The Strengths of Total Holographic Video Microscopy in Detecting Sub-Visible Protein Particles in Biopharmaceuticals: A Comparison to Flow Imaging and Resonant Mass Measurement.

Determination of subvisible particle (SVP) content in biopharmaceuticals is a prerequisite to ensure the quality of liquid biopharmaceutical products. Here, we present a comparison of the recently introduced holographic video microscopy (total holographic characterization, THC) with two orthogonal and well-established analytical technologies: micro flow imaging (MFI) and resonant mass measurement (RMM). The capabilities of the THC were investigated under conditions commonly applied in drug product development. Three different antibody products were used at different concentrations and formulations to cover a wide range of realistic use-cases. The comparison was particularly focused on protein aggregates to investigate the applicability of THC to this critical class of particles in drug product development. Protein concentrations up to 100 mg/ml were investigated covering a broad range of viscosity and refractive indices, both important parameters in particle detection. The comparison reveals that THC is highly sensitive to detect protein aggregates in a size range from 0.5 µm to 10 µm. THC shows a significant superiority to FI and RMM in detecting heterogenous protein aggregates which often appear as transparent and porous particles. Additionally, THC needs very small sample amount of about 30 µl and short measurement times, making it applicable for early development stages and high-throughput approaches. These results show that THC is a valuable supplement to the existing particle characterization method portfolio in drug product development.