(M1030-08-50) Characterizing Protein Aggregation Using Flow Imaging Microscopy with Orthogonal and Complementary Analytical Techniques

Purpose: Effective strategies for monitoring particles in biotherapeutic drug products are critical to meet regulatory requirements like USP < 788 >, mitigate the potential safety risks of particles, and ensure overall product quality. Flow imaging microscopy (FIM) is an increasingly common particle characterization approach used to monitor the number, size distribution, and types of subvisible particles (2-100 µm in size) in biotherapeutic samples. Recent innovations in FIM technologies such as FlowCam LO and FlowCam Nano allow researchers to obtain orthogonal light obscuration (LO) measurements and submicron particle characterization, respectively. Combining FlowCam LO and FlowCam Nano with complementary techniques like dynamic light scattering (DLS) is an effective method for comprehensive particle characterization in biotherapeutics. In this study, these FIM platforms and DLS were used to analyze protein aggregation in a simple accelerated stability study to determine the impact of stresses and surfactant on protein aggregation and to demonstrate the effectiveness of combining FIM with orthogonal and complementary techniques at characterizing protein aggregation over a wide size range.

Methods: A simple accelerated stability study was performed on formulations of bovine serum albumin (BSA) in phosphate-buffered saline (PBS) with and without polysorbate 80 (PS80). Samples were exposed to either a shaking stress in which samples were agitated on a plate shaker for four hours and a heating stress in which samples were heated to 60°C for four hours. The resulting protein aggregates were then analyzed on three instruments and four analytical techniques: FlowCam LO (FIM and LO), FlowCam Nano (submicron FIM), and a Dynapro NanoStar (DLS).

Results: The observed changes in particle concentrations and size distributions indicated that shaking stress generated more numerous and larger protein aggregates than heating stress. These measurements also indicated that the surfactant greatly reduced the particle count and size generated by shaking stress while having a negligible impact on heat-induced aggregation. Similar changes in particle content were observed via all four analytical techniques.

Conclusion: The observed changes in protein aggregates were consistent with all investigated instruments and the expected impacts of stresses and surfactants (e.g., shaking likely induces aggregation at interfaces; surfactants mitigate interfacial stresses). This combination of methods can help researchers analyze protein aggregates and other particles from nanometers to micrometers in size as well as validate trends observed with any analytical technique alone.

Acknowledgements: The authors are employees of Yokogawa Fluid Imaging Technologies, the manufacturer of the FlowCam LO and FlowCam Nano instruments used to collect data presented in this poster.

FlowCam LO and Nano histograms for BSA with and without PS80 exposed to shaking and heating stress. Shaking stress (blue plots) generated higher concentrations and larger sizes of subvisible and submicron particles than heating stress (green and orange plots) in BSA without PS80. Adding PS80 reduced particle concentration and size of subvisible and submicron particles generated by shaking stress, with a smaller effect on particles generated by heating stress. Particle concentrations in particles/mL for each instrument and experimental condition are shown in the table below the histograms.

DLS autocorrelation plots for BSA aggregates generated by (left) shaking stress and (right) heating stress with and without PS80. Three replicates are shown per sample.