Methods for determining active ingredients in pro-drug PEG protein conjugates with releasable PEG reagents (in vitro de-pegylation)

Patent metadata

Field	Value
Publication number	US-9274122-B2
Application number	US-58249709-A
Country	US
Kind code	B2
Filing date	Oct 20, 2009
Priority date	Oct 21, 2008
Publication date	Mar 1, 2016
Grant date	Mar 1, 2016

Abstract

The invention relates to the development of in vitro assay systems that force the release of a water-soluble polymer, such as polyethylene glycol (PEG) and polysialic acid (PSA), from proteins modified with a reversibly-linked water-soluble polymer. The invention includes methods for analyzing the release of the water-soluble polymer and measuring regained protein activity. The invention further includes methods appropriate for the quality control of proteins modified with releasable water-soluble polymers, including polymers like PEG and PSA.

First claim

What is claimed is:

1. A method of releasing a reversibly-linked water-soluble polymer from a protein modified by the water-soluble polymer comprising the step of adding free amine consisting of free lysine, histidine, or a combination thereof to a solution comprising the protein modified by the water-soluble polymer at a concentration effective to release the water-soluble polymer.

2. A method of increasing activity of a protein modified with a reversibly-linked water-soluble polymer by releasing the water-soluble polymer from the protein comprising the step of adding free amine consisting of free lysine, histidine, or a combination thereof to a solution comprising the protein modified by the water-soluble polymer at a concentration effective to release the water-soluble polymer.

3. The method of claim 1 or 2 , wherein the method further comprises increasing pH of the solution comprising the protein.

4. The method of claim 3 , wherein the method comprises increasing pH of the solution to about pH 10.

5. The method of claim 3 , wherein the method comprises increasing pH of the solution to about 9.8.

6. The method of claim 3 , wherein the method comprises increasing pH of the solution to about 8.1.

7. The method of claim 1 or 2 , wherein the method further comprises increasing temperature of the solution above about 4° C.

8. The method of claim 7 , wherein the temperature of the solution is increased to about 37° C.

9. The method of claim 1 or 2 , wherein the method further comprises incubating the protein modified by the water-soluble polymer in the solution for at least about 5 minutes.

10. The method of claim 9 , wherein the incubating step is carried out for up to about 168 hours.

11. The method of claim 9 , where the incubating step is carried out for about 48 hours.

12. The method of any one of claim 1 or claim 2 , wherein the protein is factor VIII (FVIII).

13. The method of any one of claim 1 or claim 2 , wherein the protein is von Willebrand Factor (VWF).

14. The method of claim 1 or claim 2 wherein the water-soluble polymer is reversibly linked to the protein with 9H-(f)luoren-9-yl(m)eth(o)xy(c)arbonyl, dibenzofulvene, or a derivative thereof.

15. The method of claim 14 wherein the water-soluble polymer is linked with 9H-(f)luoren-9-yl(m)eth(o)xy(c)arbonyl, or a derivative thereof.

16. The method of any one of claims 1 - 6 , 7 - 10 and 12 - 15 , wherein the water-soluble polymer is polyethylene glycol.

17. The method of any one of claims 1 - 6 , 7 - 10 and 12 - 15 , wherein the water-soluble polymer is polysialic acid.

18. The method of claim 1 or 2 , wherein the concentration of free amine is about 100 mM.

19. The method of claim 1 or 2 , wherein the concentration of free amine is about 200 mM.

Assignees

• Varadi Katalin
• Schrenk Gerald
• Rottensteiner Hanspeter
• Turecek Peter
• Weber Alfred
• Anderle Heinz
• Culbertson Sean M
• Fang Zhihao
• Zappe Harold
• Zhang Ping
• Bossard Mary J
• Baxalta Inc
• Baxalta GmbH
• Nektar Therapeutics

Inventors

• Varadi Katalin
• Schrenk Gerald
• Rottensteiner Hanspeter
• Turecek Peter
• Weber Alfred
• Anderle Heinz
• Culbertson Sean M
• Fang Zhihao
• Zappe Harold
• Zhang Ping
• Bossard Mary J

Classifications

• G01N33/86

  involving blood coagulating time {or factors, or their receptors} · CPC title

• G01N2333/76

  Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation · CPC title

• G01N33/542

  with steric inhibition or signal modification, e.g. fluorescent quenching · CPC title

• G01N33/582

  with fluorescent label · CPC title

• G01N33/68 Primary

  involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)} · CPC title