Abstract
Bioactive by-products with antithrombin activity were obtained during the production of recombinant hirudin, rHV1-Tyr63, by a biotechnological process based on a genetically-modified Saccharomyces strain. The by-products were separated using preparative high performance liquid chromatography. The molecular masses of five major and three minor analytes were determined by electrospray mass spectrometry to be in the 5060 to 6835 Da range, all within ±0.9 Da of the calculated masses of the proposed structures. Interpreted in terms of the known amino acid sequence of recombinant hirudin (6964 Da), the major by-products corresponded to residues after scissions at the 46, 49, 61, 63 and 64 amino acid positions, while the minor by-products resulted after cleavages at the 51, 53 and 62 positions.
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