whole_PatelRahulPrabhudas2008_thesis.pdf (8.16 MB)
Enoxaparin : physicochemical investigations into the effects of freezing and heating
thesisposted on 2023-05-27, 16:13 authored by Patel, Rahul Prabhudas
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 ¬¨‚àûC, -12 ¬¨‚àûC or -80 ¬¨‚àûC 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 ¬¨‚àûC. Enoxaparin sodium diluted with 4% glucose retained greater than 99% of its initial AFXa activity at 4 ¬¨‚àûC after 31 days. Enoxaparin sodium diluted with water lost almost 10% of its original activity after 31 days at 4 ¬¨‚àûC and lost more than 10% of its activity after freezing at -12 ¬¨‚àûC or -80 ¬¨‚àûC. Storage in glass or plastic containers made no difference to the loss in the activity. Heating study Enoxaparin samples were kept at 70 ¬¨‚àûC for up to 576 hours. Enoxaparin activity decreased to 74% of its initial AFXa activity after 8 hours at 70 ¬¨‚àûC 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 ¬¨‚àûC 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 ¬¨‚àûC 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 ¬¨‚àûC 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.
Rights statementCopyright 2008 the author Thesis (PhD)--University of Tasmania, 2008. Includes bibliographical references