Nucleation in Protein Aggregation in Biotherapeutic Development: A look into the Heart of the Event

In spite of extensive research, protein aggregation still remains one of the most difficult phenomena to be understood in the field of biologics research and development. Protein aggregation is a complex process which results in the formation of a variety of supramolecular protein structures. Nucleation is the core step that initiates the cascade of molecular events leading to the formation of protein aggregates. Understanding and characterizing nucleation is therefore crucial to avoid undesired protein aggregation. Here we review the state of the art on protein aggregation in biotherapeutics, primarily focusing on the nucleation events, stimulating discussions about key open questions, and clarifying the peculiarities of aggregation process relative to other protein phase separation processes, such as crystallization. We summarize recent progress in the identification of the sources of protein aggregation and in the development of analytical tools to characterize this process. Moreover, we discuss significant gaps in the analysis and understanding of nucleation in non-native aggregation of biologics.