Abstract
OBJECTIVE:
Cryo-transmission electron microscopy (cryoTEM), with its unique capability for providing exact visual information of a sample in its natural hydrated state, is a powerful new tool in the armamentarium of characterization techniques applied to complex biological therapeutics. The ability of this technique to yield useful information on size, shape, morphology, aggregation state, and structural information is demonstrated through application to a variety of biologics.
METHODS:
Samples were preserved in vitrified ice supported by holey carbon films on 400-mesh copper grids. Electron microscopy was performed using an FEI Tecnai T12 electron microscope, operating at 120keV equipped with an FEI Eagle 4k x 4k CCD camera. Vitreous ice grids were transferred into the electron microscope using a cryostage that maintains the grids at a temperature below −170 °C. Images of each grid were acquired using automated image collection software package LEGINON at multiple scales to assess the overall distribution of the specimen. After identifying potentially suitable target areas for imaging at lower magnifications, pairs of high magnification images were acquired at nominal magnifications of 52,000x (0.21 nm/pixel) and 21,000x (0.50 nm/pixel). Quantitative analysis and 3D structure determination was enabled by the automated software package APPION.
RESULTS:
CryoTEM can be used to visualize the integrity, morphology, size, and shape of a variety of biologics in their native state. Automated data collection and processing allowed quantitative analysis of shape, size, aggregation state, and concentration. Advanced methods can be used to determine the 3D structures of biologics, thereby allowing assessment of encapsulation efficiency, lamellarity, and the location of antigen binding sites.
CONCLUSIONS:
CryoTEM provides a useful orthogonal characterization tool for understanding complex biologics.