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