Sequence-specific deamidation: isolation and biochemical characterization of succinimide intermediates of recombinant hirudin.

Natural hirudin variant 2 with a lysine residue in position 47 (rHV2-Lys47) was produced in a genetically engineered strain of Saccharomyces cerevisiae as a secreted protein of 65 amino acids and purified to greater than 99% homogeneity. Only reversed-phase high-performance liquid chromatography (RP-HPLC) using very shallow acetonitrile gradients indicated the presence of a component in the final product (approximately 1% of total protein) with a slightly increased retention time. Using successive RP-HPLC purification steps, this hydrophobic impurity was isolated and separated into two constituents defined as components A1 and A2 which differed from the parent molecule by mass reductions of 17.