US 12,351,832 B2
Recombinant adeno-associated virus particle purification with multiple-step anion exchange chromatography
Nicole Brument, Nantes (FR)
Assigned to INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM), Paris (FR); UNIVERSITE DE NANTES, Nantes (FR); CHU NANTES, Nantes (FR); and ASSOCIATION FRANCAISE CONTRE LES MYOPATHIES, Paris (FR)
Filed by INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM), Paris (FR); NANTES UNIVERSITE, Nantes (FR); CHU NANTES, Nantes (FR); and ASSOCIATION FRANCAISE CONTRE LES MYOPATHIES, Paris (FR)
Filed on Nov. 15, 2021, as Appl. No. 17/526,467.
Application 17/526,467 is a continuation of application No. 15/549,279, granted, now 11,203,740, issued on Dec. 21, 2021, previously published as PCT/EP2016/052739, filed on Feb. 9, 2016.
Claims priority of application No. 15305187 (EP), filed on Feb. 9, 2015.
Prior Publication US 2022/0098618 A1, Mar. 31, 2022
Prior Publication US 2024/0060054 A9, Feb. 22, 2024
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 7/02 (2006.01); B01D 15/36 (2006.01); B01D 69/02 (2006.01); C12N 7/00 (2006.01); C12N 15/86 (2006.01); A61K 48/00 (2006.01)
CPC C12N 7/02 (2013.01) [B01D 15/363 (2013.01); B01D 69/02 (2013.01); C12N 7/00 (2013.01); C12N 15/86 (2013.01); A61K 48/00 (2013.01); B01D 2257/91 (2013.01); B01D 2325/34 (2013.01); C12N 2750/14121 (2013.01); C12N 2750/14143 (2013.01); C12N 2750/14151 (2013.01)] 8 Claims
 
1. A method for obtaining purified recombinant Adeno-Associated Virus (rAAV) particles, comprising the steps of:
a) performing a depth filtration of a starting material previously obtained from cells producing rAAV particles, the said starting material being a cell lysate or a culture supernatant, whereby a rAAV-containing clarified composition is provided;
b) submitting the rAAV-containing clarified composition to a first step of anion-exchange chromatography on a chromatographic support, wherein elution is performed by using a linear salt gradient and wherein the rAAV-containing fraction is collected, whereby a first rAAV enriched composition is provided;
c) submitting the first rAAV enriched composition at least once to a second step of anion-exchange chromatography on a chromatographic support wherein elution is performed by using a linear salt gradient and wherein the rAAV-containing fraction is collected, whereby a second rAAV enriched composition is provided;
d) submitting the second rAAV enriched composition to a step of tangential flow filtration, whereby purified recombinant Adeno-Associated Virus particles (rAAV) are provided;
wherein said rAAV particles belong to an AAV5 capsid serotype and
wherein the purified rAAV particles have an amount of residual host cell DNA equal or inferior to 50 ng per dose of 1012 vector genomes (vg) of the rAAV particles that belong to the AAV5 capsid serotype.