The development of anti-drug antibodies (ADA) against therapeutic proteins can impact upon drug safety and efficacy. This is a major challenge in the development of biotherapeutics. Various factors have the potential to contribute to protein immunogenicity and the production of ADA. Protein aggregation is one of these factors, though the mechanisms underlying aggregate immunogenicity are poorly understood. In this thesis the effect of protein aggregation on immunogenicity has been investigated. The thermal and/or mechanical stresses required in order to achieve subvisible aggregates of three test proteins were determined. Stressed preparations of proteins were characterised using a suite of biophysical techniques, including dynamic light scattering and circular dichroism. The immunogenic potential of subvisible aggregates of a humanised single chain variable fragment (scFv) and ovalbumin (OVA) was studied following intraperitoneal exposure in BALB/c strain mice. Monomeric proteins induced a T helper (Th) 2 dominant immune response, but when aggregated, the responses gained a Th1 phenotype, with a significant increase in the antigen-specific IgG2a antibody response. Cytokine profiles in supernatants taken from splenocyte-dendritic cell co-cultures were also consistent with aggregated preparations of OVA inducing a Th1-type response. Host cell protein (HCP) impurities can also contribute to immunogenicity. Mass spectrometry analysis of an scFv preparation identified the presence of the Escherichia coli (E.coli) heat shock protein DnaK, amongst other HCP, as an impurity. Protein preparations free from DnaK were spiked with recombinant E.coli DnaK to mimic the HCP impurity. The effect of DnaK on the immunogenicity of aggregated and monomeric scFv preparations was then investigated. BALB/c mice were immunised with monomeric and aggregated preparations, with and without E.coli DnaK at 0.1% by mass. Aggregation alone resulted in an enhanced IgG2a antibody response, and the presence of DnaK increased this further. Comparable investigations were also conducted using mouse albumin; here an increase in immunogenicity was observed with protein aggregation, and the presence of DnaK was found to increase the IgG2a response. Collectively, the evidence presented in this thesis shows that aggregation can impact upon the magnitude and character of induced immune responses, and that subvisible aggregation promotes a Th1 immune skewing. Additionally, E.coli HCP DnaK enhances protein aggregate immunogenicity, which indicates that heat shock proteins, as a class of HCP, could have an adjuvant-like effect on biotherapeutic aggregates.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:764545 |
| Date | January 2017 |
| Creators | Ratanji, Kirsty |
| Contributors | Derrick, Jeremy ; Dearman, Rebecca |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/investigating-the-immunogenicity-of-therapeutic-proteins-protein-aggregation-and-host-cell-protein-impurities(fda43dd8-2c1e-492b-abd9-e010774d2219).html |
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