Methods for purification of messenger RNA

• US 10,155,785 B2
• Filed: 12/04/2017
• Issued: 12/18/2018
• Est. Priority Date: 04/25/2014
• Status: Active Grant

• Current Assignee: RaNA Therapeutics Inc. (Sanofi )
• Original Assignee: Translate Bio, Inc. (Sanofi )
• Inventors: DeRosa, Frank, Dias, Anusha, Karve, Shrirang, Heartlein, Michael
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US15/831,252
• Publication Number:
  US 20180127449A1
• Time in Patent Office: 379 Days
• CPC Class Codes: C07H 1/06 Separation; Purification
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• 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 (a) precipitating mRNA from an impure preparation; (b) subjecting the impure preparation comprising precipitated mRNA to a purification process involving membrane filtration such that the precipitated mRNA is captured by a membrane; and (c) eluting the captured precipitated mRNA from the membrane by re-solubilizing the mRNA, thereby resulting in a purified mRNA solution. In some embodiments, a purification process involving membrane filtration suitable for the present invention is tangential flow filtration.

Claims

1. A method of large scale purification of messenger RNA (mRNA), comprising:

providing an impure preparation comprising in vitro synthesized mRNA greater than 1 gram and prematurely aborted RNA sequences;

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

precipitating the mRNA from the treated impure preparation;

subjecting a heterogeneous suspension comprising the precipitated mRNA to tangential flow filtration such that the precipitated mRNA is captured by a filtration membrane; and

eluting the captured precipitated mRNA from the membrane by re-solubilizing the mRNA, thereby resulting in a purified mRNA solution,

wherein the mRNA has a coding region that encodes cystic fibrosis conductance regulator protein.

2. The method of claim 1, wherein the step of precipitating mRNA comprises treating the impure preparation with a solution comprising a reagent selected from the group consisting of lithium chloride, potassium chloride, guanidinium chloride, guanidinium thiocyanate, guanidinium isothiocyanate, and ammonium acetate, or a combination thereof.

3. The method of claim 1, wherein the method further comprises a step of dialyzing the purified mRNA solution.

4. The method of claim 3, wherein the purified mRNA solution is dialyzed with 1 mM sodium citrate using a 100 kDa molecular weight cut-off (MWCO) membrane.

5. The method of claim 1, wherein the impure preparation comprises an in vitro mRNA synthesis reaction mixture.

6. The method of claim 5, wherein the impure preparation further comprises enzyme reagents used in in vitro synthesis.

7. The method of claim 1, wherein the purified mRNA solution contains less than 1% of prematurely aborted RNA.

8. The method of claim 1, wherein the in vitro synthesized mRNA is greater than 10 gram, 100 gram, 1 kg, 10 kg, or 100 kg.

9. The method of claim 1, wherein the 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, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 20 kb in length.

10. The method of claim 1, wherein the purified mRNA has an integrity of or greater than 95%.

11. A method for manufacturing messenger RNA (mRNA) having a coding region that encodes cystic fibrosis conductance regulator protein, the method comprising:

synthesizing in vitro mRNA having a coding region that encodes cystic fibrosis conductance regulator protein; and

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

12. The method of claim 1, further comprising a step of adding a cap and/or a polyA tail to the mRNA in the purified solution, thereby producing polyA tailed and capped mRNA.

13. The method of claim 12, further comprising a step of purifying the polyA tailed and capped mRNA.

14. The method of claim 11, wherein the purified mRNA has the integrity of or greater than 96%.

15. The method of claim 11, wherein the purified mRNA has the integrity of or greater than 98%.

16. The method of claim 11, wherein the purified mRNA has the integrity of or greater than 99%.