IDENTIFICATION OF RECOMBINANT HIRUDIN BY-PRODUCTS BY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

Bioactive by-products with antithrombin activity were obtained during the production of recombinant hirudin, rHV1-Tyr63, by a biotechnologic al process based on a genetically-modified Saccharomyces strain, The b y-products were separated using preparative high performance liquid ch romatography, The molecular masses of five major and three minor analy tes were determined by electrospray mass spectrometry to be in the 506 0 to 6835 Da range, all within +/-0.9 Da of the calculated masses of t he proposed structures, Interpreted in terms of the known amino acid s equence of recombinant hirudin (6964 Da), the major by-products corres ponded to residues after scissions at the 46, 49, 61, 63 and 64 amino acid positions, while the minor by-products resulted after cleavages a t the 51, 53 and 62 positions.