The increasing emergence of new protein and peptide-based drugs makes necessary the development of rapid and sensitive methods to check consistency between and within batches of biotechnology pharmaceuticals to ensure product quality. We evaluated electrospray ionization (ESI) mass spectrometry in combination with H/D isotopic exchange as a potential tool, taking as examples for this case study the four insulins used for treating insulin-dependent diabetes. Two (bovine and porcine) are produced naturally, and two are produced by recombinant biotechnology techniques (recombinant human [r-Human] and its human insulin analog [LysPro]). The extent of H/D exchange at a given time was measured with less than 2 ug (< 350 pmol) of sample and was sufficient for discriminating among the different insulins. After 60 min, bovine, porcine, r-Human and LysPro insulins exchanged on average 25, 28, 30, and 38 amide protons, respectively. After prolonged incubation with D2O for 24 hours, bovine and porcine insulins exchanged 31 protons, whereas r-Human and LysPro insulins exchanged 34 and 43 amide protons, respectively. The differences in H/D exchange are protein signatures that relate to differences in conformation and folding. Extent of exchange distinguishes among the insulin types and assures the consistency of batch preparations for a given insulin.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR000954-22
Application #
6118508
Study Section
Project Start
1998-08-01
Project End
1999-07-31
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
22
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Washington University
Department
Type
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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