4321 Recombinant versions of hirudin such as lepirudin (Refludan®) and desirudin (Iprivask®) are currently used as parenteral anticoagulants for various indications. The recombinant hirudin preparations differ from the natural hirudin in lacking a sulfate group on tyrosine at the 63rd position and a single amino acid substitution. It is hypothesized that these minor differences in the natural and recombinant versions of hirudins contribute to a differential immunogenic behavior. The purpose of this investigation was to compare the relative immunoreactivity of the three forms of hirudin to a sheep antin–hirudin antibody. Antibodies (anti-n-hirudin) were generated in sheep treated with natural hirudin isolated from the medicinal leech (Hirudo medicinalis ). SDS-PAGE immunoblot and Western transfer were conducted using the native hirudin and two recombinant hirudins against the sheep anti-n-hirudin IgG antibodies. Cross-reactivity was quantified by measuring total optical density of the bands using a UVP densitometric scanning system and chemiluminescence detection, by comparing the protein-antibody complex band density from the western blot showed a comparable behavior of the Desirudin and Lepirudin in contrast to the normal or native hirudin which exhibited a much higher band density (2-fold increase). Moreover, the native hirudin exhibited specific bands representing mono, di- and tetra-meric forms, whereas the two recombinant forms exhibited primarily monomeric and dimeric forms. Interestingly several other preclinical versions of recombinant hirudins exhibited fifferent immunoblotting patterns. Although, the structural differences and the molecular weight represent relatively minor variations in the natural and recombinant hirudins, these studies strongly suggest a differential behavior of natural and recombinant hirudins in terms of their cross-reactivity with anti-n-hirudin antibodies. Disclosures: No relevant conflicts of interest to declare.

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.