Abstract by Camilla Thorlaksen

The inherent ability of proteins to aggregate is a major challenge upon the development of therapeutic products and can occur during the entire production chain, shipping, storage and patient administration. It is widely accepted that protein aggregates may accelerate an immune response towards the therapeutic product, which has impact on efficacy and patient safety. However, there is little knowledge on the characteristics an aggregate must encompass to become an immunogenicity risk. Current research rely on the assessment of heterogeneous aggregate ensembles varying in multiple morphological characteristics, obscuring the contribution from the single feature which might elicit the immunogenic response. Thus there is a need for controlled immunogenicity studies with well-characterized aggregates varying in only a single morphological characteristic. This will allow for untangling the characteristic(s) with highest immunogenicity risk.

In this thesis we developed and optimized protocols for generating homogeneous samples of aggregates varying in few characteristics. The aggregates were characterized by a plethora of analytical techniques to obtain knowledge on their appearance, size, structure and presence of chemical modifications. Ultimately, the aggregate samples were evaluated for their immunogenicity potential in vitro in a DC:T-cell assay and in vivo in a BALB/c mouse model. The results shows that loose micronsized aggregates with highly altered structure were most immunogenic in all of our assays. While compact micronsized aggregates with native-like structure were most immunogenic in vivo. In general, we observed that micronsized aggregates were more immunogenic than sub-micron aggregates and we did not find any contribution from chemical modifications (deamidation, ethylation, covalent dimers) on the immunogenicity potential. Our approach provides a relative immunogenicity risk ranking of distinct characteristics, which could be employed in the risk mitigation strategy at early product development.