Electron microscopy-based semi-automated characterization of aggregation in monoclonal antibody products

Graphical abstract
Highlights • Size-based quantification of small heterogeneous proteins using electron microscopy. • Electron microscopy as an orthogonal tool for characterizing protein aggregates. • Quick assessment of small heterogeneous proteins via softEM, a GUI-based algorithm.
Aggregation is a critical parameter for protein-based therapeutics, due to its impact on the immunogenicity of the product. The traditional approach towards characterization of such products is to use a collection of orthogonal tools. However, the fact that none of these tools is able to completely classify the distribution and physical characteristics of aggregates, implies that there exists a need for additional analytical methods. We report one such method for characterization of heterogeneous population of proteins using transmission electron microscopy. The method involves semi-automated, size-based clustering of different protein species from micrographs. This method can be utilized for quantitative characterization of heterogeneous populations of antibody/protein aggregates from TEM images of proteins, and may also be applicable towards other instances of protein aggregation.