Methods for purification of messenger RNA

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

(a) precipitating mRNA from a solution comprising one or more contaminants from manufacturing the mRNA to provide a suspension comprising precipitated mRNA;

(b) subjecting the suspension comprising the precipitated mRNA to a depth filter, wherein the suspension flows in a direction perpendicular to the surface of the filter and precipitated mRNA is retained by the filter, comprising pore sizes ranging from 400 μm to 5 μm;

(c) dissolving the precipitated mRNA retained by the filter in step (b) thereby allowing purified mRNA to pass through the filter; and

(d) recovering the purified mRNA from step (c);

wherein each of steps (b), and (c) is conducted using normal flow filtration, and wherein at least 10 grams of mRNA is purified by the method in a single batch.

2. The method of claim 1 , wherein the manufacturing comprises in vitro transcription (IVT) synthesis of the mRNA prior to step (a).

3. The method of claim 1 , wherein the manufacturing comprises a step of 5′-capping of the mRNA prior to step (a).

4. The method of claim 1 , wherein the manufacturing comprises a step of 3′-tailing of the mRNA prior to step (a).

5. The method of claim 1 , wherein the one or more contaminants comprise an enzyme.

6. The method of claim 5 , wherein the enzyme is a polymerase or a capping enzyme.

7. The method of claim 1 , wherein the one or more contaminants comprise a salt.

8. The method of claim 1 , wherein the one or more contaminants comprise short abortive transcripts.

9. The method of claim 1 , further comprising a step of washing the precipitated mRNA retained on the filter from step (b).

10. The method of claim 1 , wherein the filter is made of an inert material.

11. The method of claim 1 , wherein no dispersant is added to the suspension.

12. The method of claim 1 , wherein the mRNA encodes a protein or a peptide.

13. The method of claim 1 , wherein the total purified mRNA recovered from the method is recovered in an amount that results in a yield of about 85%.

14. The method of claim 1 , wherein the total purified mRNA is recovered in an amount that results in a yield of about 90%.

15. The method of claim 1 , wherein the purified mRNA has a clinical grade purity without further purification.

16. The method of claim 15 , wherein the purified mRNA comprises 5% or less of protein contaminants as determined by capillary electrophoresis.

17. The method of claim 1 , wherein the purified mRNA has integrity of 95% or greater as determined by capillary electrophoresis.

18. A method of purifying a composition comprising 100 gm or more of mRNA manufactured by in vitro transcription (IVT) synthesis, the method comprising:

precipitating the IVT-transcribed mRNA comprising one or more contaminants from the IVT synthesis to generate a suspension;

subjecting the suspension comprising the precipitated mRNA and contaminants to normal flow filtration through a filter, where the precipitated mRNA is retained by the filter;

recovering the mRNA from the filter in a solution, thereby purifying the mRNA,

wherein at least 85% of the mRNA is recovered and the recovered mRNA has integrity of 90% or greater and is substantially free of protein contaminants.

19. The method of claim 1 , wherein at least 100 grams of mRNA is purified by the method in a single batch.

20. The method of claim 1 , wherein at least 250 grams of mRNA is purified by the method in a single batch.

21. The method of claim 1 , wherein at least 500 grams of mRNA is purified by the method in a single batch.

22. The method of claim 1 , wherein at least 1 kilogram of mRNA is purified by the method in a single batch.

23. The method of claim 1 , wherein the depth filter has a surface area of or greater than 264 m 2 .

24. The method of claim 1 , wherein the depth filter has a surface area of or greater than 1056 m 2 .

25. The method of claim 1 , wherein the depth filter has a surface area of or greater than 2112 m 2 .