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.
Keywords: Aggregation; Antibodies; CD, Circular Dichroism; Connected component labelling; DLS, Dynamic Light Scattering; DPBS, Dulbecco's phosphate-buffered saline; EM, Electron Microscopy; Electron microscopy; FEG, field emission electron gun; GUI, Graphical User Interface; HDX-MS, Hydrogen Deuterium Exchange Mass Spectroscopy; Heterogeneity; MS, Mass Spectroscopy; SEC, Size Exclusion Chromatography; SEC-MALS, Size Exclusion Chromatography Multi Angle Light Scattering; TEM, Transmission Electron Microscopy; TV, Total Variation; UV, Ultra Violet; mAb, monoclonal Antibody.
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