DOI
https://doi.org/10.25772/YG9M-VS98
Defense Date
2011
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Pharmaceutical Sciences
First Advisor
Umesh Desai
Abstract
Heparin is a complex mixture of sulfated polysaccharides derived from animals and one of the oldest drugs in use. While an efficacious anticoagulant, heparin is beset by side effects and pharmacokinetic difficulties. Low molecular weight heparins (LMWH) are made by depolymerizing unfractionated heparin (UFH) and present improvements in these areas. However, they still retain a phenomenally high level of complexity due to their polydispersity and the introduction of non-native structural features. This makes the structural characterization LMWHs a daunting task. This work details the development of a novel capillary electrophoretic (CE) method for fingerprinting LMWHs. Since their complexity normally results in a nearly featureless electropherogram, polyalkylamines were used as a resolving agents to yield highly resolved and reproducible fingerprints characteristic of the LMWH being investigated. Linear polyamines of resolved LMWH in a manner dependent on chain length and charge density, while cyclic polyamines were incapable of resolution. Longer length glycosaminoglycans such as UFH and chondroitin sulfate were not successfully fingerprinted as they lacked run to run consistency. Further investigation into the mode of polyamine binding showed that they bound to LMWH via a two site binding model, indicating the presence of specific sites on LMWH that tightly bind polyamines. Upon the saturation of these sites, the polyamines continue to interact via general electrostatic binding. Pentaethylenehexamine was also able to separate the known contaminant oversulfated chondroitin sulfate from UFH. In July of 2010, the US food and drug administration approved a generic for the widely used LMWH enoxaparin, a questionable move due to the difficulties of proving the equivalence of such a complex mixture. A comparison of the brand and generic batches of enoxaparin using the fingerprinting method revealed striking similarities, bolstering the generic’s claim of equivalency and providing a protocol for the evaluation of other biosimilar LMWHs. This is the first work utilizing CE in developing high resolution fingerprints of LMWH. It presents a noteworthy method for quality assessment of LMWH and provides the basis for designing other small molecule probes for the analysis of complex glycosaminoglycans.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
August 2011