Chapter 6: High-Throughput Conformational and Colloidal Stability Screening of Biologic Molecules

Abstract

A variety of biophysical techniques have been developed to characterize protein drug-like properties and assess aggregation propensity to support manufacturing unit operations and long-term storage. Conformational and colloidal mechanisms comprise the two principal pathways involved in the aggregation of proteins in solution. Either of these two pathways may dominate depending on individual protein molecule and specific formulation and stress conditions. For a high-throughput drug candidate selection and developability screening platform, it is important to assess both conformational and colloidal stability. In this study, the relationship between indicators of thermal unfolding (Tonset-DSF) and oligomer formation (Tonset-SLS or Tagg) obtained from DSF/SLS analysis and aggregation behavior at a range of concentrations were investigated and compared with aggregation results from short-term accelerated storage. Also investigated was the comparison of results between two instruments. We evaluated a randomly selected group of monoclonal antibodies (mAb) and bispecific antibodies (DVD-Ig). Both Tonset-Agg-SLS and Tonset-DSF can be determined simultaneously by differential scanning fluorimetry (DSF) coupled with static light scattering (SLS). It was revealed that the mAbs in this study with a low aggregation propensity (Δmonomer loss ≤5% at 40 °C for 21 days) display a Tonset- SLS, agg larger than 60 °C and a Tonset-DSF larger than 55 °C. Similarly, both Tonset and Tagg for bispecific antibodies with lower aggregation propensities were approximately greater than 55 °C. We found no relationship between aggregation and thermal parameters for mAbs. However, an inverse relationship between aggregation and both Tagg and Ton was evident for the DVD-Igs. Both the Abs and DVD-Igs in this study comprised sample sizes that were likely too small to identify significant relationships between parameters if any did exist. We propose displaying the data on an aggregation risk assessment diagram divided into quadrants according to severity. The boundaries are defined by rationally defined temperatures and aggregation levels informed by experimentation, prior knowledge and experience. DSF/SLS is capable of determining conformational and colloidal stability indicators simultaneously using a small amount of protein sample (~ 0.1 mg). In the early discovery stage with the limited materials, these two parameters potentially can be considered as useful indicators for high-throughput drug candidate selection and developability screening.