Transmission Electron Microscopy as an Orthogonal Method to Characterize Protein Aggregates

Abstract

Aggregation of protein-based therapeutics is a challenging problem in the biopharmaceutical industry. Of particular concern are implications for product efficacy and clinical safety due to potentially increased immunogenicity of the aggregates. We used transmission electron microscopy (TEM) to characterize biophysical and morphological features of antibody aggregates formed upon controlled environmental stresses. TEM results were contrasted with results obtained in parallel by independent methods, including size exclusion chromatography, dynamic light scattering, microflow imaging and nanoparticle tracking. For TEM, stressed samples were imaged by negative staining and in the frozen-hydrated state. In both cases, aggregates appeared amorphous but differed in fine structural detail. Specifically, negatively stained aggregates were compact and consisted of smaller globular structures that had a notable three dimensional character. Elements of the native IgG structure were retained, suggesting that the aggregates were not assembled from denatured protein. In contrast, aggregates in frozen-hydrated samples appeared as extended, branched protein networks with large surface area. Using multiple scales of magnification, a wide range of particle sizes was observed and semi-quantitatively characterized. The detailed information provided by TEM extended observations obtained with the independent methods, demonstrating the suitability of TEM as a complementary approach to submicron particle analysis.

Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma

“…These structures were present frequently and consistently in repeated experiments in the samples from all six healthy donors of the first group, while they were absent from the kit elution buffer. The irregular morphology of these particles was suggestive of protein aggregates [ 12 ]. The identity of these particles is yet unknown.…”

Particle Detection and Characterization for Biopharmaceutical Applications: Current Principles of Established and Alternative Techniques

“…Take the determination of something as fundamental as particle size, for example. Microscopy is the only visual and direct technique/principle to determine particle size, and even here, sample preparation steps, such as drying on microscopy carrier, can impair the results by producing artefacts [ 16 , 34 , 85 , 86 , 87 ]. In the absence of direct size determination approaches, most particle-by-particle measurements and higher throughput methods rely on the concept of equivalent particle diameter (EPD) [ 16 ].…”

Measurement of Average Aggregate Density by Sedimentation and Brownian Motion Analysis

“…There is evidence that protein aggregates are porous. Sung et al found that “TEM revealed that aggregates were highly irregular in shape and porous in nature suggesting that their water content is substantial 8 .” This would suggest an average density lower than the value for pure protein. There are several reports of researchers using aggregate density to estimate the amount of protein in an aggregate based on imaging results.…”