Anal. Chem. 1997, 69, 5142-5145
Monitoring Recombinant Protein Drugs: A Study of Insulin by H/D Exchange and Electrospray Ionization Mass Spectrometry Ragulan Ramanathan and Michael L. Gross*
Department of Chemistry, Washington University, One Brookings Drive, St. Louis, Missouri 63130 Walter L. Zielinski* and Thomas P. Layloff
Division of Testing and Applied Analytical Development, CDER, FDA, St. Louis, Missouri 63101
The increasing emergence of new protein- and peptidebased 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 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 insulindependent 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 µg (15 min). The exchange is quenched (for insulin, 2 min) and its extent analyzed by ESI-MS. In the case of insulins, significant differences are seen for the isomeric LysPro and r-human insulins and for porcine and bovine insulins. This assay method was tested with 10 insulin samples of different types and batches that were submitted as unknowns. Assignments based on deuterium incorporation after 60 min (Table 1) were correct in all cases, suggesting that the method is reproducible and reliable for distinguishing r-human insulin from LysPro insulin and for evaluating consistency of different batches of insulin types. The assay can be conducted at one time point for a sample size of less than 2 µg of insulin. ACKNOWLEDGMENT This research was supported in part by the NIH Grants (1PO1CA49210 and 2P41RR00954). We thank Micromass for the loan of the Maximum Entropy software and Joseph Hanig, Susan Jenney, and Charles Dieter of the FDA/CDER/LRL insulin certification program for providing the insulin samples and for helpful discussions. Received for review March 17, 1997. Accepted September 30, 1997.X AC970290A X
Abstract published in Advance ACS Abstracts, November 15, 1997.
Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
5145
