A Pharmaceutical Human Apotransferrin Product for Iron Binding Therapy

........................................................................................................................ 3 PREFACE ............................................................................................................................ 5 LIST OF ORIGINAL PUBLICATIONS....................................................................................... 9 ABBREVIATIONS .............................................................................................................. 10 INTRODUCTION ................................................................................................................ 11 1 Transferrin and serum iron .................................................................................... 11 1.1 Transferrin characteristics........................................................................... 11 1.2 Iron and role of transferrin.......................................................................... 13 1.3 Serum non-transferrin-bound iron (NTBI) ................................................. 15 1.4 Measurement of NTBI in serum ................................................................. 17 2 Iron and infection .................................................................................................. 20 2.1 NTBI, microbial growth, and risk of infection ........................................... 20 2.2 Iron acquisition of Staphylococcus epidermidis ......................................... 22 3 Methods to produce apotransferrin........................................................................ 22 AIMS OF THE STUDY......................................................................................................... 24 MATERIALS AND METHODS.............................................................................................. 25 1 Methods for production, identification and characterisation of apotransferrin (I) 25 1.1 Apotransferrin production method.............................................................. 25 1.2 Identification of transferrin ......................................................................... 25 1.3 Apotransferrin characteristics and purity.................................................... 25 2 Serum iron parameters (II-V) ................................................................................ 27 2.1 Patient serum samples................................................................................. 27 2.2 Serum iron and transferrin saturation ......................................................... 27 2.3 Determination of serum NTBI.................................................................... 27 2.4 Determination of transferrin iron forms in serum....................................... 28 3 Growth of Staphylococcus epidermidis (IV, V) .................................................... 29 3.1 Bacterial strains and preparation of inoculum ............................................ 29 3.2 Monitoring of growth with pure transferrin................................................ 29 3.3 Monitoring of growth in serum samples..................................................... 29 RESULTS .......................................................................................................................... 31 1 Pharmaceutical apotransferrin preparation (I)....................................................... 31 1.1 Large scale purification of apotransferrin................................................... 31 1.2 Identification and characterisation of the apotransferrin product ............... 32 2 Binding of NTBI with apotransferrin .................................................................... 34 2.1 Evaluation of assay for measuring NTBI (II) ............................................. 34 2.2 Binding of NTBI in patient serum (III, V).................................................. 35 3 Growth of Staphylococcus epidermidis (IV, V) .................................................... 36