the vascular endothelial growth factor (VEGF) family are the only growth factors thought to be primarily specific to vascular endothelial cells. Several eye disorders are associated with pathological neovascularization. For example, the development of age-related macular degeneration (AMD) is associated with a process called choroidal neovascularization (CNV). Leakage from CNV causes the accumulation of fluid in the submacula, leading to macular edema and vision loss. Diabetic macular edema (DME) is another eye disorder involving neovascularization. DME is the most widely recognized cause of moderate vision loss in diabetic patients and is a common complication of diabetic retinopathy, a disease that negatively affects the blood vessels of the retina. Clinically significant DME occurs when fluid leaks into the center of the macula, the photosensitive part of the retina responsible for clear, direct vision. The presence of fluid in the macula can lead to severe vision loss or blindness. For example, various VEGF inhibitors, such as the VEGF trap Eyea (aflibercept), are approved to treat these eye disorders. Summary This invention relates to anti-VEGF proteins, including aflibercept, a VEGF trap protein, which is a fusion protein. The invention also relates to novel anti-VEGF proteins, aflibercept MiniTrap, or VEGF MiniTrap (collectively referred to as MiniTrap unless otherwise specified). This specification discloses methods for producing these anti-VEGF proteins, including manufacturing modes that provide an efficient and effective means of producing the proteins of interest. In one embodiment, the invention is made for the use of a synthetic medium (CDM) for producing anti-VEGF proteins. In a particular embodiment, the CDM of interest is a CDM that, upon use, produces a protein sample, which is yellowish-brown and may contain oxidized species. Furthermore, protein variants of aflibercept and VEGF MiniTrap are disclosed in this application along with the accompanying manufacturing methods. Manufacturing of Aflibercept This disclosure describes the manufacturing of aflibercept using a cell culture medium. In one embodiment, the cell culture medium is a synthetic medium ("CDM"). CDM is often used because it is a synthetic preparation that does not contain proteins or animal-derived components and provides certainty regarding the composition of the medium. In another embodiment, the cell culture medium is a soy hydrolyzed medium. In one embodiment, a method for producing a recombinant protein comprises: (a) providing a genetically modified host cell to express a recombinant protein of interest; (b) culturing the host cell in a CDM under conditions suitable for the cell to express the recombinant protein of interest; and (c) recovering a preparation of the recombinant protein of interest produced by the cell. In one embodiment, the recombinant protein of interest is an anti-VEGF protein. In a particular embodiment, the anti-VEGF protein is selected from the group consisting of aflibercept and recombinant MiniTrap (an example of which is disclosed in U.S. Patent No. 7,279,159), aflibercept scFv, and other anti-VEGF proteins. In a preferred embodiment, the recombinant protein of interest is aflibercept. In one embodiment of this design, aflibercept is expressed in a suitable host cell. Non-limiting examples of such host cells include, but are not limited to, CHO, CHO K1, EESYR®, NICE®, NS0,