(174am) Development of Novel Stability Assays for Protein Biopharmaceuticals Using Time-Dependent Light Scattering Analysis

We are developing a rapid analytical method for determining protein stability in order to better understand protein aggregation in general, and specifically to improve the shelf life of biopharmaceuticals at high concentration. Using polyclonal immunoglobulin G (IgG) as a model protein and dynamic light scattering (DLS) as a tool to determine the size of particles present in solution, we analyze how the rate of aggregation varies with temperature and concentration. DLS measurements conducted at room temperature showed only a slight increase in apparent average hydrodynamic radius as a function of concentration in the 1.25 – 10 mg/mL range, but no change in apparent radius was observed after three weeks. To reduce the observation period, we designed a new assay for DLS analysis by performing periodic measurements while heating the protein solutions at a constant rate. While the onset of aggregation became visible with this technique, it was difficult to see differences between samples. To highlight differences more clearly, we implemented (i) a multimodal analysis comprising of a high-resolution fitting for multiple sizes present in a solution, (ii) a temperature ramp to intermediate temperature followed by a stress test at constant temperature over several hours, and (iii) 3-dimensional plots that show the time-dependent evolution of the particle size distribution at the selected temperature. The resulting 3-D plots allow us to pinpoint the onset of aggregation at different conditions. We expect that this method could enable improved understanding of the effects of parameters such as temperature and concentration on the stability of protein solutions. It can also be used as a rapid assay for determining long term stability of biopharmaceutical protein products.