Molecular Origins of Surfactant Stabilization of a Human Recombinant Factor VIII (rFVIII) J. Dill, K. Tadehara, F. von Flotow - Chemical, Biological and Environmental Engineering Introduction Acknowledgements Zeta Potential Sample Preparation Bare microspheres had zeta potential readings of -30 mV FVIII treated microspheres had readings of -25 mV Indicates the masking of surface charge due to protein adsorption Bare spheres treated with varying concentrations of F68 had zeta potentials of -32 mV to -40 mV, similar to bare spheres F68 did not appear to adsorb to the sphere surface at significant levels Microspheres with rFVIII and varying concentrations of F68 had zeta potential values similar to bare spheres and F68 spheres (-33 mV) Indicates that adsorption is blocked due to coating of the proteins and not the spheres’ surface Conclusions Figure 1: rFVIII Hemophilia A results in reduced Factor VIII (FVIII) protein activity Leads to insufficient blood clotting Current treatments use FVIII injections and cost $60K to $150K annually Figure 2: Current FVIII product Zeta potential is a measure of particle surface charge Higher magnitude = higher charge density and greater repulsion A zeta potentiometer is a machine that measures zeta potential Uses probe with a positive and negative electrode Experiments Proposed Solution Investigate stabilization properties of the surfactant Pluronic© F68 Develop superior processing and storage methods of FVIII Figure 3: F68 structure Our investigation utilizes one micron silica microspheres with uniform negative surface charge under four treatments outlined below. Controls Treatment Clean surface No adsorbed molecules on spheres Unmasked surface charge Presence of surfactant Formation of F68 aggregates Minimal adsorption to spheres Largely unmasked surface charge rFVIII adsorbed to spheres Masked surface charge Presence of rFVIII and F68 Prevention of rFVIII adsorption by surfactant F68 Largely unmasked surface charge Figure 4: Incubation step uses a slow rotation to provide consistent and thorough mixing and ample residence time for interactions. Size of FVIII makes it unstable, form aggregates and adsorb Leads to process and transportation difficulty Current stabilization methods are expensive Figure 5: Diagram of operational concept of a zeta potentiometer (L) and zeta potentiometer probe (R) Untreated microspheres have similar zeta potential to microspheres treated with F68 Microspheres treated with FVIII have masked zeta potential due to adsorption Microspheres treated with F68 and FVIII show no FVIII adsorption Zeta Potential Results Figure 6: Average zeta potential readings of microspheres with different treatments. Examine F68 interactions in solution Quantify effects of high F68 concentrations on zeta potential Reduce costs of protein pharmaceutical processing Develop ready-to-inject formulations Silica microspheres were used to model a solid-water interface for FVIII binding. The sample procedure is shown below. Future Work Bayer Healthcare Pharmaceuticals for providing rFVIII Dr. Joe McGuire for sponsoring the project and advice Dr. Michelle Bothwell for experimental insights Dr. Karl “Rat” Schilke for lab assistance Dylan Stankus for zeta potential education Dr. Phil Harding for the opportunity