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

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 17.2 Da (A1) and 17.6 Da (A2), respectively, as determined by electrospray mass spectrometry (ESMS). Proteolytic digestion with endoprotease Glu-C from Staphylococcus aureus (V8 protease) and analysis of the peptide mixture by ESMS showed that the mass difference between rHV2-Lys47 and component A1 was due to a modification between amino acids 1 and 43, while the corresponding mass difference with component A2 was the result of a modification within the peptide fragment comprising residues 50-61. Further analyses using amino acid sequencing and ESMS in combination with collision-activated dissociation (CAD) detected modifications at residues Asn33-Gly34 in component A1 and at Asn53-Gly54 in component A2. Both of these sites were previously shown to be susceptible to spontaneous deamidation under slightly basic pH conditions. Thus, the mass reductions of approximately 17 Da and the fact that both asparagines, Asn33 in component A1 and Asn53 in component A2, proved to be resistant to Edman degradation provided strong support for them being stable succinimide intermediates of the corresponding deamidation reactions. Both intermediates were shown to have inhibition constants for human alpha-thrombin on the order of 1 pM, identical to that of rHV2-Lys47. The isoelectric point of component A2 was determined to be within 0.01 pH unit of that of the parent molecule by isoelectric focusing in an immobilized pH gradient.