Characterization of the deamidated forms of recombinant hirudin

Recombinant hirudin variant rHV2-Lys 47 (MW = 6906.5) was intentionally deamidated by incubation in pH 9 phosphate buffer at 37 degrees C. Anion-exchange HPLC analysis showed that 11 forms could be generated. These were isolated and purified by combined anion-exchange and reversed-phase HPLC. Acid-catalyzed carboxyl methylation was used to introduce a mass shift of +15 amu per deamidated residue present in the molecule before analysis by liquid secondary ion mass spectrometry (LSIMS). Methylation enhanced, in particular, the abundance of the sequence ions in the LSIMS spectra. This permitted the determination of both the number (three) and the localization of the deamidated residues: Asn 52, Asn 53, and a residue located in the N-terminal 1-39 domain. Complementary sequencing techniques proved that the latter residue was Asn 33. Altogether four mono-, three di-, and four tri-deamidated forms were identified. The heterogeneity of the forms having identical deamidation positions but being chromatographically separable is thought to arise from the generation of alpha- and beta-aspartyl iso forms during the nonenzymatic deamidation process.