MONITORING RECOMBINANT PROTEIN DRUGS - A STUDY OF INSULIN BY H D EXCHANGE AND ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY/

The increasing emergence of new protein- and peptide-based drugs makes necessary the development of rapid and sensitive methods to check con sistency between and within batches of biotechnology pharmaceuticals t o ensure product quality. We evaluated electrospray ionization mass sp ectrometry in combination with H/D isotopic exchange as a potential to ol, taking as examples for this case study the four insulins used for treating insulin-dependent diabetes. Two (bovine and porcine) are prod uced naturally, and two are produced by recombinant biotechnology tech niques [recombinant human (r-human) and its human insulin analog (LysP ro)]. The extent of H/D exchange at a given time was measured with les s than 2 mu g (<350 pmol) of sample and was sufficient for discriminat ing among the different insulins. After 60 min, bovine, porcine, r-hum an, and LysPro insulins exchanged on average 25, 28, 30, and 38 amide protons, respectively. After prolonged incubation with D2O for 24 h, b ovine and porcine insulins exchanged 31 protons, whereas r-human and L ysPro insulins exchanged 34 and 43 amide protons, respectively. The di fferences in H/D exchange are protein signatures that relate to differ ences in conformation and folding. The extent of exchange distinguishe s among the insulin types and assures the consistency of batch prepara tions for a given insulin.