Total Holographic Characterization is used to distinguish individual sub-visible contaminants in model biologic pharmaceuticals, ranging in size from 0.5 μm – 10 μm. All measurements are performed on xSight. Samples flow down a channel in a disposable microfluidic sample chip 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, and breakdown products of industry standard surfactants.

Aging surfactants produce free fatty acids that can be monitored and identified using Total Holographic Characterization. Control samples of a variety of free fatty acids including oleic acid, and linoleic acid are compared with accelerated aging of polysorbate 80 (PS80) to identify the breakdown products of the surfactant using Total Holographic Characterization.

Total Holographic Characterization uses Lorenz-Mie Theory to analyze holograms of individual sub-visible particles. The analysis yields the size distribution of particles present and simultaneously determines the composition from the index of refraction of the particles. Particle symmetry provides additional information about the morphology of the particles. Spherical particles, such as those of emulsion droplets or air bubbles are distinguished from more irregularly shaped particles, by the symmetry of their holograms. We compare the size, refractive index and morphology of the control samples and the PS80 sample to identify the composition of the contaminants in the sample.

Additional studies of stressed protein solutions will also be presented. Protein aggregation behavior varies when IgG is exposed to different handling conditions. IgG solutions exposed to different mixing and handling protocols in standard buffers will be presented. Robust and repeatable concentration measurements of protein aggregates are a critical requirement for monitoring product quality in biologic manufacturing. The precision and robustness of holographic characterization measurements are demonstrated through repeated measurement of solutions of IgG and silicone oil, and through dilution studies of NIST standard particles.

Total Holographic Characterization requires 25-35 mL of sample per measurement and measurements can be performed in a matter of minutes. In addition, xSight meets all of the requirements for 21 CFR Part 11.

Fook Chiong Cheong

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