Development of In Vitro Models and Analytical Methods to Assess the In Vivo Stability of Therapeutic Proteins

Maintaining the stability of therapeutic proteins is a hallmark of successful drug product (DP) development. The stability of DPs is closely monitored during various stages such as development, manufacturing, and in clinical preparation. However, the stability of a DP can change considerably after administration (in vivo stability). Assessing protein stability under physiologic conditions can improve the safety and efficacy profile of commercial DPs.
The first part of the thesis focuses on protein aggregation and fragmentation of monoclonal antibodies (mAbs), after simulated intravenous administration. The molecules showed markedly different stability in human serum with some mAbs being subjected to substantial fragmentation, while others remained stable. Moreover, substantial sub-visible particle formation in serum was detected with developed methods such as fluorescence microscopy and flow cytometry.
In the second part of this thesis, we simulated the subcutaneous administration of mAbs without fluorescence labeling. In line with our previous observations, mAb candidates had substantial differences in their stability under physiologic conditions. Furthermore, we showed that phosphate-buffered saline does not stress mAbs to the same extent as bicarbonate-buffers and human serum. The in vivo stability of therapeutic proteins should be an integral part of holistic stability assessments during early stages of development.