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Abstract

Introduction
Low-molecular-weight heparins (LMWHs) are modified heparin fractions with a
molecular weight range of 2 OOO to 8 OOO Da prepared by chemical or enzymatic
depolymerisation of unfractionated heparin (UFH). Although UFH was the standard
anticoagulant, LMWHs constitute an effective alternative antithrombotic therapy to UFH, and
they have more favourable pharmacokinetic profiles and several clinical advantages. LMWHs
present a special set of difficulties for chemical and structural analysis because they are
highly negatively charged, structurally complex, and polydisperse in nature. Various LMWHs
are prepared by different processes and show dissimilarity in physical, chemical and
biological properties. Lack of versatile and efficient analytical techniques makes
characterisation and stability analysis of various LMWHs difficult. An earlier study of
enoxaparin stabilities showed that the ant1factor (AFXa) activity decreased upon freezing and
showed an unusual pattern of change upon heating.

Objectives
The main aim of the study was to investigate the mechanisms behind the observed
activity changes of enoxaparin upon storage at elevated and reduced temperatures, with the
potential goal of improving the stability of LMWHs. Secondary objectives were to develop new
analytical techniques in order to accomplish the above mentioned aim.
Analytical methods development
A low-volume microtitre plate assay was developed for the determination of AFXa
activity of enoxaparin. This method was validated against a standard method and equivalent
results were obtained. A simple, selective and accurate capillary electrophoresis (CE) method
was developed with a superior resolution than previously reported CE methods for the
separation and identification of various LMWHs and UFH. The developed CE method was
successfully applied to demonstrate batch-to-batch variations in enoxaparin. An efficient
ion-interaction reversed-phase high performance liquid chromatography (ion-interaction RP-HPLC) method with diode array detection was developed. Resolution of
various LMWHs was superior to any of the previously reported analytical techniques. A novel
application of ion-interaction RP-HPLC coupled to an evaporative light scattering detection
(ELSD) system was also developed.

Freezing study
Enoxaparin solutions were frozen and thawed under different conditions and the
AFXa activity was determined. Freezing adversely affected the AFXa activity of enoxaparin
solution. Physical investigations of enoxaparin solution suggested that formation of ice
crystals or glassy state transitions were not responsible for the loss in activity. Chemical
investigations of enoxaparin solution showed that the loss of AFXa activity did not involve the
loss of N-sulfate groups or breakdown of glycosidic bonds. Freezing-induced loss of AFXa
activity could be reduced by the inclusion of dimethyl sulfoxide (DMSO), by dilution with water
and by controlling the freezing and thawing rates. The activity loss could be partially reversed
by sonication and sonication was more effective in the presence of DMSO. The loss in AFXa
activity was found by high performance size exclusion chromatography (HP-SEC) to be
primarily due to aggregation.

Dilution study
Commercially prepared undiluted enoxaparin or enoxaparin diluted with sterile water
or sterile 4% glucose was aseptically transferred into plastic syringes or glass vials. Samples
were kept at 4 °C, -12 °C or -80 °C for up to 31 days. The AFXa activity of stored solutions
was determined after 0, 7, 14 and 31 days. The AFXa activity of the diluted samples was
compared with the AFXa activity of undiluted enoxaparin sodium solution stored for the same
time periods at 4 °C. Enoxaparin sodium diluted with 4% glucose retained greater than 99%
of its initial AFXa activity at 4 °C after 31 days. Enoxaparin sodium diluted with water lost
almost 10% of its original activity after 31 days at 4 °C and lost more than 10% of its activity
after freezing at -12 °C or -80 °C. Storage in glass or plastic containers made no difference to
the loss in the activity.

Heating study
Enoxaparin samples were kept at 70 °C for up to 576 hours. Enoxaparin activity
decreased to 74% of its initial AFXa activity after 8 hours at 70 °C followed by a rapid
increase in the activity after 12 hours to 94% and then a gradual decrease in the AFXa
activity. The chemical changes to enoxaparin which account for the AFXa activity changes
following thermal degradation were studied. Enoxaparin was heated at 70 °C for up to
24 days in the presence and absence of various concentrations of oxygen. Samples were
collected at regular time intervals and AFXa activity, free sulfate groups, free amino groups
and reducing capacity were determined. Samples stressed at 0, 8 and 12 hours were
fractionated by HP-SEC. The fractions were collected and analysed by ion-interaction
RP-HPLC and for AFXa activity and sulfate concentration. Enoxaparin thermal degradation
resulted in the loss of sulfation, particularly N-sulfate groups, and the breakdown of glycosidic
linkages confirmed by reducing capacity assay and CE analysis.
The initial decrease (at 8 hours) and subsequent increase (at 12 hours) of enoxaparin
AFXa activity was found to be unrelated to oxygen content. No differences between the
O hours and the 8 hours samples were observed by HP-SEC. Ion-interaction RP-HPLC
analysis of 0 hours and 8 hours treated fractions (collected by HP-SEC) clearly showed
changes in some of the 8 hours treated fractions. Ion-chromatography (IC) and AFXa activity
analyses of the fractions showed loss of sulfate groups and a corresponding decrease in the
AFXa activity. Only some of the fractions lost sulfate and AFXa activity. Other fractions
appeared to be more resistant to thermally-induced desulfation and retained their AFXa
activity. HP-SEC of the 12 hours treated sample showed the presence of extra peaks which
were confirmed by ion-interaction RP-HPLC. The increased activity after 12 hours at 70 °C
was found to be because of the fragmentation of large oligosaccharides to smaller
ohgosaccharides, as confirmed by AFXa activity analysis and increased in the number of
reducing ends.

Conclusion
CE and ion-interaction RP-HPLC methods were developed and successfully applied
to investigate the mechanisms involved behind the loss in AFXa activity of enoxaparin under
various storage temperatures and conditions. The observed loss in AFXa activity was
consistent with an aggregation hypothesis. Aggregation was reversible by sonication and
sonication was more effective in the presence of DMSO. Controlling the freezing or thawing
conditions, dilution with water or addition of a small percentage of DMSO ameliorated the loss
of enoxaparin AFXa activity. Dilution of enoxaparin with 4% glucose offers a potential method
for the preparation of stable paediatric diluted doses of enoxaparin. Chemical and AFXa
activity analysis following the heating of enoxaparin at 70 °C clearly distinguished thermally
stable fractions from the thermally labile. The generation of new active fragments was found
after heating with higher AFXa activity. The thermally stable fractions of enoxaparin offer the
potential for new LMWH formulations with greater stability and shelf life.

Item Type:	Thesis - PhD
Authors/Creators:	Patel, Rahul Prabhudas
Copyright Holders:	The Author
Copyright Information:	Copyright 2008 the author
Additional Information:	Thesis (PhD)--University of Tasmania, 2008. Includes bibliographical references
Related URLs:	Publisher Publisher Publisher
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