Protein–protein interactions controlling interfacial aggregation of rhIL‐1ra are not described by simple colloid models

Abstract

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 silicone oil-water interfaces. 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 repulsive interactions that would be expected based on uniformly charged sphere models. Interfacial shear rheometry was used to characterize rhIL-1ra interfacial layers. More attractive protein-protein interactions in bulk solution correlated with stronger interfacial gels. Thioflavin-T fluorescence measurements indicated that the intermolecular β-sheet content of rhIL-1ra incubated in the presence of silicone oil-water interfaces 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. 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 silicone oil-water interfaces.

Steric Repulsion Forces Contributed by PEGylation of Interleukin-1 Receptor Antagonist Reduce Gelation and Aggregation at the Silicone Oil-Water Interface

“…Some syringes used as controls were not re-siliconized. The procedures for the removal of the silicone oil coating as well as for the re-siliconization steps were previously described in Sorret et al 1 4 mm diameter borosilicate glass beads (Sigma-Aldrich) were also cleaned, and half of those were siliconized using the same method as the siliconization of the syringes. Two beads, appropriately siliconized or un-siliconized, were added to each siliconized or un-siliconized syringe, respectively, prior to filing with protein solution to provide the capability for mixing in the syringes without introducing confounding air-water interfaces.…”

“…1% w/v silicone oil emulsions (SOE) were prepared in water as previously described. 1, 37 To decrease the silicone oil droplet size, the pressure of the emulsifier was gradually increased to 7,000 psi for a total of 7 passes at that final pressure. The SOE was then stored for a minimum of 4 days at 4 °C prior to use and was assumed to be stable for 3 weeks, as determined previously.…”

“…Adsorption of proteins to interfaces may cause structural changes that can lead to interfacial aggregation and gel formation. 1–5 Interfacial protein gel layers are extensive 3D elastic networks that are stabilized by protein-protein intermolecular interactions, such as hydrogen bonding, that can result in the formation of non-native intermolecular β-sheet structures. 6, 7 Mechanical disturbances at interfaces (e.g., generated by fluid shear exerted when protein solutions are pumped or expelled from syringes, interfacial contraction and dilation caused by agitation of interfaces, or by the motion of bubbles within containers and pre-filled syringes) may cause gelled protein aggregates to detach and be transported into bulk solutions.…”

“…6, 7 Mechanical disturbances at interfaces (e.g., generated by fluid shear exerted when protein solutions are pumped or expelled from syringes, interfacial contraction and dilation caused by agitation of interfaces, or by the motion of bubbles within containers and pre-filled syringes) may cause gelled protein aggregates to detach and be transported into bulk solutions. 1, 8–15 Such protein aggregates may elicit harmful immune responses after injection into patients. 16–18 Furthermore, protein aggregates can become large enough to form insoluble particles which may impact product quality and efficacy, making the product unacceptable for clinical use.…”

“…We showed previously that protein aggregation at the silicone oil-water interface could be decreased by reducing attractive electrostatic forces between recombinant human interleukin-1 receptor antagonist (rhIL-1ra) molecules. 1 Here, we hypothesize that rhIL-1ra interfacial aggregation can also be reduced by introducing repulsive steric interactions between protein molecules through the covalent addition of a 20 kDa PEG polymer to rhIL-1ra. Since PEGylation may affect rhIL-1ra structural integrity, we also examined the role of conformational stability in the interfacial aggregation of rhIL-1ra and PEG rhIL-1ra.…”