Main content area

In-Solution Microscopic Imaging of Fractal Aggregates of a Stressed Therapeutic Antibody

Senga, Yukako, Imamura, Hiroshi, Ogura, Toshihiko, Honda, Shinya
Analytical chemistry 2019 v.91 no.7 pp. 4640-4648
analytical methods, antibodies, biopharmaceuticals, drying, electron microscopy, fractal dimensions, image analysis, immunoglobulin G, light scattering, manufacturing, monitoring, neutralization, pharmaceutical industry, protein aggregates, staining, therapeutics
Aggregates of therapeutic proteins that can contaminate drug products during manufacture is a growing concern for the pharmaceutical industry because the aggregates are potentially immunogenic. Electron microscopy is a typical, indispensable method for imaging nanometer- to micrometer-sized structures. Nevertheless, it is not ideal because it must be performed with ex situ monitoring under high-vacuum conditions, where the samples could be altered by staining and drying. Here, we introduce a scanning electron-assisted dielectric microscopy (SE-ADM) technique for in-solution imaging of monoclonal immunoglobulin G (IgG) aggregates without staining and drying. Remarkably, SE-ADM allowed assessment of the size and morphology of the IgG aggregates in solution by completely excluding drying-induced artifacts. SE-ADM was also beneficial to study IgG aggregation caused by temporary acid exposure followed by neutralization, pH-shift stress. A box-counting analysis of the SE-ADM images provided fractal dimensions of the larger aggregates, which complemented the fractal dimensions of the smaller aggregates measured by light scattering. The scale-free or self-similarity nature of the fractal aggregates indicated that a common mechanism for antibody aggregation existed between the smaller and larger aggregates. Consequently, SE-ADM is a useful method for characterizing protein aggregates to bridge the gaps that occur among conventional analytical methods, such as those related to in situ/ex situ techniques or size/morphology assessments.