Process for the production of purified cytidinemonophosphate-sialic acid-polyalkylene oxide (CMP-SA-PEG) as modified nucleotide sugars via anion exchange chromatography

• US 9,493,499 B2
• Filed: 06/12/2008
• Issued: 11/15/2016
• Est. Priority Date: 06/12/2007
• Status: Active Grant

• Current Assignee: Ratiopharm GmbH (Teva Pharmaceutical Industries Limited)
• Original Assignee: Novo Nordisk A/S
• Inventors: DeFrees, Shawn, Bowe, Caryn
• Primary Examiner(s): Jiang, Shaojia Anna
• Assistant Examiner(s): Miller, Dale R
• Application Number:
  US12/663,748
• Publication Number:
  US 20100174059A1
• Time in Patent Office: 3,078 Days
• US Class Current: 1/1
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids

Abstract

The current invention provides methods (e.g., large-scale processes) for the production of nucleotide sugars, which are modified with a polymeric modifying group, such as poly(alkylene oxide) moieties (e.g., poly(ethylene glycol) or poly(propylene glycol)) moieties. A typical process of the invention includes anion exchange chromatography followed by an ultrafiltration procedure, such as tangential flow filtration. The process of the invention provides modified nucleotide sugars in unexpectedly high purity and high overall yields.

Claims

1. A method of making a composition comprising a modified nucleotide sugar covalently linked to a polymeric modifying group, said polymeric modifying group comprising at least one linear or branched poly(alkylene oxide) moiety, said method comprising:

(i) contacting a reaction mixture comprising said modified nucleotide sugar with a Q-sepharose anion exchange resin;

(ii) eluting said modified nucleotide sugar from said Q-sepharose anion exchange resin using a bicarbonate buffer, wherein said eluting comprises a first step wherein said buffer comprises between about 0.01 mM and about 10 mM of bicarbonate and a second step wherein said buffer comprises between about 10 mM and about 30 mM of bicarbonate, thereby forming an eluate fraction comprising said modified nucleotide sugar; and

(iii) desalting said eluate fraction,

wherein said method does not comprise ultrafiltration prior to step (i), thereby forming said composition, wherein said modified nucleotide sugar comprises cytidine-monophospho-sialic acid (CMP-SA), wherein said method provides said modified nucleotide sugar in a purity between about 80% and about 100% (w/w) and wherein the linear or branched poly(alkylene oxide) moiety has a molecular weight in the range of 10-40 kDa.

2. The method of claim 1, wherein said reaction mixture has a salt conductivity of less than about 5 mS/cm.

3. The method of claim 1, wherein said desalting is accomplished using membrane filtration.

4. The method of claim 3, wherein said membrane filtration is tangential flow filtration (TFF).

5. The method of claim 3, wherein said membrane filtration reduces the volume of said eluate fraction.

6. The method of claim 3, wherein said membrane filtration results in reduced salt conductivity of said eluate fraction compared to said salt conductivity prior to said membrane filtration.

7. The method of claim 6, wherein said reduced salt conductivity of said eluate fraction is between about 10 μ

S/cm and about 200 μ

S/cm.

8. The method of claim 6, wherein said reduced salt conductivity of said eluate fraction is less than about 50 μ

S/cm.

9. The method of claim 1, further comprising reducing the volume of said mixture prior to step (i).

10. The method of claim 9, wherein said reducing is accomplished using rotary evaporation.

11. The method of claim 1, wherein said eluate fraction comprising said modified nucleotide sugar is subjected to freeze drying or spray drying.

12. The method of claim 1, wherein said poly(alkylene oxide) moiety is a linear or branched poly(ethylene glycol) moiety.

13. The method of claim 1, wherein said nucleotide sugar is cytidine-monophospho-sialic acid (CMP-SA) covalently linked to a linear or branched poly(ethylene glycol) moiety.

14. The method of claim 13, wherein said branched poly(ethylene glycol) moiety comprises a glycerol backbone.

15. The method of claim 1, wherein said modified nucleotide sugar is a member selected from the group consisting of:

16. The method of claim 1, wherein said method provides said modified nucleotide sugar in an overall yield from about 50% to about 90%.

17. The method of claim 1, wherein said reaction mixture comprising said modified nucleotide sugar is formed by contacting a nucleotide sugar derivative comprising a nucleotide sugar and a primary amino group with an activated poly(alkylene oxide) moiety under conditions sufficient to form a covalent bond between said amino group of said nucleotide sugar derivative and said poly(alkylene oxide) moiety.

18. The method of claim 17, wherein said nucleotide sugar derivative is CMP-SA-glycine.

19. The method of claim 17, wherein said activated poly(alkylene oxide) moiety comprises a p-nitrophenyl carbonate moiety.

20. The method of claim 17, wherein said nucleotide sugar derivative and said poly(alkylene oxide) reagent are contacted in the presence of an aqueous solvent having a pH between about 8.0 and about 8.8.

21. The method of claim 17, wherein said poly(alkylene oxide) reagent is a poly(ethylene glycol)-p-nitrophenyl carbonate.

22. A method of making a composition comprising a modified nucleotide sugar covalently linked to a polymeric modifying group, said polymeric modifying group comprising at least one linear or branched poly(alkylene oxide) moiety, said method comprising:

(i) contacting a nucleotide sugar derivative comprising a primary amino group with an activated poly(alkylene oxide) moiety comprising a p-nitrophenyl carbonate moiety under conditions sufficient to form a covalent bond between said amino group of said nucleotide sugar derivative and said poly(alkylene oxide) moiety, wherein said contacting occurs in the presence of an aqueous solvent having a pH between about 8.0 and about 8.8, thereby forming a reaction mixture comprising said modified nucleotide sugar;

(ii) contacting said reaction mixture comprising said modified nucleotide sugar with a Q-sepharose anion exchange resin;

(iii) eluting said modified nucleotide sugar from said Q-sepharose anion exchange resin using a bicarbonate buffer, wherein said eluting comprises a first step wherein said buffer comprises between about 0.01 mM and about 10 mM of bicarbonate and a second step wherein said buffer comprises between about 10 mM and about 30 mM of bicarbonate thereby forming an eluate fraction comprising said modified nucleotide sugar;

(iv) desalting said eluate fraction using membrane filtration; and

(v) removing water from said eluate fraction,

wherein said method does not comprise ultrafiltration prior to step (i), thereby forming said composition wherein said modified nucleotide sugar comprises cytidine-monophospho-sialic acid (CMP-SA), wherein said method provides said modified nucleotide sugar in a purity between about 80% and about 100% (w/w), and wherein the linear or branched poly alkylene oxide) moiety has a molecular weight in the range of 10-40 kDa.

23. The method of claim 22, wherein said nucleotide sugar derivative is CMP-SA-glycine.

24. The method of claim 22, wherein said modified nucleotide sugar is CMP-SA covalently linked to a linear or branched poly(ethylene glycol) moiety.