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JP-7857059-B2 - Anti-VEGF protein composition and method for producing the same

JP7857059B2JP 7857059 B2JP7857059 B2JP 7857059B2JP-7857059-B2

Inventors

  • タスティアン アンドリュー
  • ヴァルタク アンキット
  • ダリー トーマス
  • パイルズ エリカ
  • パラッカル ニシャ
  • 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
20260512
Application Date
20200818
Priority Date
20191206

Claims (11)

  1. A method for producing aflibercept, (a) A step of producing a clarified recovery product of cells cultured in synthetic medium (CDM), (b) A step of binding aflibercept from the clarified recovered material to a protein A resin , wherein the aflibercept comprises a variant having at least one oxidized amino acid residue selected from the group consisting of methionine, tryptophan, histidine, phenylalanine, tyrosine, and combinations thereof , (c) A step of eluting the aflibercept of step (b) to form an affinity eluate, wherein the eluate has a first color, (d) The step of subjecting the eluate containing aflibercept to an anion exchange chromatography (AEX) column , (e) A method comprising the step of collecting a flow-through fraction, wherein the flow-through fraction has a second color, and when the protein concentrations of the eluate and the flow-through fraction are normalized, the first color of the affinity eluate is a darker yellowish-brown than the second color of the flow-through fraction.
  2. The method according to claim 1, wherein the cells are selected from the group consisting of CHO, NS0, Sp2/0, fetal kidney cells, and BHK.
  3. The method according to claim 1 , wherein the oxidized amino acid residue is histidine.
  4. The method according to claim 1 , wherein the oxidized amino acid residue is tryptophan.
  5. A method for producing aflibercept from a clarified recovery of cells cultured in a synthetic medium (CDM), (a) A step of binding aflibercept from the clarified recovered material to a protein A resin, (b) A step of eluting the aflibercept of step (a) to form an affinity eluate, wherein the eluate contains an acidic species of aflibercept, (c) The step of subjecting the eluted aflibercept to an anion exchange chromatography (AEX) column , A method comprising the step of collecting one or more flow-through fractions, wherein when the protein concentrations in the affinity eluate and the flow-through fractions of step (b) are normalized to 10.0 g/L, the percentage of acidic species of aflibercept in the affinity eluate is greater than the percentage of acidic species of aflibercept in the one or more flow-through fractions of step (d) , the acidic species of aflibercept corresponds to a peak that elutes earlier than the main peak in the cation exchange chromatography (CEX) chromatogram of aflibercept, the chromatogram being produced using a first mobile phase of 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.7) and a second mobile phase of 40 mM sodium phosphate and 100 mM sodium chloride (pH 9.0) (mobile phase B), and the chromatogram being produced using detection at 280 nm.
  6. The method according to claim 5 , wherein the acidic species of aflibercept comprises aflibercept having at least one oxidized amino acid residue selected from the group consisting of methionine, tryptophan, histidine, phenylalanine, tyrosine, and combinations thereof.
  7. The method according to claim 5, further comprising the step of binding aflibercept from the clarified recovered product, followed by subjecting the aflibercept to one or more further chromatographic steps selected from the group consisting of cation exchange chromatography ( CEX ), hydrophobic interaction chromatography, size exclusion chromatography, and combinations thereof.
  8. The method according to claim 5, wherein the cells are selected from the group consisting of CHO, NS0, Sp2/0, fetal kidney cells, and BHK.
  9. The method according to claim 6, wherein the cells are selected from the group consisting of CHO, NS0, Sp2/0, fetal kidney cells, and BHK.
  10. The method according to claim 6, wherein the acidic species of aflibercept comprises aflibercept having at least one oxidized amino acid residue which is tryptophan.
  11. The method according to claim 6, wherein the acidic species of aflibercept comprises aflibercept having at least one oxidized amino acid residue which is histidine.

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 070816-02201_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,