Date of Award

Spring 1-1-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Theodore W. Randolph

Second Advisor

Daniel K. Schwartz

Third Advisor

Joel L. Kaar

Fourth Advisor

John F. Carpenter

Fifth Advisor

John P. Gabrielson

Abstract

The synergic exposure to silicone oil-water interfaces and to agitation has been shown to promote the aggregation of therapeutic proteins. Silicone oil is typically used as a lubricant for manufacturing surfaces and pharmaceutical containers such as glass prefilled syringes. This is a problem as protein aggregates formed at the silicone oil-water interface may shed into bulk solutions, leading to product recalls or adverse immunogenicity upon injections into patients.

Protein aggregation in solution occurs as a result of attractive protein-protein interactions (PPI). PPI can be characterized using techniques such as dynamic light scattering which measures the dependence of molecular diffusivity on protein concentration. However, numerous factors unrelated to intermolecular forces can also impact protein diffusion. We therefore investigated the influence of multicomponent diffusion in a ternary protein-salt-water system on protein diffusion. This analysis demonstrated that large changes in protein diffusivity with protein concentration could result even in the absence of PPI. Therefore, careful interpretation is needed if protein diffusivity is used to measure PPI and colloidal stability of protein formulations.

PPI between proteins adsorbed at interfaces currently cannot be measured directly and the forces driving interface-induced aggregation are poorly understood. Therefore, we investigated the effects of PPI measured in solution on aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) at silicone oil–water interfaces. Attractive PPI measured in solution correlated with stronger interfacial aggregate gels while when PPI were decreased, weaker interfacial aggregate gels were formed. Upon mechanical perturbation in siliconized syringes, the strongest interfacial gels correlated with the most monomeric loss.

In addition, we suggested that interfacial aggregation could be reduced by decreasing attractive electrostatic PPI through the covalent addition of poly(ethylene)glycol (PEG) to rhIL-1ra. Monomeric loss in siliconized syringes was reduced upon PEGylation although PEGylation decreased protein’s conformational stability. PEGylation reduced rhIL-1ra’s net attractive PPI, causing the formation of weaker aggregate gels at the silicone oil-water interface. Therefore, attractive PPI measured in solution dominated the aggregation mechanism at the silicone oil-water interface for rhIL-1ra and those forces could be decreased upon PEGylation.

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