Enoxaparin: Physicochemical Investigations into the Effects of Freezing and Heating Rahul P Patel, B .Phann. (MPharmSc) Submitted in fulfilment of the requirement for the degree of Doctor of Philosophy SCHOOL OF PHARMACY UNIVERSITY OF TASMANIA DECEMBER 2007 DECLARATION This thesis contains no material that has been accepted for the award of any other degree or graduate diploma in any tertiary institution, except by way of background information and duly acknowledged in the text of the thesis. To the best of my knowledge and belief, this contains no material previously published or written by another person, except when the due reference is made in the text of the thesis. (Rahul P Patel) i AUTHORITY OF ACCESS This thesis many be available for loan and limited copy in accordance with the Copyright Act 1968. (Rahul P Patel) ii CONTENTS ACKNOWLEDGEMENTS ................................................................................................... viii LIST OF ABBREVIATIONS .................................................................................................. x SUMMARY .......................................................................................................................xii CHAPTERl General Introduction ...................................................................... 1 1.1 :HEPARIN .................................................................................... l 1.2 :HEPARIN: STRUCTURE AND BIOLOGICAL ACTIVITIES ................................................................................. 2 1.3 PREPARATION OF COMMERCIAL HEPARIN ............................................ 3 1.4 PHARMACOKINETICS OF HEPARIN .................................................... .4 1.5 LOW MOLECULAR WEIGHT :HEPARINS (LMWHs) .............................. 5 1.6 DIFFERENT METHODS FOR T:HE PREPARATION OF LMWHs ............... 6 1.6.1 Oxidative depolymerisation .. ................................................................................... 6 1.6.2 Deaminative Cleavage ...................................................................... 6 1.6.3 fJ-eliminative Cleavage ..................................................................... 8 1.7 STRUCTURAL DIFFERENCES BETWEEN AND WITHIN LMWHs ............ 9 1.8 MECHANISM OF ACTION OF :HEPARIN AND LMWHs ......................... 10 1.9 DETERMINATION OF T:HE ANTICOAGULANT ACTIVITY OF LMWHs ... 12 1.9.1 Clotting AFXa assay ...................................................................... .. 12 1.9.2 Chromogenic AFXa assay .................................................................. 13 1.10 COMPARISON OF LMWHs AND UNFRACTIONATED :HEPARIN ............. 14 1.11 ANALYSIS OF:HEPARIN ANDLMWHs .............................................. 14 CHAPTER2 Development of a Modified Low-volume Microtitre Plate Assay Method for the Determination of AFXa activity ofEnoxaparin .................. 18 2.1 SUMMARY ................................................................................. 18 2.2 INTRODUCTION ......................................................................... 18 2.3 EXPERIMENTAL .......................................................................... 20 2.3.1 Materials ..................................................................................... 20 2.3.2 Analysis ofAFXa Activity ................................................................... 21 2.3.3 Linearity ...................................................................................... 22 2.3.4 Reproducibility ............................................................................... 22 2.3.5 Precision ...................................................................................... 22 2.3.6 Accuracy ...................................................................................... 23 iii 2.4 RESULTS AND DISCUSSION ........................................................... 23 2.5 CONCLUSION .............................................................................. 25 CHAPTER3 A simple Capillary Electrophoresis Method for the Rapid Separation and Determination oflntact Low-Molecular-Weight and Unfractionated Heparins ................................................................. 27 3.1 SUMMARY .................................................................................. 27 3.2 INTRODUCTION ........................................................................... 27 3.3 EXPERIMENTAL .......................................................................... 29 3.3.1 Materials ..................................................................................... 29 3.3.2 Instrumentation .............................................................................. 30 3.3.3 CE analysis ................................................................................... 30 3.3.4 Electrolyte preparation ..................................................................... 31 3.3.5 Method development .................................................................... ..... 31 3.3.6 Assay performance .......................................................................... 31 3.3.7 Degradation of dalteparin .................................................................. 32 3.3.8 Preparation ofunfractionated heparin and various LMWH samples .................... 32 3.4 RESULTS AND DISCUSSION ........................................................... 32 3.4.1 Influence of separation parameters on dalteparin analysis ........................... 32 3.4.2 Effect of electrolyte pH on the separation efficiency .................................... 33 3.4.3 Influence of electrolyte concentration .................................................... 35 3.4.4 Effect of sodium chloride on the separation ofdalteparin ............................. 36 3.4.5 Assay performance .......................................................................... 37 3.4.6 Analysis of stressed LMWHs ............................................................... 39 3.4.7 CE analysis ofdifferent LMWHs and unfractionated heparin ........................ 41 3.5 CONCLUSION .............................................................................. 44 CHAPTER4 An Effective Reversed-Phase Ion-Interaction High-Performance Liquid Chromatography Method for the Separation and Characterisation of Intact Low-Molecular-Weight Heparins ............................................... 46 4.1 SUMMARY ................................................................................. 46 4.2 INTRODUCTION ........................................................................... 46 4.3 EXPERIMENTAL .......................................................................... 50 4.3.1 Materials ...................................................................................... 50 4.3.2 HPLC instrumentation ......................................................................50 4.3.3 Ion-interaction RP-HPLC separation .................................................... 51 4.3.4 Method development ........................................................................ 51 4.3.5 HP-SEC separation ......................................................................... 51 IV 4.3.6 Assay performance .......................................................................... 52 4.3.7 Analysis of stressed enoxaparin ........................................................... 52 4.3.8 Preparation of various LMWHs samples ................................................ 52 4.4 RESULTS AND DISCUSSION ........................................................... 53 4.4.1 Effect of acetonitrile concentration ....................................................... 53 4.4.2 Effect of the ion-interaction reagent ................................................... .... .55 4.4.3 Influence ofTBAH concentration on the separation parameters .. ................... 57 4.4.4 Influence ofpH and ammonium acetate concentration ...... ......................... 59 4.4.5 Ion-interaction RP-HPLC analysis ofHP-SEC enoxaparin fractions ............... 59 4.4.6 Assay performance .......................................................................... 64 4.4.7 Ion-interaction RP-HPLC analysis using ELSD ...... ................................. 64 4.4.8 Analysis of stressed enoxaparin solution ................................................. 66 4.4.9 Ion-interaction RP-HPLC analysis of different LMWHs .............................. 68 4.5 CONCLUSION .............................................................................. 70 CHAPTERS Investigation of Freezing- and Thawing-Induced Biological, Chemical and Physical Changes to Enoxaparin Solution ....................................... 72 5.1 SUMMARY ................................................................................... 72 5.2 INTRODUCTION ........................................................................... 72 5.3 EXPERIMENTAL.......................................................................... 75 5.3.1 Materials ...................................................................................... 75 5.3.2 Analysis ofAFXa activity .................................................................. 76 5.3.3 Freezing of enoxaparin solution at different temperatures ..................... ...... 76 5.3.4 Repeated freeze/thaw cycles ............................................................... 77 5.3.5 Different freezing and thawing conditions ............................................... 77 5.3.6 Dilution of enoxaparin solution prior to freezing