Sub-micron Aggregate Characterization: IgG and IgM


The Challenge: Sub-micron Aggregate Characterization

Direct determination of protein or protein complex morphology for therapeutic proteins is critical to characterization during pharmaceutical and biotechnology process development and formulation.  While a myriad of techniques exist for characterization of large aggregate particulates, the analysis of sub-micron aggregates remains a challenge.   

The Solution: Negative Stain Molecular Microscopy

Negative stain Molecular Microscopy allows for the direct visualization of sub-micron aggregates, avoiding the artifacts associated with standard aggregate characterization methods such as DLS (dynamic light scattering) and size exclusion chromatography (SEC) analysis.  

Negative Stain TEM of Individual IgG and IgM Molecules:

Negative stain Molecular Microscopy images of IgG (left), IgM (middle), and IgG/IgM molecules.  Yellow insets are IgG (left) and IgM (middle) at higher magnification.  Blue insets are averages of IgG, each containing hundreds of individual IgG molecules that were aligned to a common origin.

Fast and Slow Freeze-Thaw Cycles of IgG/IgM Solution:
A 0.01mg/mL solution of IgG/IgM was subjected to 3 freeze-thaw cycles and analyzed by negative stain Molecular Microscopy.  Shown here are representative micrographs from Cycle 1(left) and Cycle 3 (right) for a fast (top) and slow (bottom) freeze-thaw method.

Aggregate Characterization:
Gallery of submicron aggregates detected by negative stain Molecular Microscopy after multiple freeze-thaw cycles of a solution of IgG/IgM. Submicron particle size per unit area for 1-3 cycles using the fast (red) and slow(blue) freeze-thaw methods.
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