US 12,168,777 B2
Means and method for producing and purifying viral vectors
Brian K. Kaspar, New Albany, OH (US); James Michael Hatfield, Gurnee, IL (US); Joseph Balleydier, Holly Springs, NC (US); Allan Arman Kaspar, Carlsbad, CA (US); and Robert Emil Hodge, Libertyville, IL (US)
Assigned to Novartis AG, Basel (CH)
Appl. No. 16/761,869
Filed by Novartis AG, Basel (CH)
PCT Filed Nov. 1, 2018, PCT No. PCT/US2018/058744
§ 371(c)(1), (2) Date May 6, 2020,
PCT Pub. No. WO2019/094253, PCT Pub. Date May 16, 2019.
Claims priority of provisional application 62/583,035, filed on Nov. 8, 2017.
Prior Publication US 2021/0317474 A1, Oct. 14, 2021
Int. Cl. C12N 15/86 (2006.01); A61K 9/00 (2006.01); A61P 25/28 (2006.01); C07K 14/47 (2006.01); C12N 15/113 (2010.01); C12N 15/64 (2006.01)
CPC C12N 15/86 (2013.01) [A61K 9/0019 (2013.01); A61P 25/28 (2018.01); C07K 14/47 (2013.01); C12N 15/1137 (2013.01); C12N 15/64 (2013.01); C12Y 115/01001 (2013.01)] 54 Claims
 
1. A method of manufacturing an AAV viral vector, comprising:
a. acidifying and clarifying a cell lysate from cells expressing the AAV viral vector;
b. purifying the product of (a) using cation exchange chromatography (CEX);
c. filtering the product of (b) using a first tangential flow filtration (TFF) step;
d. ultracentrifuging the product of (c) in a cesium chloride (CsCl) buffer; and
e. collecting the AAV viral vector from the product of (d);
wherein a composition comprising the AAV viral vector from (d) comprises less than 2×105 pg/mL residual host cell DNA per 1×1013 vg/mL.