Method qualification and application of diffusion interaction parameter and virial coefficient

摘要

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 (B_(22)),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 B_(22) 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 B_(22) in understanding protein interactions. Moreover,B_(22) 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~(-4)L mmol/g~2 in contrast to >30% when it approached 10~(-5) Lmmol/g~2. In this research,we also made an attempt to study protein-protein interactions in concentrated MAb fragment solutions (e.g. >50 mg/mL). Our data suggested that such interactions could be empirically modeled by high-order virial expansions.