Nucleotide sugar purification using membranes
View Patent ↗The invention provides methods of removing contaminants from a mixture of a desired product and contaminants by pH adjustments and molecular weight cut-offs. The contaminants include phosphate groups, magnesium sulfate, sodium pyruvate and tetrasodium pyrophosphate groups. The desired product includes nucleotide sugars, glycolipids, LnNT, sialyl lactose, and salts.
1. A method of removing essentially all of a contaminant from a mixture comprising the contaminant and a nucleotide sugar comprising a branched PEG structure, the method comprising:
contacting the mixture with a first membrane for a length of time sufficient to allow essentially all of the contaminant to pass through the first membrane wherein
the mixture has a pH such that the first membrane and the nucleotide sugar comprising a branched PEG structure have a net charge of the same sign and the contaminant has a net charge which is neutral or a sign which is opposite of the sign of the net charge for the first membrane; and
the first membrane has a molecular weight cut-off that is less than the molecular weight of the nucleotide sugar comprising a branched PEG structure; and
wherein the branched PEG structure has a formula of:
wherein m and n are integers independently selected to provide a PEG moiety having a molecular weight of 2 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa, or 40 kDa;
thereby removing essentially all of the contaminant from the mixture.
2. The method according to claim 1 , wherein the nucleotide sugar and the first membrane each have a net negative charge and the contaminant has a net charge which is neutral or a net positive charge.
3. The method according to claim 1 , wherein the contaminant is a member selected from the group consisting of phosphate, pyrophosphate, nucleotide monophosphate, nucleotide diphosphate, nucleotide triphosphate, sodium phosphate, manganese chloride, sodium pyruvate, GlcNAc, magnesium sulfate, tetrasodium pyrophosphate, lactose, benzoic acid, LNT-2, LNnT, sialic acid, cytidine, CMP, benzyl alcohol, CyLac, cylexin, cytilene and sodium chloride.
4. The method according to claim 1 , wherein the first membrane is contacted with at least about 500 mg of the nucleotide sugar.
5. The method according to claim 1 , wherein the nucleotide sugar is a member selected from the group consisting of CMP-Sialic Acid, CMP-Nan, GDP-Man, GDP-Fuc, UDP-Glc, UDP-Gal, UDP-GlcNAc, UDP-GalNAc, UDP-GlcA, UDP-IdoA, and UDP-Xyl.
6. The method according to claim 1 , wherein, prior to the contacting, the method further comprises forming the mixture by contacting a nucleotide with a sugar and a nucleotide sugar synthetase capable of ligating the nucleotide and the sugar.
7. The method according to claim 1 , wherein, prior to the contacting, the method further comprises forming the mixture by subjecting a cell system to conditions whereby the cell system produces a nucleotide sugar.
8. The method according to claim 1 , wherein the mixture is not further purified prior to the contacting.
9. The method according to claim 1 , wherein the nucleotide sugar is not further purified after the contacting.
10. The method according to claim 1 , wherein the nucleotide sugar is further purified after the contacting.
11. The method according to claim 1 , wherein, prior to the contacting, the method further comprises contacting the mixture with a second membrane for a length of time sufficient to allow the nucleotide sugar to pass through the second membrane and to allow molecules with a molecular weight greater than about 500 kDa to be retained in the second membrane.
12. The method according to claim 1 , wherein, prior to the contacting, the method further comprises contacting the mixture with a third membrane for a length of time sufficient to allow the nucleotide sugar to pass through the third membrane and to allow molecules with a molecular weight greater than about 10 kDa to be retained in the third membrane.
13. A method of purifying a nucleotide sugar comprising a branched PEG structure from a mixture comprising a phosphorus-containing contaminant and the nucleotide sugar comprising a branched PEG structure, the method comprising:
contacting the mixture with a first membrane for a length of time sufficient to allow essentially all of the phosphorus-containing contaminant to pass through the first membrane
wherein
the mixture has a pH such that the first membrane and the nucleotide sugar comprising a branched PEG structure have a net negative charge and the contaminant has a net charge which is a neutral or a positive charge; and
the first membrane has a molecular weight cut-off that is less than the molecular weight of the nucleotide sugar comprising a branched PEG structure; and
wherein the branched PEG structure has a formula of:
wherein m and n are integers independently selected to provide a PEG moiety having a molecular weight of 2 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa, or 40 kDa;
thereby purifying the nucleotide sugar comprising a branched PEG structure from the mixture.
14. The method according to claim 13 , wherein the first membrane is contacted with at least about 500 mg of the nucleotide sugar.
15. The method according to claim 13 , wherein the nucleotide sugar is a member selected from the group consisting of CMP-Sialic Acid, CMP-Nan, GDP-Man, GDP-Fuc, UDP-Glc, UDP-Gal, UDP-GlcNAc, UDP-GalNAc, UDP-GlcA, UDP-IdoA and UDP-Xyl.
16. A method of removing essentially all of a contaminant from a mixture comprising the contaminant and a nucleotide sugar comprising a branched PEG structure, the method comprising:
contacting the mixture with a first membrane for a length of time sufficient to allow essentially all of the contaminant to pass through the first membrane wherein
the mixture has a pH such that the first membrane and the nucleotide sugar comprising a branched PEG structure have a net charge of the same sign and the contaminant is at its isoelectric point; and
the first membrane has a molecular weight cut-off that is less than the molecular weight of the nucleotide sugar comprising a branched PEG structure; and
wherein the branched PEG structure has a formula of:
wherein m and n are integers independently selected to provide a PEG moiety having a molecular weight of 2 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa, or 40 kDa;
thereby removing essentially all of the contaminant from the mixture.
17. The method according to claim 1 , wherein prior to the contacting, a reactive solution comprising the nucleotide sugar is contacted with a nanofiltration membrane, thereby removing a nucleotide monophosphate and a sugar from the reactive solution while retaining the nucleotide sugar in the reactive solution.
18. The method of claim 17 , further comprising: passing the reactive solution comprising the nucleotide sugar over a charged media depth filter.
19. The method according to claim 1 , wherein the nucleotide sugar is CMP-Sialic Acid.
20. The method according to claim 13 , wherein the nucleotide sugar is CMP-Sialic Acid.
21. The method according to claim 16 , wherein the nucleotide sugar is CMP-Sialic Acid.