Book Description

Therapeutic proteins are inherently unstable. Stresses such as freeze-thawing and agitation can induce protein aggregation, and consequently reduce drug efficacy or cause adverse immunogenicity. The aims of this thesis were to better understand stress-induced protein aggregation through novel findings in other disciplines such as biophysics and interfacial sciences. We studied the effects of pH and additives on freezing-induced perturbations of tertiary structure of a monoclonal antibody (mAb) by intrinsic tryptophan (Trp) fluorescence spectroscopy. We found freezing-induced protein aggregation may or may not first involve the perturbation of its native structure, followed by the assembly processes to form aggregates. Depending on the solution conditions, either step can be rate-limiting. This study demonstrates the potential of fluorescence spectroscopy as a valuable tool for screening therapeutic protein formulations subjected to freeze-thaw stress. We investigated the effects of excipients on protein aggregation during agitation as well as the effects of same compounds on interfacial shear rheology of the protein at air-liquid interface. Heparin, sucrose, and Polysorbate 80 (PS80) alone could not effectively inhibit a model protein keratinocyte growth factor 2 (KGF2, FGF10) aggregation during non-agitated and agitated incubation. The combination of PS 80 and heparin or sucrose substantially inhibited aggregation during both protocols. Interfacial shear rheology provides insight regarding the rate of gel formation and the role of non-ionic surfactants at the interface. There is a correspondence between formulations that exhibited interfacial gelation and formulations that exhibited agitation-induced aggregation. We also found filters used to remove particles from protein solutions actually shed particles, which stimulated protein aggregation during stresses such as agitation. Particles shedding from syringe filters varied greatly among the filters types from the manufacturers. Furthermore, particles shed from the filters may change the rate of protein aggregation during agitation. Last but not least, we found bevacizumab repackaged in plastic syringes could contain protein aggregates and was contaminated by silicone oil microdroplets. Freeze-thawing or other mishandling can further increase levels of particle contaminants. This study may help to reduce mishandling of repackaged bevacizumab caused adverse effects in patients with eye diseases.