US 12,487,152 B2
Scalable method for producing transfection reagents
Jan Panteli, Cambridge, MA (US); and Ying Jing, Reinach (CH)
Assigned to Ultragenyx Pharmaceutical Inc., Novato, CA (US)
Appl. No. 16/612,002
Filed by Ultragenyx Pharmaceutical Inc., Novato, CA (US)
PCT Filed May 9, 2018, PCT No. PCT/US2018/031865
§ 371(c)(1), (2) Date Nov. 8, 2019,
PCT Pub. No. WO2018/208960, PCT Pub. Date Nov. 15, 2018.
Claims priority of provisional application 62/503,660, filed on May 9, 2017.
Prior Publication US 2020/0124505 A1, Apr. 23, 2020
Int. Cl. G01N 1/28 (2006.01); C12M 1/00 (2006.01); C12M 3/06 (2006.01); C12N 7/00 (2006.01); C12N 15/85 (2006.01)
CPC G01N 1/28 (2013.01) [C12M 23/14 (2013.01); C12M 23/26 (2013.01); C12M 23/28 (2013.01); C12M 27/16 (2013.01); C12N 7/00 (2013.01); C12N 15/85 (2013.01); C12N 2750/14111 (2013.01)] 13 Claims
OG exemplary drawing
 
1. A method of preparing a transfection master mix comprising the steps of:
(1) introducing a DNA solution and a transfection reagent solution into a mixing container to create a transfection master mix, wherein the volume of the transfection master mix is 1 L or greater;
(2) rocking the mixing container with a mechanical rocking device for a period of about 1 minute to about 10 minutes at 10 rpm to 15 rpm and about 7 degrees to about 12 degrees rocking angle to reach a steady state concentration of DNA within the transfection master mix; and
(3) incubating the transfection master mix for an incubation period of about 20 minutes to about 30 minutes, wherein the transfection master mix is substantially still during the incubation period;
wherein the transfection master mix consists essentially of the DNA solution and the transfection reagent solution;
wherein the DNA solution comprises plasmids selected from the group consisting of recombinant adeno-associated virus (rAAV) cis plasmids, rAAV trans-rep-cap plasmids, and helper plasmids; and
wherein the transfection master mix is for co-transfection of rAAV packaging cells for rAAV production.