US 12,460,259 B2
Fed-batch in vitro transcription process
Joseph Elich, Cambridge, MA (US); Amy E. Rabideau, Waltham, MA (US); Michael Shamashkin, Cambridge, MA (US); Rosalie Philpot, Cambridge, MA (US); Brian Fritz, Norwood, MA (US); and Peter Wojciechowski, Cambridge, MA (US)
Assigned to ModernaTX, Inc., Cambridge, MA (US)
Appl. No. 17/438,049
Filed by ModernaTX, Inc., Cambridge, MA (US)
PCT Filed Mar. 11, 2020, PCT No. PCT/US2020/021955
§ 371(c)(1), (2) Date Sep. 10, 2021,
PCT Pub. No. WO2020/185811, PCT Pub. Date Sep. 17, 2020.
Claims priority of provisional application 62/816,734, filed on Mar. 11, 2019.
Prior Publication US 2022/0145381 A1, May 12, 2022
Int. Cl. C12Q 1/6865 (2018.01); C12N 15/10 (2006.01); C12N 15/67 (2006.01)
CPC C12Q 1/6865 (2013.01) [C12N 15/10 (2013.01); C12N 15/67 (2013.01)] 19 Claims
 
1. A method of fed-batch in vitro transcription (IVT) of a messenger ribonucleic acid (mRNA) of interest comprising:
(a) conducting an IVT reaction with an initial reaction mixture that comprises (i) deoxyribonucleic acid (DNA) encoding an mRNA of interest, (ii) an RNA polymerase, (iii) an initial concentration of nucleoside triphosphates (NTPs) comprising adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), and guanosine triphosphate (GTP), and (iv) an RNA cap analog; and
(b) delivering a feed stock mixture at a continuous flow rate to an ongoing IVT reaction mixture that comprises the initial reaction mixture, wherein the feedstock
(i) comprises NTPs comprising ATP, CTP, UTP, and GTP at a molar ratio based on percent consumption values that are specific to the mRNA of interest and are calculated separately for each of the NTPs,
wherein the percent consumption values are determined using an initial nucleotide empirical balancing reaction, and
(ii) is delivered in an amount that maintains a total concentration of NTPs in the ongoing reaction mixture that is at least between 5% to 50% of the initial concentration NTPs, thereby producing a transcribed mRNA of interest.