Methods for purification of messenger RNA

• US 9,957,499 B2
• Filed: 03/14/2014
• Issued: 05/01/2018
• Est. Priority Date: 03/14/2013
• Status: Active Grant

• Current Assignee: Translate Bio, Inc. (Sanofi )
• Original Assignee: Translate Bio, Inc. (Sanofi )
• Inventors: Heartlein, Michael, DeRosa, Frank, Dias, Anusha, Karve, Shirang
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US14/775,915
• Publication Number:
  US 20160040154A1
• Time in Patent Office: 1,509 Days
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1017 by filtration, e.g. using filters, frits, membranes

Abstract

The present invention provides, among other things, methods of purifying messenger RNA (mRNA) including the steps of subjecting an impure preparation comprising in vitro synthesized mRNA to a denaturing condition, and purifying the mRNA from the impure preparation from step (a) by tangential flow filtration, wherein the mRNA purified from step (b) is substantially free of prematurely aborted RNA sequences and/or enzyme reagents used in in vitro synthesis.

Claims

1. A method of purifying messenger RNA (mRNA), comprising

(a) providing an impure preparation comprising in vitro synthesized mRNA and prematurely aborted RNA sequences,

(b) treating the impure preparation with a denaturing agent under conditions that facilitate separation of the prematurely aborted RNA sequences from the mRNA;

and

(c) subjecting the treated impure preparation from step (b) to tangential flow filtration, thereby purifying mRNA;

wherein purified mRNA from step (c) is substantially free of prematurely aborted RNA sequences and has an integrity greater than 95%.

2. The method of claim 1, wherein the denaturing agent is selected from the group consisting of urea, guanidinium thiocyanate, KCl, sodium dodecyl sulfate, sarcosyl, and combination thereof.

3. The method of claim 1, wherein step (b) comprises incubating the impure preparation with guanidinium thiocyanate at a concentration of about 4 M or greater.

4. The method of claim 1, wherein step (b) comprises incubating the impure preparation with KCl at a concentration of about 1 M or greater.

5. The method of claim 1, wherein the tangential flow filtration is performed using only aqueous solvents.

6. The method of claim 5, wherein said aqueous solvent is water.

7. The method of claim 1, wherein the prematurely aborted RNA sequences comprise less than 15 bases.

8. The method of claim 1, wherein the impure preparation further comprises enzyme reagents used in in vitro synthesis comprise T7 RNA polymerase, DNAse I, pyrophosphatase, and/or RNAse inhibitor.

9. The method of claim 1, wherein the tangential flow filtration is performed before a cap and poly-A tail are added to the in vitro synthesized mRNA.

10. The method of claim 1, wherein the tangential flow filtration is performed after a cap and poly-A tail are added to the in vitro synthesized mRNA.

11. The method of claim 1, wherein the tangential flow filtration is performed both before and after a cap and poly-A tail are added to the in vitro synthesized mRNA.

12. The method of claim 1, wherein the in vitro synthesized mRNA is greater than about 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb in length.

13. The method of claim 1, wherein the in vitro synthesized mRNA comprises one or more modifications to enhance stability.

14. The method of claim 13, wherein the one or more modifications are selected from modified nucleotide, modified sugar phosphate backbones, 5′

and/or 3′

untranslated region.

15. A method for manufacturing messenger RNA (mRNA) comprising:

synthesizing mRNA in vitro; and

purifying the in vitro synthesized mRNA using a method according to claim 1.

16. The method of claim 1, wherein the substantially free of prematurely aborted RNA sequences is less than 1.0% prematurely aborted RNA sequences.

17. The method of claim 1, wherein the substantially free of prematurely aborted RNA sequences is less than 0.1% prematurely aborted RNA sequences.

18. The method of claim 1, wherein the substantially free of prematurely aborted RNA sequences is that the prematurely aborted RNA sequences is undetectable by Coomassie staining.

19. The method of claim 1, wherein step (b) comprises incubating the impure preparation with urea at a concentration of about 5 M or greater.

20. The method of claim 1, wherein the tangential flow filtration is performed using a feed rate between 50 and 500 mL/minute.

21. The method of claim 1, wherein the tangential flow filtration is performed using a flow rate between 10 and 100 mL/minute.

22. The method of claim 1, where the tangential flow filtration is performed using a filter having a nominal molecular weight (NMWL) limit between 100 and 1,000 kDa.

23. The method of claim 1, wherein the purified mRNA has the integrity greater than 96%.

24. The method of claim 1, wherein the purified mRNA has the integrity greater than 98%.

25. The method of claim 1, wherein the purified mRNA has the integrity greater than 99%.

26. The method of claim 1, wherein the impure preparation further comprises DNA templates.