Application of HPLC-ESI-ITMS in the quality control of carboxyterminal sequence confirmation for the recombinant DNA product Hirudin Variant 3.

摘要: In the present study, HPLC-ESI-IT (ion-trap) MS was used for carboxyterminal (C-terminal) amino acid sequence confirmation of intact recombinant Hirudin Variant 3 (HV3) and alkylated HV3. The C-terminal HV3 determined by use carboxypeptidase P (CPP), combined Y (CPY). with 1,4-dithiothreitol (DTT) reduction also confirmed CPP CPY (abbreviated to CPP-CPY). Up 19 residues were in nanomolar concentration range analyzing molecular weights truncated peptides Another five acids DTT reduction. For sequencing reduction, reduced followed alkylation iodoacetamide. reaction mixture, which included HV3, DTT, iodoacetamide, then directly sequenced without any further pre-treatment. designed a time-, concentration-dependent manner obtain maximum information. results showed that HPLC-ESI-ITMS cannot only determine but gives important information about enzymatic degradation subsequent release