Experimental characterisation of protein-protein and protein-excipient interactions and their impact on protein conformation and aggregation

Abstract

In the last decades, biopharmaceutical proteins, especially monoclonal antibodies (mAbs), have become one of the fastest growing classes of pharmaceuticals, due to their high affinity, specificity, non-toxicity, and low immunogenicity. Despite their versatility and wide applicability, their production is costly and poses unique challenges due to their high molecular weight and structural complexity. One of the major challenges in formulation development is thus prevention of protein self-association that may lead into formation of potentially immunogenic aggregates. To achieve this, excipients are often added in relatively high concentrations to the final formulations. However, there is a lack of information available on the impact of co-solutes, including excipients on protein stability. In light of these inherent problems of mAbs, alternative systems, such as protein aptamers, have been developed for high affinity binders for specific targets. Some advantages of peptide aptamers are their smaller size, versatility, specificity for chosen targets, and ease of production. However, the insertion of binding loops may cause destabilisation of protein, leading to formation of inactive oligomeric species. In the first part of the Thesis we have used a stefin A derived scaffold protein, SQT, as a model to study solution behaviour and self-association of such systems. We have determined its crystal structure, and investigated possible pathways of oligomer formation. We studied the oligomerisation kinetics and determined the limiting steps of dimer and tetramer formation. Using mutagenesis, we created a more stable variant of the SQT scaffold. In the second part of this Thesis we have applied solution NMR spectroscopy to comprehensively evaluate protein-excipient interactions between seven biotherapeutically relevant proteins and a set of eleven commonly used excipients. Additionally, we evaluated the effect of excipients on thermal and colloidal protein stability, the aggregation kinetics at elevated temperatures and on the protein storage stability at accelerated conditions. In the last part of the Thesis we have combined both protein and ligand-observed NMR approaches to evaluate interactions between the stabilised SQT variant and various excipients. We used isotopically labelled protein to evaluate the potential interaction sites on the protein surface and estimated the binding constants for individual excipients. Furthermore, we compared the sensitivity of both protein- and ligand-observed methods and discussed how they could be applied to biopharmaceutical formulations.

Date of Award	31 Dec 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	James Warwicker (Supervisor) & Alexander Golovanov (Supervisor)