Scalable chromatography process for purification of human cytomegalovirus
AssigneeMerck Sharp & Dohme Corp.
InventorsAdam Kristopeit, Janelle Konietzko, Wanli MA, Katherine Phillips, Andrew Swartz, Sheng-Ching Wang, Marc D. Wenger, Matthew Woodling, Tiago Matos
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Abstract
The present invention relates to a scalable process for the purification of human cytomegalovirus particles from cell culture medium. In particular, the process involves a two step chromatography process starting with an anion exchange chromatography step followed by a polishing chromatography step selected from mixed mode chromatography or cation exchange chromatography.
Claims23
A method of purifying human cytomegalovirus (HCMV) from a cell culture medium, the method comprising:
- a) contacting the cell culture medium comprising HCMV to an anion exchange chromatography medium under conditions that allow the HCMV to bind to the anion exchange chromatography medium;
- b) eluting the HCMV from the anion exchange chromatography medium to obtain an eluate;
- c) contacting the eluate with a polishing chromatography medium;
- and
- d) collecting the HCMV from the polishing chromatography medium to obtain purified HCMV.
12-14. (canceled)
17-19. (canceled)
A method for the purification of HCMV from a cell culture medium comprising the steps of:
- a) harvesting cell culture medium from a culture of ARPE-19 cells infected with HCMV;
- b) subjecting the cell culture medium to nuclease treatment using an endonuclease at a concentration of 10-160 U/mL;
- c) clarifying the nuclease-treated cell culture medium using a 1.2 μm glass fiber filter to obtain a clarified cell culture medium;
- d) contacting the clarified cell culture medium comprising HCMV with an anion exchange chromatography membrane under conditions that allow the HCMV to bind to the anion exchange chromatography membrane and then eluting the HCMV from the anion exchange chromatography membrane to obtain an eluate;
- e) contacting the eluate with a mixed-mode chromatography resin which is a hydrophobic anion exchange chromatography resin having a molecular weight exclusion of about 700 kDa and then collecting the HCMV from the mixed mode chromatography resin to obtain purified HCMV;
- and
- f) performing tangential flow filtration on the purified HCMV collected from step e) to adjust to the desired concentration and buffer.
(canceled)
(canceled) 28 A process for sterilizing a large virus comprising:
- a) applying unpurified material comprising said virus onto a 0.2 μm membrane made of PVDF or cellulose acetate;
- and
- b) collecting purified virus.
Dependencies are inferred from the claim text.