Modification-dependent activity assays

1. A method for detecting the presence of a recombinant polypeptide comprising a modification, the method comprising the steps of:

incubating a sample including the recombinant polypeptide comprising the modification with a capture agent that selectively binds the modification under conditions allowing the selective binding of the capture agent to the modification, thereby forming a polypeptide-agent complex;

purifying the polypeptide-agent complex from the sample; and

assaying for the presence of the recombinant polypeptide and/or a polypeptide activity, wherein detection of the recombinant polypeptide and/or the polypeptide activity is indicative of the presence of the recombinant polypeptide comprising the modification.

2. The method according to claim 1 , wherein the sample includes a polypeptide without the modification and/or a polypeptide with a different pattern of degree of modification.

3. The method according to claim 1 , wherein the recombinant polypeptide is a therapeutic polypeptide.

4. The method according to claim 1 , wherein the recombinant polypeptide is a coagulation factor.

5. The method according to claim 1 , wherein the coagulation factor is a Factor II, a Factor IIa, a Factor VII, a Factor VIIa, a Factor VIII, a Factor VIIIa, a Factor IX, a Factor IXa, a Factor X, or a Factor Xa.

6. The method according to claim 1 , wherein the capture agent has an association rate constant for a polypeptide comprising the modification of more than 1×10 5 M −1 s −1 .

7. The method according to claim 1 , wherein the capture agent has a disassociation rate constant for a polypeptide comprising the modification of less than 1×10 −3 s −1 .

8. The method according to claim 1 , wherein the capture agent has an equilibrium disassociation constant for a polypeptide comprising the modification of less than 0.500 nM.

9. The method according to claim 1 , wherein the capture agent has an association rate constant for a polypeptide without a modification or a polypeptide with a different pattern or degree of modification of less than 1×10 4 M −1 s −1 .

10. The method according to claim 1 , wherein the capture agent has an association rate constant (Ka) for the recombinant polypeptide comprising a modification that is more than 1×10 0 M −1 s −1 relative to the association rate constant (Ka) of the capture agent for a recombinant polypeptide without such a modification and/or the association rate constant (Ka) of the capture agent for a recombinant polypeptide with a different pattern or degree of modification.

11. The method according to claim 1 , wherein the capture agent has an association rate constant (Ka) for the recombinant polypeptide comprising a modification that is at least 2-fold more then the association rate constant (Ka) of the capture agent for a recombinant polypeptide without such a modification and/or then the association rate constant (Ka) of the capture agent for a recombinant polypeptide with a different pattern or degree of modification.

12. The method according to claim 1 , wherein the capture agent has a binding specificity ratio for a recombinant polypeptide comprising a modification relative to a recombinant polypeptide without such a modification and/or relative to a recombinant polypeptide with a different pattern or degree of modification of at least 2:1.

13. The method according to claim 1 , wherein the capture agent distinguishes the recombinant polypeptide comprising a modification from the same polypeptide but without the modification.

14. The method according to claim 1 , wherein the capture agent distinguishes the recombinant polypeptide comprising a modification from the same polypeptide but with a different pattern or degree of the same modification.

15. The method according to claim 1 , wherein the recombinant polypeptide comprising the modification is a PEGylation Factor II, a PEGylation Factor IIa, a polysialylation Factor II, a polysialylation Factor IIa, a HESylation Factor II, a HESylation Factor IIa, a Sylation Factor II, or a Sylation Factor IIa.

16. The method according to claim 1 , wherein the recombinant polypeptide comprising the modification is a PEGylation Factor VII, a PEGylation Factor VIIa, a polysialylation Factor VII, a polysialylation Factor VIIa, a HESylation Factor VII, a HESylation Factor VIIa, a Sylation Factor VII, or a Sylation Factor VIIa.

17. The method according to claim 1 , wherein the recombinant polypeptide comprising the modification is a PEGylation Factor VIII, a PEGylation Factor Villa, a polysialylation Factor VIII, a polysialylation Factor Villa, a HESylation Factor VIII, a HESylation Factor Villa, a Sylation Factor VIII, or a Sylation Factor VIIIa.

18. The method according to claim 1 , wherein the recombinant polypeptide comprising the modification is a PEGylation Factor IX, a PEGylation Factor IXa, a polysialylation Factor IX, a polysialylation Factor IXa, a HESylation Factor IX, a HESylation Factor IXa, a Sylation Factor IX, or a Sylation Factor IXa.

19. The method according to claim 1 , wherein the capture agent is an anti-PEG antibody, an anti-PSA antibody, an anti-HES antibody, or an anti-S antibody.

20. The method according to claim 1 , wherein the capture agent is attached to a solid support.

21. The method according to claim 20 , wherein the solid support is a multi-well plate, a film, a tube, a sheet, a column, or a microparticle.

22. The method according to claim 1 , wherein the assaying step is performed using a qualitative assay or a quantitative assay.

23. The method according to claim 1 , wherein the assaying step is performed using an in vitro assay, a cell-based assay, or an in vivo assay.

24. The method according to claim 1 , wherein the assaying step is performed using a non-specific polypeptide assay or a specific polypeptide assay.

25. The method according to claim 24 , wherein the non-specific polypeptide assay is a UV absorption, a biuret assay, or a Bradford assay.

26. The method according to claim 24 , wherein the specific polypeptide assay is a chromogenic assay, a colorimetirc assay, a chronometric assay, a chemiluminescense assay, an electrochemiluminescence assay, a bioluminescence assay, a fluorogenic assay, a resonance energy transfer assay, a plane polarization assay, a flow cytometry assay, an immuno-based assay or an activity assay.

27. The method according to claim 24 , wherein the activity assay is an enzymatic activity assay, an inhibitory activity assay, a coagulation activity assay, or a polymerization activity assay.

28. The method according to claim 1 , wherein selective binding of the capture agent occurs at a neutral to alkaline pH.