RNA purification methods
Methods for purifying RNA from a sample, comprising one or more steps of tangential flow filtration, hydroxyapatite chromatography, core bead flow-through chromatography, or any combinations thereof. These techniques are useful individually, but show very high efficiency when used in combination, or when performed in particular orders. The methods can purify RNA in a highly efficient manner without unduly compromising potency or stability, to provide compositions in which RNA is substantially cleared of contaminants. Moreover, they can be performed without the need for organic solvents.
1. A method for purifying desired RNA from an in vitro transcription (IVT) reaction sample, comprising the following steps:
(i) loading RNA containing desired RNA from an in vitro transcription sample to a core bead flow-through column;
(ii) collecting a flow-through containing the desired RNA and undesired components from the core bead flow-through column; and
(iii) passing the flow-through from step (ii) through a tangential flow filtration system having a filter membrane having a sample side and a filtrate side with a specific pore size and passing a final formulation buffer tangentially across the sample side of the filter membrane of the tangential flow filtration system such that the undesired components from the flow-through from step (ii) pass through the filter membrane to the filtrate side and the desired RNA is retained on the sample side of the filter membrane, thereby purifying the desired RNA.
2. The method according to claim 1 , wherein step (i) is conducted using a buffer containing a potassium salt.
3. The method according to claim 1 , wherein the desired RNA is purified on a preparative scale.
4. The method according to claim 1 , wherein each step of the method does not comprise the use of lithium chloride or organic solvents, each step of the method is not performed at temperatures greater than 70° C., and each step of the method does not involve enzymatic digestion of DNA.
5. The method according to claim 1 , wherein the method further includes discarding materials which do not contain the RNA or the desired RNA species.
6. The method according to claim 1 , wherein the sample contains the RNA and one or more of: plasmid DNA, deoxy-oligonucleotides, deoxynucleoside monophosphates, ribonucleoside triphosphates and protein; and wherein the sample does not contain genomic DNA and/or a cell membrane or fragments thereof.
7. The method according to claim 1 , wherein the sample contains the RNA and not more than four of: plasmid DNA, deoxy-oligonucleotides, deoxynucleoside monophosphates, ribonucleoside triphosphates and protein; and wherein the sample does not contain genomic DNA and/or a cell membrane or fragments thereof.
8. The method according to claim 1 , wherein the desired RNA is a single-stranded mRNA.
9. The method according to claim 1 , wherein the desired RNA comprises a linear sequence of at least 1,000 nucleotides.
10. The method according to claim 1 , wherein the tangential flow filtration system has a hydrophilic stationary phase, a hydrophilic membrane, or both.
11. The method of claim 1 , wherein the sample side of said filter membrane is held at positive pressure relative to said filtrate side of the filter membrane.
12. The method according to claim 1 , wherein the in vitro transcription sample is made by an in vitro transcription of an RNA sample.
13. The method according to claim 1 , wherein the desired RNA is at least 99% pure.
14. The method according to claim 1 , wherein the desired RNA is free from DNA, pyrophosphates, and free nucleotides.
15. The method of claim 1 , wherein both said passing the flow-through from step (ii) through a tangential flow-filtration system having a filter membrane having a sample side and a filtrate side with a specific pore size and said passing a final formulation buffer tangentially across said sample side of the filter membrane of the tangential flow filtration system occur at the same time during step (iii).
16. The method of claim 1 , wherein the tangential flow filtration system is in a single closed system.
17. The method of claim 1 , wherein the tangential flow filtration system is a tangential flow filtration system that comprises a cartridge that contains the filter membrane.
18. The method of claim 1 , step (iii) further comprising a step of recovering the desired RNA.
19. The method of claim 1 , wherein said undesired components comprise free nucleotides.
20. A method for preparing a pharmaceutical composition comprising the steps of:
(a) purifying desired RNA from an in vitro transcription (IVT) reaction sample by a process comprising
(i) loading RNA containing desired RNA from an in vitro transcription sample to a core bead flow-through column;
(ii) collecting a flow-through containing the desired RNA and undesired components from the core bead flow-through column; and
(iii) passing the flow-through from step (ii) through a tangential flow filtration system having a filter membrane having a sample side and a filtrate side with a specific pore size and passing a final formulation buffer tangentially across the sample side of the filter membrane of the tangential flow filtration system such that the undesired components from the flow-through from step (ii) pass through the filter membrane to the filtrate side and the desired RNA is retained on the sample side of the filter membrane, thereby purifying the desired RNA; and
(b) preparing said pharmaceutical composition by formulating the desired RNA purified from step (a).
21. The method of claim 20 , wherein the sample side of said filter membrane is held at positive pressure relative to said filtrate side of the filter membrane.
22. The method of claim 20 , wherein said final formulation buffer is exchanged for a purification buffer which the flow-through from step (ii) is present in.
23. The method of claim 20 , wherein said undesired components comprise free nucleotides.