The doctoral dissertations of the former Helsinki University of Technology (TKK) and Aalto University Schools of Technology (CHEM, ELEC, ENG, SCI) published in electronic format are available in the electronic publications archive of Aalto University - Aaltodoc.
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A Pharmaceutical Human Apotransferrin Product for Iron Binding Therapy

Leni von Bonsdorff

Dissertation for the degree of Doctor of Science in Technology to be presented with due permission of the Department of Chemical Technology for public examination and debate in Auditorium KE 2 (Komppa Auditorium) at Helsinki University of Technology (Espoo, Finland) on the 14th of November, 2003, at 12 o'clock noon.

Overview in PDF format (ISBN 952-5457-06-0)   [1262 KB]
Dissertation is also available in print (ISBN 952-5457-05-2)

Abstract

Transferrin is the major iron binding protein in human plasma. It binds iron with high affinity in a redox inactive form and delivers it to growing cells. Each molecule is capable of binding two molecules of ferric iron. Normally, transferrin is only about 30 % saturated with iron. In certain clinical conditions, the iron concentration in serum is increased so that the iron binding capacity is exceeded and non-transferrin-bound iron (NTBI) is formed in serum. NTBI is potentially toxic because it generates free radical formation and can be taken up by tissues, leading to excess deposits that can potentiate tissue damage. It is also known that iron enhances the growth of bacteria and fungi, and can predispose patients to septic infections.

This thesis describes the development of an efficient process for producing pharmaceutical grade iron-free apotransferrin. The biochemical efficacy of apotransferrin for iron binding therapy was studied in early phase clinical trials in haematological stem cell transplant (SCT) patients. The scope of this work did not include studying the clinical efficacy of apotransferrin.

The manufacturing method used fraction IV of the Cohn cold ethanol human plasma fractionation process as starting material. Apotransferrin was purified in two ion exchange chromatography steps and ultrafiltration with over 90 % recovery. In order to obtain a virus-safe product, the process comprised solvent detergent treatment as the main virus inactivation step and virus filtration and polyethylene glycol precipitation to remove physico-chemically resistant infectious agents. The purity of the product was at least 98 %, main impurities being IgG, IgA and hemopexin. Methods for studying the iron binding capacity, the transferrin conformation and its iron forms, and the glycosylation variants were developed and used to study the quality of the finished product batches. The product had intact iron binding capacity and a native conformation. The results of several production batches indicated that the manufacturing could be carried out reproducibly. Product characterisation by electrospray and MALDI-TOF mass spectrometry indicated no other chemical modifications than N-linked glycan chains and disulphide bonds, except minor oxidation. A stable liquid formulation suitable for intravenous infusion was developed.

The biochemical binding of NTBI to apotransferrin in vivo was studied by several methods. The bleomycin method for NTBI determination was modified for microwell measurement and evaluated. The bleomycin assay was reproducible and NTBI was found in serum samples only when transferrin saturation was > 80 % and haemolysed samples were excluded. The bleomycin assay that measures redox-active iron underestimated the true concentration of NTBI. The concentration of NTBI could be calculated from the shift of transferrin iron forms found in vivo after intravenous infusion of apotransferrin to patients. It could also be determined with a chelation based method, which, however, had a lower specificity than the bleomycin method. In haematological SCT patients, the concentration of NTBI could be as high as 20 µmol / l. Apotransferrin given in single intravenous doses to six patients bound NTBI effectively, although in most cases temporarily. With repeated high dose regimens, the appearance of NTBI was prevented in 5 of 8 patients.

The influence of NTBI on the growth of the opportunistic pathogen Staphylococcus epidermidis was studied both with purified transferrin and in serum milieu. In both cases, growth was critically dependent on NTBI and on a high transferrin saturation. Only at high initial bacterial concentrations could growth be detected with partially saturated transferrin. Apotransferrin administered to SCT patients bound NTBI and restored the growth inhibitory effect of serum. Exogenous apotransferrin might protect the patients against infections by S. epidermidis and other opportunistic pathogens whose growth is dependent on NTBI.

In conclusion, the apotransferrin was pure and safe and showed in vivo the biochemical effects that could be expected of a functional human apotransferrin product. In SCT patients it was possible to prevent the appearance of NTBI and maintain the bacterial growth inhibitory effect in serum.

This thesis consists of an overview and of the following 5 publications:

  1. von Bonsdorff L., Tölö H., Lindeberg E., Nyman T., Harju A. and Parkkinen J., 2001. Development of a pharmaceutical apotransferrin product for iron binding therapy. Biologicals 29, No. 1, pages 27-37. © 2001 Elsevier Science. By permission.
  2. von Bonsdorff L., Lindeberg E., Sahlstedt L., Lehto J. and Parkkinen J., 2002. Bleomycin-detectable iron assay for non-transferrin-bound iron in hematologic malignancies. Clinical Chemistry 48, No. 2, pages 307-314. © 2002 American Association for Clinical Chemistry (AACC). By permission.
  3. Sahlstedt L., von Bonsdorff L., Ebeling F., Ruutu T. and Parkkinen J., 2002. Effective binding of free iron by a single intravenous dose of human apotransferrin in haematological stem cell transplant patients. British Journal of Haematology 119, No. 2, pages 547-553. © 2002 Blackwell Publishing. By permission.
  4. Matinaho S., von Bonsdorff L., Rouhiainen A., Lönnroth M. and Parkkinen J., 2001. Dependence of Staphylococcus epidermidis on non-transferrin-bound iron for growth. FEMS Microbiology Letters 196, No. 2, pages 177-182. © 2001 Elsevier Science. By permission.
  5. von Bonsdorff L., Sahlstedt L., Ebeling F., Ruutu T. and Parkkinen J., 2003. Apotransferrin administration prevents growth of Staphylococcus epidermidis in serum of stem cell transplant patients by binding of free iron. FEMS Immunology and Medical Microbiology 37, No. 1, pages 45-51. © 2003 Elsevier Science. By permission.

Keywords: apotransferrin, non-transferrin-bound iron, Staphylococcus epidermidis, plasma protein

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© 2003 Helsinki University of Technology

Last update 2011-05-26