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EP-3997123-B1 - REFINING METHOD OF OPHTHALMIC PROTEIN PHARMACEUTICAL

EP3997123B1EP 3997123 B1EP3997123 B1EP 3997123B1EP-3997123-B1

Inventors

  • HA, BYUNG-JHIP
  • PARK, Yong-Seop
  • SEO, Mee-Ran
  • LEE, JAE-HO
  • KIM, DONG-KYU
  • JEONG, JAE-IN

Dates

Publication Date
20260506
Application Date
20190909

Claims (10)

  1. A method for refining aflibercept with an isoelectric point in a range of 6.0-8.3 by performing an anion exchange chromatography after a cation exchange chromatography, wherein the steps of cation exchange chromatography includes the following steps: (a-1) loading an aflibercept mixture onto a cation exchange resin equilibrated with a buffer solution of pH 4.5-6.6; (a-2) washing with a buffer solution containing 0-40 mM sodium chloride or potassium chloride in the condition of pH 4.5-6.6; and (a-3) collecting aflibercept with an isoelectric point in a range of 6.0-9.0 by elution with a buffer solution containing sodium chloride or potassium chloride of 40-120 mM, which is higher than the concentration in step (a-2) in the condition of pH 4.5-6.6; and wherein the steps of anion exchange chromatography includes the following steps: (b-1) loading the aflibercept with an isoelectric point in a range of 6.0-9.0 in step (a-3)onto an anion exchange resin equilibrated with a buffer solution of pH 7.5-9.0; (b-2) washing with a buffer solution containing 0-20 mM sodium chloride or potassium chloride in the condition of pH 7.5-9.0; and (b-3) collecting aflibercept with an isoelectric point in a range of 6.0-8.3 by elution with a buffer solution containing sodium chloride or potassium chloride of 20-150 mM, which is higher than the concentration in step (b-2) in the condition of pH 7.5-9.0.
  2. A method for refining aflibercept with an isoelectric point in a range of 6.0-8.3 by performing a cation exchange chromatography after an anion exchange chromatography, wherein the steps of anion exchange chromatography includes the following steps: (b-1) loading an aflibercept mixture onto an anion exchange resin equilibrated with a buffer solution of pH 7.5-9.0; (b-2) washing with a buffer solution containing 0-20 mM sodium chloride or potassium chloride in the condition of pH 7.5-9.0; and (b-3) collecting aflibercept with an isoelectric point in a range of 6.0-8.3 by elution with a buffer solution containing sodium chloride or potassium chloride of 20-150 mM, which is higher than the concentration in step (b-2) in the condition of pH 7.5-9.0; and wherein the steps of cation exchange chromatography includes the following steps: (a-1) loading the aflibercept with an isoelectric point in a range of 6.0-8.3 in step (b-3) onto a cation exchange resin equilibrated with a buffer solution of pH 4.5-6.6; (a-2) washing with a buffer solution containing 0-40 mM sodium chloride or potassium chloride in the condition of pH 4.5-6.6; and (a-3) collecting aflibercept with an isoelectric point in a range of 6.0-8.3 by elution with a buffer solution containing sodium chloride or potassium chloride of 40-120 mM, which is higher than the concentration in step (a-2) in the condition of pH 4.5-6.6.
  3. The method of claim 1 or 2, wherein the buffer solution in the steps (a-1), (a-2) and (a-3) is at least one selected from the group consist of sodium acetate and sodium phosphate buffer, and wherein the buffer solution in the steps (b-1), (b-2) and (b-3) is at least one selected from the group consist of sodium phosphate and Tris buffer.
  4. The method of claim 1 or 2, wherein a ligand for the cation exchange resin is a carboxymethyl (CM) group.
  5. The method of claim 1 or 2, wherein a ligand for the anion exchange resin is a quaternary ammonium (Q) group.
  6. The method of claim 1 or 2, wherein at least one selected from the group consisting of protein A affinity chromatography, mixed-mode or multimodal chromatography, and desalting ultrafiltration is applied before the cation exchange chromatography is performed on the aflibercept mixture.
  7. The method of claim 1 or 2, wherein at least one selected from the group consisting of protein A affinity chromatography, mixed-mode or multimodal chromatography, and desalting ultrafiltration is applied before the anion exchange chromatography is performed on the aflibercept mixture.
  8. The method of claim 1 or 2, wherein the buffer solution in step (b-3) contains 20-90 mM sodium chloride or potassium chloride.
  9. The method of claim 1 or 2, wherein the buffer solution in step (b-3) contains 20-90 mM sodium chloride or potassium chloride and pH is 7.5-8.5.
  10. A method for formulating a final refined aflibercept to have any one of the following three compositions so as to be suitable for intravitreal injection: (a) a composition of 10mM sodium acetate, 7% sucrose, 0.03% polysorbate 20, pH 5.5; (b) a composition of 10mM sodium acetate, 6.5% sucrose, 15mM sodium chloride, 0.03% polysorbate 20, pH 5.5; and (c) a composition of 50mM sodium acetate, 6% sucrose, 0.03% polysorbate 20, pH 5.5, wherein the aflibercept is refined by the method of any one of claims 1 to 9.

Description

Technical Field The present invention relates to a method for refining aflibercept, which is used as an active ingredient of an ophthalmic protein pharmaceutical. According to the present invention, aflibercept with an isoelectric point value in a particular range can be purified by performing cation exchange chromatography and anion exchange chromatography, leading to an improvement in quality and productivity of ophthalmic protein pharmaceuticals. Background Art Angiogenesis is known to be involved in pathogenesis of various diseases, such as solid tumors, proliferative retinopathies, age-related macular degeneration (AMD), and rheumatoid arthritis. Vascular endothelial growth factor (VEGF), one of the factors needed for the angiogenesis, is expressed in human cancer and also plays a key role in tumor neoangiogenesis. It is also known that a high concentration of VEGF in the eye fluid is highly correlated with angiogenesis activity in diabetic and other ischemic retinopathy patients (Aiello LP, et al., N Engl J Med 1994; 331:1480-1487) and leads to localization of growth factors in the choroidal neovascular membranes of age-related macular degeneration (AMD) patients (Amin R1, et al. Invest Ophthalmol Vis Sci. 1994 Jul; 35(8):3178-88). Therefore, anti-VEGF antibodies or VEGF inhibitors may be promising candidates for the treatment of solid tumors and intraocular angiogenesis-related diseases. Aflibercept, one of the VEGF inhibitors, is a recombinant fusion protein composed of vascular endothelial growth factor (VEGF)-binding portions originated from extracellular domains of human VEGF receptors 1 and 2, which are fused to the Fc portion of the human IgG1 immunoglobulin. Aflibercept has been approved in the United States and Europe for the treatment of wet macular degeneration under the trade name Eylea™. Aflibercept may be expressed in animal cells, such as CHO cells and NS0 cells, by using gene recombination technology. Aflibercept is present in a dimeric form and shows biological activity. The expressed dimer is known to show a form in which glycan structures are bound to a total of ten asparagine residues, and negatively charged sialic acid may be contained in a terminal portion of each of the glycan structures, and thus the aflibercept substances expressed through cell culture show a form of a composite having an isoelectric point value in a very broad range of pH 6.0-10 due to the presence of various contents of sialic acid sugars. To use, on the human body, protein pharmaceuticals produced using gene recombination technology, extremely strict safety and efficacy verification procedures are needed, and thus such procedures require an effective purification process capable of attaining purification at an extremely high purity level. That is, the culture produced through cell culture contains various contents of the sialic acid sugars as well as impurities originated from a variety of processes, and therefore, a process of refining, therefrom, only proteins with isoelectric point values distributed within a standard set for strict verification of safety and efficacy is required. Furthermore, such proteins can be industrially used only when economic efficiency is secured by ensuring a sufficient refinement yield. Conventional purification process schemes (or sequences) of antibody drugs or Fc fusion proteins often include a chromatography step of testing the ability of antibody molecules to preferably bind to or be held on a solid phase of a chromatography column when compared with the binding or maintaining strength of various impurities. These chromatography processes are characterized in that a series of chromatography methods including various techniques, such as protein A or protein G immobilized affinity chromatography, immobilized metal affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, size exclusion gel filtration chromatography, mixed-mode or multimodal chromatography, and hydroxyapatite chromatography are performed under predetermined conditions. However, particular conditions of chromatography performed to purify aflibercept (or aflibercept biosimilar) substances at high purity have not been yet revealed. WO 2018/116198 discloses a purification method for aflibercept comprising a cation exchange chromatography and an anion chromatography. Disclosure of Invention Technical Problem The present inventors tried to complete a series of processes for developing high-purity medicines usable for humans by effectively removing multimers, endotoxins, heterologous proteins and nucleic acids, impurities in the form of high-isoelectric-point analogs, and the like from aflibercept produced from cell culture using animal cell lines. The present inventors excluded size exclusion chromatography that may have a negative effect on productivity due to limited injectable volumes of samples, and tried to complete a process having an adva