Wood, Victoria Elizabeth; (2019) Use of in silico and biophysical techniques to predict protein stability. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A protein’s structure dictates its function; which in turn governs its role of maintaining the cascade of activities that characterise all living systems. In solution, proteins are labile structures that move and flex naturally or in response to external stimuli, which is also important for function. However, this movement of proteins, collectively termed protein dynamics, can also have a negative impact on stability. During manufacturing, biotherapeutic proteins are exposed to a variety of stressors that can cause unfolding and lead to aggregation, which can cause loss of activity and increased immunogenicity. Therefore, implementing strategies to increase resistance to stress is paramount to ensuring protein efficacy and safety. Hydrogen/deuterium exchange (HDX) has become a valuable tool for the study of protein dynamics. The method involves labile hydrogens in the protein backbone exchanging with deuterium atoms when the protein is placed in a deuterium oxide solution. The subsequent increase in protein mass over time can be measured with high‐resolution mass spectrometry (MS). The rate of deuterium exchange is a function of solvent accessibility, hydrogen bonding and protein flexibility. Dynamics can be studied at a local level via the inclusion of a peptide digestion step after the labeling reaction to monitor peptic fragment exchange. This thesis explored the use of peptide level HDX‐MS to monitor changes in local protein dynamics of the recombinant cytokine, granulocyte colony stimulating factor (GCSF). Three common stabilising strategies were assessed: site‐directed mutagenesis, excipient formulation and lyophilisation. Data found all strategies reduced GCSF flexibility in similar regions of the protein, which was linked to improvements in stability and minimal effect on function. Additionally, regions of increased flexibility were linked with protein destabilisation. Computational design during biopharmaceutical development is becoming routine for both protein conformation and formulation to reduce the number of screening candidates. In silico protein‐excipient docking and site‐directed mutagenesis data was performed alongside HDX‐MS experiments for in vitro validation of outputs. Consequently, both in silico and biophysical analysis methods provided advancements towards the rational manipulation of the physical, chemical and biological stability of proteins.

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