Method for separating viruses from a contaminant-containing liquid
AssigneeSartorius Stedim Biotech GmbH
InventorsRene Faber, Martin Leuthold
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Abstract
The present invention relates to a method for separating viruses from a contaminant-containing liquid medium using two adsorbents having cationic groups, wherein the viruses are adsorbed to the first adsorbent and subsequently eluted and wherein the contaminants present in the resulting eluate are subsequently adsorbed to the second adsorbent. The yield and purity of the viruses obtained as per the method according to the invention is increased by the addition of multivalent anions during the adsorption of the contaminants to the second adsorbent.
Claims15
A method for separating viruses from a virus and contaminant-containing liquid medium, comprising:
- diluting the virus and contaminant-containing liquid medium with a binding buffer solution;
- contacting the virus and contaminant-containing liquid medium with a first adsorption membrane that includes cationic groups, then
- separating the contaminant-containing liquid medium from the first adsorption membrane, then
- quantitatively eluting the viruses from the first adsorbent with a monovalent salt and binding buffer solution, and thereby forming a second liquid medium that includes the viruses, and then
- adding multivalent anions consisting of divalent and/or trivalent anions to the second liquid medium that includes the viruses, then
- flowing the second liquid medium that includes the viruses and that has the multivalent anions therein through a second adsorption membrane that includes cationic groups, and then
- collecting the second liquid medium with the viruses, wherein:
- the charge density of the cationic groups on the first adsorption membrane is less than 50 μmol/ml;
- and
- the first adsorption membrane has, as cationic groups, trimethylammonium groups and the second adsorption membrane has, as cationic groups, trimethylammonium groups or ammonium groups based on polyallylamine ligands.
A method comprising:
- providing a liquid medium that includes viruses and contaminants in a binding buffer;
- forming a virus-adsorbed membrane by contacting the liquid medium with a virus-adsorbent microporous membrane that includes cationic groups;
- separating the liquid medium from the virus-adsorbed membrane;
- washing the virus-adsorbed membrane with the binding buffer;
- quantitatively eluting the viruses from the adsorbent membrane with a monovalent anion solution to provide a virus-containing eluent;
- and then
- adding a phosphate (PO43−) and/or citrate (C6H5O73−) trivalent anion to the virus-containing eluent to provide a trivalent anion solution with a trivalent anion concentration of at least 30 mM;
- filtering the trivalent anion solution through a non-binding microporous membrane that includes ammonium groups based on polymers selected from polyallylamine, polytheylene imine, and polyvinylamine;
- wherein:
- the eluted trivalent anion solution from the filtering of the trivalent anion solution, includes the viruses and less than 1% of nonenveloped nucleic acids:
- the charge density of the cationic groups on the virus-adsorbent microporous membrane is less than 50 μmol/ml;
- and
- the virus-adsorbent microporous membrane has, as cationic groups, trimethylammonium groups and the second adsorption membrane has, as cationic groups, trimethylammonium groups or ammonium groups based on polyallylamine ligands.
Dependencies are inferred from the claim text.