Complete Molecular Weight Profiling of Low-Molecular Weight Heparins Using Size Exclusion Chromatography-Ion Suppressor-High-Resolution Mass Spectrometry

Low-molecular
weight heparins (LMWH) prepared by partial depolymerization
of unfractionated heparin are used globally to treat coagulation disorders
on an outpatient basis. Patent protection for several LMWH has expired
and abbreviated new drug applications have been approved by the Food
and Drug Administration. As a result, reverse engineering of LMWH
for biosimilar LMWH has become an active global endeavor. Traditionally,
the molecular weight distributions of LMWH preparations have been
determined using size exclusion chromatography (SEC) with optical
detection. Recent advances in liquid chromatography–mass spectrometry
methods have enabled exact mass measurements of heparin saccharides
roughly up to degree-of-polymerization 20, leaving the high molecular
weight half of the LMWH preparation unassigned. We demonstrate a new
LC–MS system capable of determining the exact masses of complete
LMWH preparations, up to dp30. This system employed an ion suppressor
cell to desalt the chromatographic effluent online prior to the electrospray
mass spectrometry source. We expect this new capability will impact
the ability to define LMWH mixtures favorably.