US 11,718,835 B2
Scalable purification method for AAV8
Martin Lock, Southampton, PA (US); and Mauricio Alvira, Philadelphia, PA (US)
Assigned to Ths Trustees of the University of Pennsylvania, Philadelphia, PA (US)
Filed by The Trustees of the University of Pennsylvania, Philadelphia, PA (US)
Filed on May 24, 2021, as Appl. No. 17/328,686.
Application 17/328,686 is a continuation of application No. 16/060,408, granted, now 11,015,174, previously published as PCT/US2016/065976, filed on Dec. 9, 2016.
Claims priority of provisional application 62/322,098, filed on Apr. 13, 2016.
Claims priority of provisional application 62/266,341, filed on Dec. 11, 2015.
Prior Publication US 2021/0277364 A1, Sep. 9, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 7/02 (2006.01); C12N 7/00 (2006.01); B01D 15/38 (2006.01); B01J 20/281 (2006.01); C12N 15/864 (2006.01); G01N 21/33 (2006.01); B01J 41/05 (2017.01); B01D 15/16 (2006.01); B01D 15/36 (2006.01); B01J 41/20 (2006.01)
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.