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Title: Stability, structure, and formulation of antibody fragments during bioprocessing
Author: Uye, U. O.
ISNI:       0000 0004 5351 6828
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2014
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Despite their huge potential as therapeutic agents, protein molecules are not naturally optimised for stability and are susceptible to aggregation during bioprocessing. A good understanding the mechanisms that drive protein aggregation is therefore important to enable the development of strategies for eliminating protein aggregation bioprocessing. To better understand the factors affecting aggregation, a detailed kinetic and biophysical analysis of the Af-X molecule when subjected to a range of pH and NaCl concentrations was carried out. At pH 4.0, a salt-induced dimer was found to accumulate at low NaCl concentrations, but less so at higher salt where the rate of protein precipitation increased. Kinetic studies showed that this salt-induced dimer is not an intermediate on the Af-X precipitation pathway. Surface plasmon resonance, circular dichroism, and fluorescence spectroscopy highlighted the biological inactivity of the salt-induced dimer and confirmed its structural and functional similarity to a previously observed cell culture dimer. Analysis of the crystal structures of both dimers confirmed the biophysical characterisation. The post induction time and cellular location of Af-X dimer formation, as well as the effect of IPTG concentration, temperature, and post-induction feed rate on the propensity for dimerisation of the Af-X molecule, were investigated using fed-batch fermentation. While a decrease in both the IPTG concentration and the post-induction feed rate resulted in a 2% increase and a 5% decrease (respectively) in dimer content of the total Af-X yield, a reduction in the post-induction temperature resulted in a 12% increase in the dimer content of the total Af-X yield. This unexpected increase in dimer concentration was found to agree with the proposed effect of temperature on the Af-X mechanism of aggregation. Site-directed mutagenesis was used to generate L-XX-KVRST single and H-XX-Y/L-XX-KVRST double mutants that were evaluated for their propensity for aggregation both in solution and during E. coli cell culture. While the L-XX-K single mutant was found to be destabilising to the Af-X molecule, the L-XX-R single mutant was found to be stabilising. These contrasting observations can be explained by taking into account possible physical distance limitations or angular orientation of the lysine side chain that is less than optimal for salt bridge formation with the aspartic acid residue at position XX. Finally, samples of Af-X were incubated for 8 weeks at 25 °C without shaking, and compared to samples incubated for 6 days at 25 °C with shaking in order to establish a correlation between agitation and the long term stability studies commonly used in formulation development. The effect of ultrasonication on the propensity for aggregation of the Af-X molecule was also assessed using a time-course experiment. Both agitation and sonication had negligible effects on the propensity for Af-X dimerisation. In addition to providing useful insights into aggregation in the Af-X molecule, this work will serve as a starting point for future investigation of aggregation in antibody fragments in general. A number of potential future directions that build on these insights are also discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available