Protein-based therapeutics play an increasingly important role in medicine, and the exquisite bio-recognition properties exhibited by antibodies has also led the their use in a number of other fields apart from medicine. The increasing use of these molecules requires more efficient methods of purification. A review of the current purification strategies was conducted. Of all the purification methods studied, peptide epitope/mimotope affinity chromatography proved to be the method of choice - resulting in antibodies exhibiting high specific immunoreactivity. The need to tailor unique affinity ligands for each antibody to be purified by peptide epitope/ mimotope affinity chromatography was identified as the major problem with this technique. A review of the technologies available to regulate the antibody-antigen interaction was conducted. Little was published on the use of phage display for the discovery of peptide ligands for use in peptide mimotope affinity chromatography. Experiments were conducted using a polyvalent phage display library to identify novel peptide ligands for the purification of the therapeutic monoclonal antibody C595. A novel peptide was isolated which demonstrated improved chromatographic performance compared to the standard epitope peptide used to purify mAb C595 from biological supernatants. Circular dichroism showed that the novel peptide had a more highly ordered structure at 4oC and room temperature than the epitope peptide, and fluorescence quenching revealed a higher equilibrium association constant. A method for the optimisation of peptide mimotopes derived from phage display, cross-reactive with an anti-steroid antibody was investigated. Improvements relating to the selection of lead peptide sequences are described, and the use of mimotopes in an assay to determine concentrations of steroid in solution has been demonstrated. The optimised mimotope was used as an effective paratope-specific affinity ligand. A novel method for selecting high affinity antibody fragments in vivo is described. The C595 scFv gene was fused to a gene encoding green fluorescent protein and incorporated into the phagemid vector pCANTAB 5E. The fusion protein could not be expressed at high levels and could only be detected using epitope affinity chromatography in combination with ELISA.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:395580 |
| Date | January 2002 |
| Creators | Smith, Richard Gary |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/10053/ |
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