CPC C12N 7/02 (2013.01) [B01D 15/3804 (2013.01); B01J 20/281 (2013.01); C12N 7/00 (2013.01); C12N 15/8645 (2013.01); G01N 21/33 (2013.01); B01D 15/166 (2013.01); B01D 15/363 (2013.01); B01J 41/05 (2017.01); B01J 41/20 (2013.01); C12N 2750/14143 (2013.01); C12N 2750/14151 (2013.01)] | 24 Claims |
1. A method for separating recombinant adeno-associated virus (AAV) 8 (rAAV8) viral particles having packaged genomic sequences from genome-deficient AAV8 capsid intermediates, said method comprising:
(a) performing filtration through a series of depth filters to afford a clarified rAAV8 production culture harvest;
(b) treating the clarified rAAV8 production culture harvest of (a) with a nuclease or a combination of nucleases to digest contaminating high molecular weight nucleic acid and to afford a mixture comprising rAAV8 viral particles and genome-deficient AAV8 capsid intermediates;
(c) concentrating the nuclease-treated mixture of (b) via tangential flow filtration (TFF) to afford a concentrated rAAV8 feedstream;
(d) contacting the concentrated rAAV8 feedstream of (c) comprising the rAAV8 viral particles and genome-deficient AAV8 capsid intermediates with a high-performance AAV8-capsid binding affinity resin to afford purified mixture;
(e) performing anion exchange chromatography by anion exchange resin on the affinity purified mixture of (d) containing the rAAV8 viral particles at a pH of 10.0 to 10.4;
(f) monitoring an eluate of (e) for ultraviolet absorbance at 260 nm (A260) and at 280 nm (A280); and
(g) collecting rAAV8 viral particles from fractions eluted when the ratio of A260 to A280 (A260/A280) reaches an inflection point.
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