Method qualification and application of diffusion interaction parameter and virial coefficient

This research focused on evaluation and application of two methods in studying weak protein-protein interactions, i.e. diffusion interaction parameter (K_D) and second virial coefficient (B₂₂), both of which are first-order coefficients of protein interactions. Although the plate-based K_D method successfully distinguished K_D values with relatively large difference in a pH ranging study, it failed to make a consistent statistical decision to determine close interactions as shown by the comprehensive ANOVA analysis. We also validated the DLS-based B₂₂ method by using a model protein lysozyme. The dramatic change of solution appearance for lysozyme as a function of NaCl concentration highlighted the importance of B₂₂ in understanding protein interactions. Moreover, B₂₂ measurement for a MAb fragment suggested a more repulsive protein interaction in histidine buffer than in citrate buffer. The coefficient of variation was <10% when B₂₂ was on an order of magnitude of 10^-4Lmmol/g² in contrast to >30% when it approached 10^-5Lmmol/g². In this research, we also made an attempt to study protein-protein interactions in concentrated MAb fragment solutions (e.g. >50mg/mL). Our data suggested that such interactions could be empirically modeled by high-order virial expansions.