Transmission electron microscopy as an orthogonal method to characterize protein aggregates

Area of Science:

• Biopharmaceutical industry
• Protein aggregation
• Antibody therapeutics

Background:

• Protein aggregation is a significant challenge in biopharmaceutical development.
• Aggregates can impact therapeutic efficacy and patient safety due to immunogenicity.
• Characterizing aggregate morphology is essential for quality control.

Purpose of the Study:

• To characterize the biophysical and morphological features of antibody aggregates.
• To compare transmission electron microscopy (TEM) with other analytical techniques.
• To assess TEM's suitability for submicron particle analysis.

Main Methods:

• Controlled environmental stresses were applied to induce antibody aggregation.
• Transmission electron microscopy (TEM) was used in negative staining and frozen-hydrated states.
• TEM results were compared with size-exclusion chromatography, dynamic light scattering, microflow imaging, and nanoparticle tracking.

Main Results:

• TEM revealed amorphous antibody aggregates with differing fine structural details based on imaging state.
• Negatively stained aggregates appeared compact with 3D globular substructures, retaining native IgG features.
• Frozen-hydrated aggregates showed extended, branched networks with large surface areas.
• TEM provided detailed information across a wide range of particle sizes, complementing other methods.

Conclusions:

• Transmission electron microscopy (TEM) is a valuable complementary technique for submicron particle analysis in biopharmaceuticals.
• TEM offers detailed morphological insights into antibody aggregates that extend observations from other methods.
• Understanding aggregate structure is key to ensuring the safety and efficacy of protein-based therapeutics.