Date of Award

Spring 1-1-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemical & Biochemical Engineering

First Advisor

Theodore W. Randolph

Second Advisor

John F. Carpenter

Third Advisor

Raul M Torres

Fourth Advisor

Jack A. Ragheb

Fifth Advisor

Kristi S. Anseth

Abstract

Subvisible particles in therapeutic protein products may act as adjuvants to provoke unwanted immune responses against administered proteins. Silicone oil is used as a lubricant in prefilled syringes, and microdroplets of silicone oil are frequently detected in protein formulations expelled from prefilled syringes. In this work, we investigated the potential of silicone oil microdroplets to act as an adjuvant to elicit immune responses against formulations containing foreign or self-protein in vivo.

First, we tested the immunogenicity of formulations that contained a foreign protein and silicone oil microdroplets. A foreign model protein, ovalbumin (OVA), was added to emulsions of silicone oil microdroplets. Antibody responses in mice to subcutaneous injections of protein formulations that contained silicone oil microdroplets were measured using enzyme-linked immunosorbent assays. These responses were compared against responses to oil-free protein formulations and to protein formulations that contained microparticulate aluminum hydroxide (alum), the common vaccine adjuvant. When administered with silicone oil microdroplets, OVA formulations elicited robust anti-OVA IgG1 and IgG2a antibody responses in mice relative to oil-free OVA formulations. These responses were equivalent to those observed when alum microparticles were added to OVA formulations, indicating that silicone oil can act as a potent adjuvant against proteins containing foreign epitopes.

Mice were also injected with similar formulations wherein recombinant murine growth hormone (rmGH), a recombinant self-protein, was substituted in place of OVA. In this study, we found that immunological tolerance was broken in mice when formulations of rmGH that contained silicone oil microdroplets were administered daily. Thus, silicone oil microdroplets can promote a breakage in immunological tolerance and induce antibody responses against recombinant self-proteins.

Furthermore, we investigated the capacity of silicone oil microdroplets to induce antibody responses against a model protein known to form agglomerates and undergo structural perturbations in the presence of silicone oil microdroplets, hen egg lysozyme (HEL). Silicone oil microdroplets elicited robust antibody responses against HEL in non-transgenic mice where HEL was recognized as foreign, but silicone oil microdroplets did not promote a break in tolerance in immune tolerant transgenic mice. Thus, structural perturbations in proteins adsorbed to silicone oil microdroplets may not increase the immunogenicity of proteins.