Protein Chip Array Profiling of Molecular Variants of Hirudins Using Surface Enhanced Laser Desorption Ionization (SELDI) Technique

Abstract

Various molecular variants of natural hirudin are developed for pharmaceutical purposes. Most of these are molecular variants, their conjugates with PEG Hirudin and fragment conjugates. Desirudin and Refludin represent recombinant hirudin variants with minor structural differences. PEG Hirudin represents a polyethylene glycol conjugate. Angiomax represents a terminal decapeptide of hirudin which is complex with a tripeptide and linker polypeptide. Although these products are homogeneous, their immunogenic responses reportedly vary. The purpose of this investigation was to investigate the molecular profile of each of these agents utilizing the SELDI technique employing a gold chip. Desirudin showed a single peak at 6.97kda, where is Refludin showed a single peak at 6.98kda. PEG Hirudin showed a heterogeneous peak at 17.8kda Angiomax showed a major peak at 2.18kda and a minor peak at 2.39kda. All of these products were free of other minor components below 1kda. These results show that ProteinChip array can be used to determine the molecular weight profile of each of these proteins and to check the presence of other impurities such as peptide fragment and other contaminants.