MOLECULAR-WEIGHT PROFILING OF LOW-MOLECULAR-WEIGHT HEPARINS UTILIZING A HEPARINASE DEGRADED OLIGOSACCHARIDE MIXTURE AS A CALIBRATOR

Heparinase degraded heparin fragments (HDHF) are enriched with a UV chromophore and can be utilized as calibrators to determine the molecular weight profile of low molecular weight heparins (LMWH's). In a standard protocol (1), the second to the last peak is assumed to be a hexasaccharide with a molecular weight of 1.8 kDa. All other peak molecular weights in the elution profile are assigned based upon this assumption. In this study, synthetic analogues of heparin with a defined molecular weight have been used to investigate the validity of this assumption. These compounds included a sulfated bis-lactobionic acid amide (2.4 kDa), a pentasaccharide (1.7 kDa), and lactose polysulfate (1.2 kDa). With reference to these internal standards, the second to the last peak of the HDHF mixture was found to elute after the pentasaccharide and close to the lactose polysulfate suggesting that it is a tetrasaccharide. For additional validation, calibration curves were constructed using individual calibrators as the reference method as well as the HDHF method. Several low molecular weight heparins were profiled for their molecular weight using these methods. In comparison to the reference method, the molecular weights obtained with the HDHF method were 25-40% higher. When the results were recalculated assuming the molecular weight of the second to the last peak as 1.2 kDa, the HDHF results compared well with the reference method. Additionally, it is seen that the HDHF method does not identify molecular components greater than 8.0 kDa.