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Title: Self-interaction chromatography and the aggregation of biopharmaceuticals
Author: Hedberg, Sarah
ISNI:       0000 0004 7657 6268
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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Protein aggregation is a critical problem in the manufacture of biopharmaceutical, effecting process efficiency as well as being linked to adverse immunologically related responses in patients. Protein-protein intermolecular interactions are known to be involved in protein solution aggregation behaviour which is a common issue for the manufacture of therapeutic proteins such as monoclonal antibodies (mAbs). Much effort has been employed to gain a better understanding of protein aggregation, however, the mechanisms leading to protein aggregation are still not fully understood. The osmotic second virial coefficient (B22), or in its dimensionless form (B2), is a fundamental physiochemical property that describes protein-protein interactions in solution, which can be a useful tool for not only predicting the aggregation propensity of proteins, but also protein solubility, crystallisation and phase behaviour. An automatic, rapid and high-throughput way of determining B22 is self-interaction chromatography (SIC). This thesis reports on how SIC has been refined in order for it to be successfully deployed for studying mAbs. These studies include correction of the dead volume (the non-interacting reference volume), scale-down approaches, identification of the critical experimental parameters involved in SIC and improved immobilisation reaction conditions. The results of these studies have allowed for more accurate determinations of B22 for mAbs, as well as more efficient use of SIC in terms of time and amounts of protein required. The colloidal and conformational stability of mAbs were studies both over time and at increasing temperatures using SEC and DLS. The B22/ B2 values reported here to correlate well with aggregation propensity for two different mAb types, IgG1 and IgG4, when exposed to a wide variety of solution conditions that would be very useful in a process development study. In this thesis it is shown that strongly negative B2 values predicted fast aggregation kinetics, weakly negative B2 values predicted slow aggregation kinetics and weakly positive B2 values predicted almost no aggregation. Finally, a cross-interaction chromatography (CIC) study suggested that for different mAbs their B22 values could be reliably estimated by simply eluting these proteins through other mAb or polyclonal (pIgG) immobilised columns, thus significantly simplifying and accelerating the process by which B22 values can be obtained.
Supervisor: Heng, Jerry ; Williams, Daryl Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral