Student thesis: Doctoral ThesisDoctor of Philosophy


Recombinant human erythropoietin as well as the other synthetic analogs
darbepoetin alpha and methoxy-polyethyleneglycol-epoetin-β, are protein-based drugs which have the ability to stimulate erythrocyte production that leads to their abuse as blood doping agents in both human and equine sports. These epoetins may produce anti-rhEPO antibodies that can cause severe inhibition of
erythropoiesis, leading to anemia and even death. Thus, it is essential to develop an efficient screening method to detect these doping agents in equine plasma at low concentration.
A fast and sensitive method was explored in this project to differentiate and identify recombinant human erythropoietin and darbepoetin in equine plasma by LC- MSMS for doping control. Various approaches were investigated to improve the sensitivity, differentiating the unique peptide fragments of the synthetic analogs, and by creating more on-line techniques to reduce the sample handling time and avoid contamination.
Capillary flow and nano flow chromatography were study and compared with
conventional high flow LC. Results of this study showed that capillary flow is capable of detecting immunoaffinity-extracted plasma at 0.2 ng. Nano flow has also been shown to give better sensitivity when compared with capillary flow. I had also explored the fabrication of an internal tapered capillary tip packed with commercial C18 silica to use as a nano-electrospray tip. This application was shown to improve the sensitivity for detecting the peptides.
To differentiate the synthetic analogs, requires the identifying of the unique peptide fragments, which involves enzyme digestion and deamination. Immobilised enzyme reactors were fabricated to reduce the handling time, avoiding autolysis and sample contamination. The on-line enzyme reactors for both the trypsin digestion and deglycosylation were shown to be as effective as the off-line in-solution digestion. It also showed better sensitivity for some peptides.
A novel organo cross-linker was synthesised and polymerised with a polar
zwitterionic monomer to form a HILIC monolith. The morphology of fabricating the HILIC monolith was studied. Different polymerisation times were studied, with a shorter polymerisation time resulting in a monolith that had a significant proportion of both mesopores and macropores and this allowed rapid and high resolution separation of large biomolecules.
Finally, with the configuring of the orthogonal set-up, solved the incompatibility of the loading buffer, which is highly aqueous, and the high organic buffer used for HILIC phase. This configuration enables an on-line concentration and desalting, which minimised the preparation steps, therefore achieving good sensitivity.
Date of Award2014
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorNorman Smith (Supervisor)

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