Distinguishing protein aggregates from contaminants in viscous mixtures with holographic video microscopy*

We demonstrate how holographic video microscopy detects, counts, and characterizes individual sub-visible protein aggregates ranging in diameter from 0.5 μm – 10 μm in concentrations and viscosities typical of biologic pharmaceutical formulations. Protein aggregates are distinguished from other contaminants that are found in pharmaceutical manufacturing, including silicon oil emulsion droplets, air bubbles, tungsten mental particles and breakdown products of industry standard surfactants. A study was also performed comparing the holographic signatures of protein aggregates and other contaminants in solvents of different viscosities. These results differentiates uniform spherical particles from particles composed of an irregularly shaped aggregation of proteins. Holographic characterization’s unique ability to measure refractive index provides a basis for differentiating protein aggregates from contaminants. For example, silicone oil droplets are clearly distinguished from protein aggregates on the basis of refractive index, even when they have the same size.