Nucleotide sugar purification using membranes

• US 8,841,439 B2
• Filed: 08/23/2011
• Issued: 09/23/2014
• Est. Priority Date: 11/03/2005
• Status: Expired due to Fees

• Current Assignee: Novo Nordisk A/S
• Original Assignee: Novo Nordisk A/S
• Inventors: Felo, Michael, DeFrees, Shawn
• Primary Examiner(s): Fetterolf, Brandon
• Assistant Examiner(s): Shiao, Yih-Horng
• Application Number:
  US13/215,439
• Publication Number:
  US 20120083600A1
• Time in Patent Office: 1,127 Days
• Field of Search: 536/26.8
• US Class Current: 536/26.8
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C12P 19/305 Pyrimidine nucleotides
• CPC Class Codes: Y02P 20/582 Recycling of unreacted starting or intermediate materials

Abstract

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.

Claims

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;

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.

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.

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;

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.

20. The method according to claim 13, wherein the nucleotide sugar is CMP-Sialic Acid.

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;

21. The method according to claim 16, wherein the nucleotide sugar is CMP-Sialic Acid.