Image Analysis Algorithm-Based Platform for Determining Micron and Higher Aggregate Size Distribution of Therapeutic IgG Using Brightfield and Fluorescence Microscope Images.

A platform for determining size distribution of micron (1-100 μm) and larger (> 100 μm) aggregates of therapeutic IgG has been established by using image processing algorithms for brightfield and fluorescence microscope images. The algorithm for brightfield images involved conversion to grayscale followed by pixel-based and size-based thresholding. Morphological operations were then applied and the size distribution of aggregates were extracted. Fluorescence images of the aggregates of mAb tagged by a fluorescent dye were captured using widefield fluorescence microscope, confocal laser scanning microscope, and Cytell Cell Imaging System and the images were processed using a series of denoising steps followed by thresholding and morphological operations. The samples were subjected to different stresses, among which the aggregates were visible in the microscope for sample subjected to bubbling, stirring, and temperature. The images of these aggregates were effectively denoised and the size distribution of aggregates was analyzed using the algorithm. The overall aggregate size distribution obtained by image processing ranged in the micron and higher size range. The size obtained from brightfield image processing was validated using images of liquid chromatography resins. Further, the aggregate size distribution obtained using image processing was compared with experimental techniques such as Mastersizer 2000 and Micro Flow Imaging. It was found that analysis of IgG aggregates using image processing could serve as an orthogonal methodology to the existing approaches.