Abstract

Type 1 Diabetes (T1D) is characterized by autoreactive immune responses to insulin and its precursor, proinsulin. Soluble antigen arrays (SAgAs) displaying the metabolically inactive proinsulin variant, proinsulin(F25D), represent a promising antigen‑specific immunotherapy candidate for T1D. However, the production of these platforms is hindered by substantial practical challenges, including protein aggregation, inefficient refolding, and the lack of sensitive analytical tools to verify quality and dosage. This work establishes an integrated, scalable pipeline for the purification, conjugation, and quantification of a proinsulin(F25D) SAgA. First, we optimized the refolding of recombinant methionine-proinsulin(F25D) (M-PI(F25D)) from inclusion bodies using controlled buffer systems and created a robust chromatography capture strategy which significantly improved process reproducibility and yield. Second, we optimized the conjugation of M-PI(F25D) to hyaluronic acid (HA) via reductive amination and mitigated oxidative degradation to preserve antigenicity. Finally, we developed and validated a sandwich enzyme‑linked immunosorbent assay (ELISA) capable of selectively quantifying M-PI(F25D) within buffer and complex biological matrices.

Collectively, this workflow provides a reproducible biochemical and analytical foundation to produce proinsulin‑based therapeutics for T1D. By integrating refined refolding and purification steps with optimized conjugation and quantification, this study addresses critical bottlenecks in protein processing and supports the progression of this engineered antigen array toward preclinical investigation.

Committee Chair

Cory Berkland

Committee Members

Stephen Sykes, Michael Vahey

Degree

Master of Science (MS)

Author's Department

Biomedical Engineering

Author's School

McKelvey School of Engineering

Document Type

Thesis

Date of Award

Spring 5-6-2026

Language

English (en)

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