초록 ▼

This invention discloses a production process for recombinant human endostatin adenovirus in order to optimize the procedure for small batch and mass industrialization. Exemplary process include steps of: (1) fermentation of eukaryotic cells (HEK293 cells) in the condition of 37° C. and 5% CO2; (2)

This invention discloses a production process for recombinant human endostatin adenovirus in order to optimize the procedure for small batch and mass industrialization. Exemplary process include steps of: (1) fermentation of eukaryotic cells (HEK293 cells) in the condition of 37° C. and 5% CO2; (2) adenovirus infection; (3) collection of diseased cells; (4) freezing and thawing; (5) concentration by ultrafiltration; and (6) preparation and packaging of recombinant human endostatin adenovirus. The process is controllable and easy to operate. The concentration of adenovirus titers can reach 1.0×1012−3.0×1012 vp/ml.

대표청구항 ▼

1. A process for producing recombinant human endostatin adenovirus useful for injective administration, the process comprising: fermenting eukaryotic cells in a culture;performing adenovirus infection of the eukaryotic cells to yield diseased eukaryotic cells having recombinant human endostatin aden

1. A process for producing recombinant human endostatin adenovirus useful for injective administration, the process comprising: fermenting eukaryotic cells in a culture;performing adenovirus infection of the eukaryotic cells to yield diseased eukaryotic cells having recombinant human endostatin adenovirus;harvesting the diseased eukaryotic cells;causing cell lyses of the diseased eukaryotic cells; andpurifying the resulting recombinant human endostatin adenovirus. 2. The process of claim 1, wherein the eukaryotic cells are Human Embryonic Kidney 293 (HEK293) cells. 3. The process of claim 2, wherein fermenting eukaryotic cells is carried out in a DMEM medium comprising glucose at a concentration greater than about 1 g/L, fetal bovine serum concentration from about 8% to about 12%, and an adenovirus culture medium with about 4% to about 6% serum. 4. The process of claim 1, wherein fermenting is carried out in a NBS bioreactor for cell culture. 5. The process of claim 2, wherein fermenting eukaryotic cells is performed under conditions comprising: at a cell density from about 2×105 mL to about 5×105/mL, at a temperature from about 36° C. to about 37° C., with a CO2 concentration of about 5%, with a pH in the range from about 7.2 to about 7.4, with an oxygen concentration of about 30% to about 70%, and at a stirring speed of about 30 rpm to about 100 rpm. 6. The process of claim 1, wherein fermenting eukaryotic cells comprises a gradual increase in a medium perfusion rate according to glucose consumption during the culturing process to maintain a concentration of glucose equal to or greater than about 1 g/L. 7. The process of claim 2, wherein adenovirus infection of the eukaryotic cells is carried to a MOI in the range from about 10 to about 30; and harvesting the diseased eukaryotic cells is carried out in about 48 to about 72 hours from virus infection. 8. The process of claim 2, wherein adenovirus infection of the eukaryotic cells is carried out with a pH in the range from about 7.0 to about 7.4, at a temperature from about 36° C. to about 37° C., and with an oxygen concentration of about 30% to about 70%. 9. The process of claim 1, wherein harvesting the diseased eukaryotic cells is carried out when about 95% of the eukaryotic cells have been infected by adenovirus. 10. The process of claim 1, wherein causing cell lyses is by freezing-thawing of the diseased eukaryotic cells. 11. The process of claim 1, wherein purifying the resulting recombinant human endostatin adenovirus comprises: isolating the human endostatin adenovirus by chromatography; andseparating the human endostatin adenovirus by centrifugation or ultra-filtration. 12. The process of claim 1, further comprising allocating the purified recombinant human endostatin adenovirus into aliquots. 13. The process of claim 11, wherein isolating the human endostatin adenovirus by chromatography comprises using anion exchange fillings selected from Q Sepharose XL Virus Licensed, Q Sepharose XL, DEAE-Sephacel and DEAE-Biogel P for chromatography. 14. The process of claim 13, wherein chromatography is carried out at a pH from about 7.5 to about 8.5. 15. The process of claim 14, wherein chromatography is carried out in a buffer comprising from about 1 mmol/L to about 100 mmol/L of phosphate, a Tris solution, from about 0.1 mmol/L to about 50 mmol/L of MgCl2, from about 1 mmol/L to about 2000 mmol/L of NaCl, and from about 1% to about 15% of glycerol. 16. The process of claim 13, wherein chromatography is carried out at a sample loading speed from about 10 mL/min to 20 mL/min and an elution speed from about 15 mL/min to about 25 mL/min. 17. The process of claim 11, wherein ultrafiltration is carried out with a 0.22 μM filter. 18. The process of claim 12, wherein the aliquots comprise human endostatin adenovirus at about (1±0.1)×1012 VP/mL. 19. A process for large-scale production of recombinant human endostatin adenovirus, the process comprising: fermenting HEK293cells in a culture under conditions of a cell density from about 2×105 mL to about 5×105/mL, a temperature from about 36° C. to about 37° C., a CO2 concentration of about 5%, a pH in the range from about 7.2 to about 7.4, an oxygen concentration of about 30% to about 70%, and a stirring speed of about 30 rpm to about 100 rpm;performing adenovirus infection of HEK293 cells to yield diseased eukaryotic cells having recombinant human endostatin adenovirus, wherein the adenovirus infection is carried out to a MOI in the range from about 10 to about 30 and under conditions of a pH in the range from about 7.0 to about 7.4, a temperature from about 36° C. to about 37° C., and an oxygen concentration of about 30% to about 70%;harvesting the diseased HEK293 cells in about 48 to about 72 hours from virus infection;causing cell lyses by at least three cycles of freezing-thawing of the diseased HEK293 cells;isolating the human endostatin adenovirus by anion exchange chromatography carried out at a pH from about 7.5 to about 8.5 with a buffer comprising from about 1 mmol/L to about 100 mmol/L of phosphate, a Tris solution, from about 0.1 mmol/L to about 50 mmol/L of MgCl2, from about 1 mmol/L to about 2000 mmol/L of NaCl, and from about 1% to about 15% of glycerol;separating the human endostatin adenovirus by ultra-filtration with a 0.22 μM filter; andallocating the purified recombinant human endostatin adenovirus into aliquots comprising human endostatin adenovirus from about 1.0×1012 to about 3.0×1012 VP/mL. 20. The process of claim 19, wherein anion exchange chromatography uses fillings selected from Q Sepharose XL Virus Licensed, Q Sepharose XL, DEAE-Sephacel and DEAE-Biogel P for chromatography.