Methods for detecting protein in plasma
The present invention provides methods for detecting protein in plasma comprising contacting said plasma with a protease capable of digesting said protein into at least one detectable fragment and detecting said at least one detectable fragment using high performance liquid chromatography and mass spectroscopy.
1. A method for detecting at least one therapeutic protein in plasma from a mammal comprising contacting said plasma with a first protease capable of digesting said at least one therapeutic protein into at least one detectable fragment and detecting said at least one detectable fragment using high performance liquid chromatography and mass spectroscopy, wherein said at least one therapeutic protein comprises at least two GLP-1(7-36(A8G)) tandemly and genetically fused to human serum albumin.
2. The method of claim 1 , wherein the at least two GLP-1(7-36(A8G)) are genetically fused at the N-terminus of the human serum albumin.
3. The method of claim 1 , wherein said therapeutic protein comprises SEQ ID NO:1.
4. The method of claim 1 , wherein said first protease is selected from the group of: Lys-C, Trypsin, Asp-N, Arg-C, Asn-C, and Post-Proline cleaving enzyme.
5. The method of claim 1 , wherein said mammal is a human.
6. The method of claim 1 , further comprising preparing a stable detectable standard peptide.
7. The method of claim 6 , wherein said stable detectable standard peptide is prepared in a separate container from said at least one therapeutic protein.
8. The method of claim 6 , wherein said stable detectable standard peptide is contacted with said plasma.
9. The method of claim 8 , wherein said stable detectable standard peptide is further digested with a second protease.
10. The method of claim 9 , wherein said second protease is selected from the group of: Lys-C, Trypsin, Asp-N, Arg-C, Asn-C, and Post-Proline cleaving enzyme.
11. The method of claim 10 , wherein said first and second protease are the same.
12. The method of claim 6 , wherein said stable detectable standard peptide is isotopically labeled.
13. The method of claim 1 , further comprising generating a peptide library of said therapeutic protein.
14. The method of claim 13 , further comprising selecting a fragment of said at least one protein that will be produced upon digesting with said protease.
15. The method of claim 14 , wherein said fragment can be separated from all other fragments of said therapeutic protein produced by said protease based on one or more of the following characteristics: mass, charge, size, secondary structure, and tertiary structure.
16. The method of claim 1 , wherein said method is a high throughput method.
17. The method of claim 1 , wherein said method is quantitative.
18. The method of claim 1 , further comprising calculating at least one pharmacokinetic parameter of said at least one therapeutic protein in plasma.
19. The method of claim 1 , further comprising calculating at least one pharmacodynamic parameter of said at least one therapeutic protein in plasma.
20. The method of claim 1 , wherein sample reproducibility is confirmed by ELISA.