Investigation of Factors Affecting Opalescence and Phase Separation in Protein Solutions
Opalescence and liquid liquid phase separation reduce physical stability of a therapeutic protein formulation and have been recently reported for high concentration monoclonal antibody solutions. Liquid liquid phase separation resulting in opalescence can be attributed to attractive intermolecular interactions; formulation factors that mitigate these interactions reduce solution opalescence and tendency to phase separate. Protein protein interactions in solution have also been reported to enhance reversible/irreversible aggregation and viscosity of the solution. These formulation challenges are further enhanced for the second generation of antibodies due to the increased complexity of the molecules. Current work focuses on understanding the formulation factors and nature of intermolecular interactions that result in opalescence and liquid liquid phase separation for monoclonal antibody (mAb) and Dual variable domain immunoglobulin antibody (DVD Ig TM protein solutions. Opalescence for protein solution was measured as a function of formulation factors and correlated to interactions in dilute and concentrated solutions. Results show that presence of attractive interactions (enthalpy effect) and strong temperature dependence (entropy effect) result in liquid liquid phase separation in solution. On undergoing phase separation, solutions attain thermodynamic equilibrium, and protein does not exhibit structural changes. Other than the traditional non specific interactions, the presence of Ashlesha S. Raut, University of Connecticut, 2015 specific protein interactions also affects phase separation and was investigated for DVD Ig TM protein. Determining the nature of these attractive protein protein interactions is of utmost significance so as to facilitate the selection of appropriate excipients. The effect of different excipients to suppress phase separation and irreversible aggregation in solution was also studied. The mechanism by which these excipients affect aggregation and liquid liquid phase separation in solution is different, as both these phenomena show a strong and opposite temperature dependence. The effect of size of the molecule with respect to intrinsic viscosity and excluded volume effect, electroviscous effect and interactions, on the viscous behavior of DVD Ig TM protein was investigated. Interplay of more than one factor contributes to significantly high viscosity of DVD Ig TM protein compared to mAb solutions. The overall results demonstrated that opalescence and liquid liquid phase separation are a concern in formulation development of therapeutic protein solutions. Excipients and solution conditions (pH, ionic strength, concentration and temperature) that reduce noncovalent interactions in solution provide physically stable products. Investigation of Factors Affecting Opalescence and Phase Separation in Protein Solutions Ashlesha Shirish Raut M.Pharm. (Pharmaceutics) Institute of Chemical Technology, 2008 B. Pharm. Mumbai University, 2006 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2015