US 11,713,449 B2
Scalable purification method for AAVRH10
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 Apr. 21, 2021, as Appl. No. 17/236,448.
Application 17/236,448 is a continuation of application No. 16/060,404, granted, now 11,028,372, previously published as PCT/US2016/066013, filed on Dec. 9, 2016.
Claims priority of provisional application 62/322,055, filed on Apr. 13, 2016.
Claims priority of provisional application 62/266,347, filed on Dec. 11, 2015.
Prior Publication US 2021/0238560 A1, Aug. 5, 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) rh10 (rAAVrh10) viral particles having packaged genomic sequences from genome-deficient AAVrh10 capsid intermediates, said method comprising:
(a) performing filtration through a series of depth filters to afford a clarified rAAVrh10 production culture harvest;
(b) treating the clarified rAAVrh10 production culture harvest with a nuclease or a combination of nucleases to digest contaminating high molecular weight nucleic acid and to afford a mixture comprising rAAVrh10 viral particles and genome-deficient AAVrh10 capsid intermediates;
(c) concentrating the nuclease-treated mixture of (b) via tangential flow filtration (TFF) to afford a concentrated rAAVrh10 feedstream;
(d) contacting the concentrated rAAVrh10 feedstream of (c) comprising the rAAVrh10 viral particles and genome-deficient AAVrh10 capsid intermediates with a high-performance AAVrh10-capsid binding affinity resin to afford purified mixture;
(e) performing anion exchange chromatography with an anion exchange resin on the affinity purified mixture of (d) containing the rAAVrh10 viral particles at a pH of 9.8 to 10.2;
(f) monitoring an eluate of (e) for ultraviolet absorbance at 260 nm (A260) and at 280 nm (A280); and
(g) collecting rAAVrh10 viral particles from fractions eluted when the ratio of A260 to A280 (A260/A280) reaches an inflection point.