Method of preparing recombinant adeno-associated virus compositions
AssigneeUniversity of Florida
InventorsSergei Zolotukhin, Barry J. Byrne, Nicholas Muzyczka
Life of this patent
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Abstract
Disclosed are methods for the isolation and purification of high-titer recombinant adeno-associated virus (rAAV) compositions. Also disclosed are methods for reducing or eliminating the concentration of helper adenovirus in rAAV samples. Methods are disclosed that provide highly-purified rAAV stocks having titers up to about 10.sup.13 particles/ml at particle-to-infectivity ratios of less than 100 in processes that are accomplished about 24 hours or less.
Claims29
A method of isolating a recombinant adeno-associated virus, comprising applying a sample containing recombinant adeno-associated virus to an iodixanol gradient, and collecting said recombinant adeno-associated virus from said gradient.
A method of isolating a recombinant adeno-associated virus, comprising the steps of:
- a) centrifuging a sample containing recombinant adeno-associated virus through an iodixanol gradient;
- b) collecting from said iodixanol gradient at least a first fraction comprising said recombinant adeno-associated virus;
- c) contacting said at least a first fraction comprising said recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of said virus to said matrix;
- d) removing non-bound species from said matrix;
- and
- e) eluting said virus from said matrix.
A method of isolating a recombinant adeno-associated virus, comprising the steps of:
- a) centrifuging a sample containing recombinant adeno-associated virus through an iodixanol gradient;
- b) collecting from said iodixanol gradient at least a first fraction comprising said recombinant adeno-associated virus;
- c) contacting said at least a first fraction comprising said recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of said virus to said matrix;
- d) removing non-bound species from said matrix;
- e) eluting said virus from said matrix;
- f) contacting the eluted virus with a hydrophobic matrix, under conditions effective to permit interaction of hydrophobic species with said hydrophobic matrix;
- and
- g) collecting the non-interacting virus from said hydrophobic matrix.
A method for reducing or eliminating adenovirus from a recombinant adeno-associated virus composition contaminated with adenovirus, comprising applying a sample containing recombinant adeno-associated virus and adenovirus to an iodixanol gradient, and collecting from said gradient at least a first fraction comprising said recombinant adeno-associated virus.
A method of producing a recombinant adeno-associated virus having a particle-to-infectivity ratio of less than about 100 to 1, comprising the steps of:
- a) centrifuging a sample containing recombinant adeno-associated virus through an iodixanol gradient;
- b) collecting from said iodixanol gradient at least a first fraction comprising said recombinant adeno-associated virus;
- c) contacting said at least a first fraction comprising said recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of said virus to said matrix;
- d) removing non-bound species from said matrix;
- and
- e) eluting said virus from said matrix.
A kit comprising, in a suitable container, iodixanol, a matrix comprising heparin and instructions for isolating recombinant adeno-associated virus.
Dependencies are inferred from the claim text.