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Discriminating Heparin from Chondroitin Sulfate by Charge:Mass Ratio

Characterizing key parameters of different heparin lots requires multiple techniques. Weight-average molar mass measured by multi-angle static light scattering (MALS) and hydrodynamic radius measured by dynamic light scattering (DLS) can be used to characterize the size and polydispersity of the molecule. These quantities can also be used for lot-to-lot comparison and to qualify heparin from different suppliers. Massively-parallel phase analysis light scattering (MP-PALS) and DLS are used to calculate the net charge and characterize the purity of the sample. In this study, we compared heparin from two suppliers, including one lot modified with super-sulfated material.

Weight-average molar mass (Mw) for the different hep-arin samples and for heparin mass standards was deter-mined by size exclusion chromatography (SEC) coupled with MALS. The difference in Mw and hydrodynamic radius (rh) between the two suppliers was <10%. Very little difference in Mw and rh (<5%) was observed be-tween unmodified heparin and heparin modified with super-sulfated material. Thus, size alone could not be used to qualify contaminated heparin samples.

Using the Wyatt Möbiu, z-averaged electrophoretic mobility and rh were measured simultaneously to yield net charge. The net charge for a set of heparin standards obeyed a linear relationship between as a function of Mw, indicating a constant charge:mass ratio. Unmodified heparin from both suppliers obeyed the same linear relationship, but the super-sulfated material exhibited a 30% increase in negative charge. The increase in negative charge per unit mass, is consistent with an in-crease in sulfate groups in the modified heparin sample.

Thus, the combination of Mw by SEC-MALS and net charge by MP-PALS provides clear differentiation be-tween unmodified and contaminated heparin. This multi-technique approach for determining the charge:mass ratio enables rapid, nondestructive characterization of different lots of heparin.