Method for influenza virus purification
Inventors
Peterka, Matjaz • Strancar, Ales • Banjac, Marko • Kramberger, Petra • Roethl, Elisabeth • Muster, Thomas
Assignees
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Publication Number
US-10570376-B2
Publication Date
2020-02-25
Expiration Date
Abstract
A process for the purification of influenza virus or derivative thereof requires providing a source of influenza virus or derivative thereof, optionally subjecting the source to a pre-purification step, followed by subjecting the source to at least one chromatographic step on chromatographic material selected from the group consisting of porous particles having mean pore sizes of at least 20 nm, perfusion particles, gel-in-a-shell particles, tentacle like particles, membrane adsorbers, and monoliths, and collecting fractions eluted from the chromatographic material that contain the influenza virus or derivative thereof, excluding sulfuric ester of cellulose or cross-linked polysaccharides.
Core Innovation
The invention relates to a process for the purification of infective influenza virus selected from the group consisting of influenza virus wild type and influenza virus containing mutations. A source containing the influenza virus in an infectious state is provided and then subjected to a pre-purification step selected from centrifugation, tangential flow filtration ultrafiltration, selective precipitation, expanded bed chromatography, batch chromatography utilizing magnetic beads, and combinations thereof.
The process proceeds by more than one anion exchange chromatographic step on chromatographic anion exchange monoliths arranged in parallel. The monoliths have positively charged functional groups on the monolith surface and are selected from monoliths having ethylene diamine (EDA) groups, monoliths having quaternary amine (QA) groups, and monoliths having diethylaminoethyl (DEAE) groups, and each chromatographic step utilizes a loading buffer having a pH of 7-8 and an elution buffer containing about 0.3M-1M NaCl.
After the anion exchange steps, a fraction with infective virus is obtained in a yield of at least about 40%, and the obtained fraction is then subjected to size exclusion chromatography. The process targets the capture of infective virus and reduction or removal of impurities including nucleic acids and proteins, while excluding sulfuric ester of cellulose and sulfuric ester of cross-linked polysaccharides.
Claims Coverage
The claims define a multi-step purification process with four inventive features: pre-purification, parallel anion exchange on positively charged monoliths under specified buffer conditions, an infective virus yield threshold before size exclusion chromatography, and a specific exclusion proviso.
Pre-purification step selection before anion exchange
Subjecting the source to a pre-purification step selected from centrifugation, tangential flow filtration ultrafiltration, selective precipitation, expanded bed chromatography, batch chromatography utilizing magnetic beads, and combinations thereof.
Parallel anion exchange chromatography on positively charged monoliths with specified buffer conditions
More than one anion exchange chromatographic step on chromatographic anion exchange monoliths arranged in parallel and having positively charged functional groups on the monolith surface, selected from ethylene diamine (EDA), quaternary amine (QA), and diethylaminoethyl (DEAE) groups, with loading buffer having a pH of 7-8 and elution buffer containing about 0.3M-1M NaCl.
Infective virus yield threshold prior to size exclusion chromatography
Obtaining a fraction with infective virus in a yield of at least about 40% and subjecting the obtained fraction to size exclusion chromatography.
Exclusion of sulfuric ester of cellulose and sulfuric ester of cross-linked polysaccharides
A proviso excluding sulfuric ester of cellulose and sulfuric ester of cross-linked polysaccharides.
Overall, the claims define purification of infective influenza virus by pre-purification, multiple parallel anion exchange monolith chromatographic steps using EDA, QA, or DEAE monoliths under specified pH and NaCl conditions, a yield of at least about 40%, and subsequent size exclusion chromatography, with the stated exclusion proviso.
Stated Advantages
Fast, scalable, mild-condition purification and manufacturing workflow for influenza virus.
Yield of infective virus of at least about 40%.
Reduction or removal of impurities including nucleic acids and proteins.
Documented Applications
Downstream use as a vaccine.
Viral vector.
A vaccine that includes an influenza virus purified by the process described in claim 1.
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