In this study, we investigated the effects of protein-protein interaction strength on interfacial viscoelastic properties and aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) at the silicone oil-water interface. This interface is typically found in glass prefilled syringes which serve as both delivery devices and storage containers for therapeutic proteins and peptides. Protein adsorption to silicone oil-water interfaces contributes to the formation of protein aggregates that may be shed from interfaces into bulk solutions, leading to product recalls or adverse immunogenicity if particles are injected into patients. Osmotic second virial coefficients determined by static light scattering were used to quantify protein-protein interactions in bulk solution. Attractive protein-protein interactions dominated at low ionic strengths and their magnitude decreased with increasing ionic strength, in contrast to the repulsive interactions that would be expected based on electrical double layer (Gouy-Chapman) theory. Interfacial shear rheometry was used to characterize rhIL-1ra interfacial layers by measuring interfacial loss and storage moduli as a function of aging time. Notably, we found that more attractive protein-protein interactions in bulk solution correlated with stronger interfacial gels at the silicone oil-water interface. Thioflavin T fluorescence measurements indicated that the interfacial rhIL-1ra gels contained intermolecular β-sheet structures whose content correlated with gel strength. Siliconized syringes were used to probe the effects of mechanical perturbation of the interfacial gel layers. When rhIL-1ra solutions in siliconized glass syringes were subjected to end-over-end rotation, monomeric rhIL-1ra was lost from solution, and particles containing aggregated protein were released into the bulk aqueous phase. Importantly, the loss of monomeric rhIL-1ra in response to mechanical perturbation was highest under the conditions where the strongest gels were observed. Aggregation of rhIL-1ra was strictly interface-induced and growth of aggregates in the bulk solution was not observed, even in the presence of particles released from the silicone oil-water interface.

Protein-protein interactions control aggregation at silicone oil-water interfaces

255th National Meeting (2018)

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