PURIFICATION AND RECYCLING OF mRNA NUCLEOTIDE CAPS
This disclosure relates to purification and recycling nucleotide messenger RNA (mRNA) caps from the preparation of mRNA, which comprises isolating and purifying excess nucleotide mRNA caps from mRNA synthesis.
1 . A method of recycling mRNA nucleotide cap or a salt thereof, from an mRNA preparation, comprising:
collecting and combining one or more mixtures comprising the nucleotide mRNA cap, or a salt thereof, and one or more contaminants; and
removing the contaminants from the combined mixtures.
2 . The method of claim 1 , wherein the mRNA nucleotide cap, or a salt thereof, is:
3 . The method of claim 1 , wherein the mRNA nucleotide cap, or a salt thereof, is:
4 . The method of claim 1 , wherein the mRNA nucleotide cap, or a salt thereof, is:
5 . The method of claim 3 or 4 , wherein the cation is ammonium.
6 . The method of claim 3 or 4 , wherein the cation is dimethyloctylammonium, dimethylhexylammonium or triethylammonium.
7 . The method of any one of claims 1-6 , wherein the removing comprises:
removing macromolecules and proteins from the combined mixture comprising the mRNA nucleotide cap, or a salt thereof, to provide a first mixture.
8 . The method of claim 7 , wherein the removing of macromolecules and proteins comprises filtration.
9 . The method of claim 8 , wherein the filtration used to remove macromolecules and proteins is tangential flow filtration.
10 . The method of claim 8 or 9 , wherein the filtration used to remove macromolecules and proteins comprises a cassette filter or spiral-wound filter.
11 . The method of any one of claims 8-10 , wherein the filtration used to remove macromolecules and proteins comprises a cellulose based membrane filter.
12 . The method of any one claims 8-10 , wherein the filtration used to remove macromolecules and proteins comprises a polyamide thin film composite filter.
13 . The method of any one of claims 8-12 , wherein the filtration used to remove macromolecules and proteins comprises a filter having a molecular weight cut off of about 5 kDa to about 15 kDa.
14 . The method of any one of claims 7-13 , wherein the pH is adjusted to about 6.0 to about 6.5.
15 . The method of any one of claims 1-6 , wherein the removing further comprises:
concentrating and de-salting the first mixture to provide a second mixture.
16 . The method of claim 15 , wherein the de-salting comprises filtration.
17 . The method of claim 16 , wherein the filtration used to de-salt the first mixture is tangential flow filtration.
18 . The method of claim 16 or 17 , wherein the filtration used to de-salt the first mixture comprises a cassette filter or spiral-wound filter.
19 . The method of any one of claims 16-18 , wherein the filtration used to de-salt the first mixture comprises a cellulose based membrane filter.
20 . The method of any one of claims 16-18 , wherein the filtration used to de-salt the first mixture comprises a polyamide thin film composite filter.
21 . The method of any one of claims 16-20 , wherein the filtration used to de-salt the first mixture comprises a filter having a molecular weight cut off of about 50 Da to about kDa.
22 . The method of any one of claims 16-20 , wherein the filtration used to de-salt the first mixture comprises a filter having a molecular weight cut off of about 2 kDa.
23 . The method of any one of claims 1-6 , wherein the removing further comprises:
removing nucleotide triphosphates (NTPs) and ion exchanging from the second mixture to provide a third mixture.
24 . The method of claim 23 , wherein the removing of NTPs comprises passing the second mixture through an ion exchange chromatography system.
25 . The method of claim 23 or 24 , wherein the ion exchange chromatography system is an anion exchange system.
26 . The method of claim 25 , wherein the anion exchange chromatography system comprises a mobile phase comprising water, an aqueous solution, or a buffered aqueous solution.
27 . The method of claim 25 or 26 , wherein the mobile phase comprises water and an aqueous solution of NaCl.
28 . The method of claim 27 , wherein the aqueous solution of NaCl has a concentration of about 0.25 M to about 2.0 M.
29 . The method of claim 25 or 26 , wherein the mobile phase comprises water and an aqueous solution of NH 4 Cl.
30 . The method of claim 29 , wherein the aqueous solution of NH 4 Cl has a concentration of about 0.5 M to about 1.5 M.
31 . The method of any one of claims 25-30 , wherein the anion exchange chromatography comprises exchanging N,N-dimethyloctylammonium (DMOA) for NH 4 + .
32 . The method of any one of claims 25-31 , wherein the anion exchange chromatography system comprises a stationary phase comprising a strong base or a weak base.
33 . The method of claim 32 , wherein the stationary phase comprises a functional group comprising dimethylamine, triethylamine, polyamine, a tertiary amine, a quaternary amine, dimethylethanolamine, or trimethylbenzylammonium.
34 . The method of any one of claims 24-33 , wherein the pump flow of the ion exchange chromatography system is about 50 mL/min to about 10 L/min.
35 . The method of any one of claims 1-6 , wherein the removing further comprises:
concentrating and de-salting the third mixture to provide a fourth mixture.
36 . The method of claim 35 , wherein the de-salting of the third mixture comprises passing the third mixture through a chromatography system or filtration, or combination thereof.
37 . The method of claim 36 , wherein the chromatography system used for de-salting the third mixture is a reverse phase chromatography.
38 . The method of claim 37 , wherein the reverse phase chromatography comprises a stationary phase comprising poly(styrene divinylbenzene) or C18 resin.
39 . The method of claim 37 or 38 , wherein the reverse phase chromatography comprises a mobile phase comprising a DMOA buffer solution.
40 . The method of claim 39 , wherein the DMOA buffer solution of the mobile phase of the reverse phase chromatography has a concentration of about 5 mM to about 15 mM.
41 . The method of claim 37 or 38 , wherein the reverse phase chromatography comprises a mobile phase comprising acetonitrile.
42 . The method of any one of claims 37-41 , wherein the reverse phase chromatopgrahy further comprises a salt exchange.
43 . The method of any one of claims 37-42 , wherein the reverse phase chromatography comprises exchanging Na + for N,N-dimethyloctylammonium (DMOA).
44 . The method of any one of claims 37-43 , wherein the pump flow of the reverse phase chromatography system is about 50 mL/min to about 10 L/min.
45 . The method of claim 36 , wherein the filtration of the third mixture is tangential flow filtration.
46 . The method of claim 36 or 45 , wherein the filtration of the third mixture comprises a cassette filter or spiral-wound filter.
47 . The method of any one of claims 36, 45, and 46 , wherein the filtration of the third mixture comprises a cellulose based membrane filter.
48 . The method of any one of claims 36, 45, and 46 , wherein the filtration of the third mixture comprises a polyamide thin film composite filter.
49 . The method of any one of claims 36 and 45-48 , wherein the filtration of the third mixture comprises a filter having a molecular weight cut off of about 50 Da to about 5 kDa.
50 . The method of any one of claims 1-6 , wherein the removing further comprises:
filtering, and adjusting the concentration and pH of the fourth mixture.
51 . The method of claim 50 comprising filtering the fourth mixture through a polyvinylidene filter, polyethylene filter, polypropylene filter, polytetrafluoroethylene filter, cellulose ester filter, or polyethersulfone filter.
52 . The method of claim 50 or 51 , wherein the filtering of the fourth mixture comprises using a filter having a size of about a 0.1 μm to about 1 μm.
53 . The method of claim 50 , wherein the concentration of the fourth mixture is adjusted to about 1000 mM to about 25 mM.
54 . The method of claim 50 , wherein the concentration of the fourth mixture is adjusted to about 100 mM.
55 . The method of claim 50 , wherein the concentration of the fourth mixture is adjusted to about 50 mM.
56 . The method of any one of claims 50 and 53-55 , wherein the concentration of the fourth mixture is adjusted with a basic solution.
57 . The method of claim 56 , wherein the basic solution used to adjust the concentration of the fourth mixture comprises NH 4 OH and water.
58 . The method of claim 56 , wherein the basic solution used to adjust the concentration of the fourth mixture comprises about 3.5% w/v to 4.5% w/v NH 4 OH in water.
59 . The method of any one of claims 50 and 53-58 , wherein the pH of the fourth mixture is adjust to about 5.3 to about 7.3.