Feg tem

The pixel size was converted to nm based on the scan parameters of the camera (FEI Eagle 4 k × 4 k CCD attached to a 200 KV FEI-Tecnai FEG-TEM). The characterized antibody aggregates were clustered based on their equivalent radius (r_e) defined as $r_e=\sqrt{\frac{A}{\pi }}$ where the area A = n_p × area of each pixel for the system, where n_p is the number of pixels occupied by the aggregate. This is the equivalent radius of a circle having the same area. The aggregated species were clustered and distributed into bins of equivalent radii.

4 µl of therapeutic mAbs, at a concentration of 400 μg/ml, was placed on the carbon face of 200 mesh glow discharged carbon coated copper grids (Agar Scientific) and allowed to adsorb for 1 min. Grids were glow discharged for 60 s at 20 mA (SC7620 Mini Sputter Coater/Glow Discharge system, Quorum Technologies), prior to usage. Grids were washed 3 times with 10 µl of double distilled water, followed by blotting with Whatmann filter paper. For staining, 4 µl of 2% uranyl acetate, pH ~ 7.0 was applied onto the grid twice, for a time period of 20 s and 40 s, respectively, and the surplus stain was removed by blotting in each case. The grid was dried at room temperature for 2 min and visualized at a magnification of 100,000x in a FEG-TEM (FEI Tecnai) operating at 200 kV. Three micrographs from different grid squares were collected for each sample.