Christine Wurth

Excipient Effects on Humanized Monoclonal Antibody Interactions with Silicone oil Emulsions

The interfacial adsorption of three humanized monoclonal antibodies to emulsions of microdroplets of silicone oil was examined using indirect measurement via integrated peak areas in size-exclusion high-performance liquid chromatograms. The level of silicone oil far exceeded the typical levels used in prefillable syringes. The three antibodies rapidly adsorbed to silicone oil-water interfaces in various buffer formulations. Addition of 140 mM NaCl to solutions buffered with 10 mM l-histidine, pH 6.0, increased the amount of protein adsorbed. Conversely, the extent of adsorption was significantly decreased by the addition of 0.03% (w/v) Tween® 20. Stern-Volmer constants determined from intrinsic tryptophan fluorescence quenching by acrylamide suggested that the tertiary structure of the adsorbed antibodies was significantly perturbed. However, no aggregation or precipitation of the antibodies was detected. Flow cytometric analysis of emulsions of fluorescently stained silicone oil in solutions containing fluorescently labeled antibodies and light microscopy experiments suggested that agglomeration of silicone oil droplets in the emulsions occurred. Zeta potentials measured for silicone oil microdroplets with adsorbed antibodies suggested that droplet agglomeration was probably the result of reduced electrostatic energy barriers to droplet collisions.

Adsorption of monoclonal antibodies to glass microparticles

Microparticulate glass represents a potential contamination to protein formulations that may occur as a result of processing conditions or glass types. The effect of added microparticulate glass to formulations of three humanized antibodies was tested. Under the three formulation conditions tested, all three antibodies adsorbed irreversibly at near monolayer surface coverages to the glass microparticles. Analysis of the secondary structure of the adsorbed antibodies by infrared spectroscopy reveal only minor perturbations as a result of adsorption. Likewise, front-face fluorescence quenching measurements reflected minimal tertiary structural changes upon adsorption. In contrast to the minimal effects on protein structure, adsorption of protein to suspensions of glass microparticles induced significant colloidal destabilization and flocculation of the suspension.