Dynamic light scattering can be used to measure the diffusivity of a protein within a formulation. The dependence of molecular diffusivity on protein concentration (traditionally expressed in terms of the interaction parameter k) is often used to infer whether protein-protein interactions are repulsive or attractive, resulting in solutions that are colloidally stable or unstable, respectively. However, a number of factors unrelated to intermolecular forces can also impact protein diffusion, complicating this interpretation. Here, we investigate the influence of multicomponent diffusion in a ternary protein-salt-water system on protein diffusion and k in the context of Nernst-Planck theory. This analysis demonstrates that large changes in protein diffusivity with protein concentration can result even for hard-sphere systems in the absence of protein-protein interactions. In addition, we show that dynamic light scattering measurements of diffusivity made at low ionic strength cannot be reliably used to detect protein conformational changes. We recommend comparing experimentally determined k values to theoretically predicted excluded-volume contributions, which will allow a more accurate assessment of protein-protein interactions.

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.

Silicone oil, used as a lubricating coating in pharmaceutical containers, has been implicated as a cause of therapeutic protein aggregation. After adsorbing to silicone oil-water interfaces, proteins may form interfacial gels, which can be transported into solution as insoluble aggregates if the interfaces are perturbed. Mechanical interfacial perturbation of both monomeric recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and PEGylated rhIL-1ra (PEG rhIL-1ra) in siliconized syringes resulted in losses of soluble monomeric protein. However, the loss of rhIL-1ra was twice that for PEG rhIL-1ra, even though in solution, PEG rhIL-1ra had a lower ΔGunf and exhibited a more perturbed tertiary structure at the interface. Net proteinprotein interactions in solution for rhIL-1ra were attractive, but increased steric repulsion due to PEGylation led to net repulsive interactions for PEG rhIL-1ra. Attractive interactions for rhIL-1ra were associated with increases in intermolecular β-sheet content at the interface, whereas no intermolecular β-sheet structures were observed for adsorbed PEG rhIL-1ra. rhIL-1ra formed interfacial gels that were five times stronger than those formed by PEG rhIL-1ra. Thus, the steric repulsion contributed by the PEGylation resulted in decreased interfacial gelation and in the reduction of aggregation, in spite of the destabilizing effects of PEGylation on the protein’s conformational stability.