Structural Characterization and Physicochemical Stability Profile of a Double Mutant Heat Labile Toxin Protein Based Adjuvant

Vishal M Toprani,John M Hickey,Neha Sahni,Ronald T Toth,George A Robertson,C Russell Middaugh,Sangeeta B Joshi,David B Volkin

doi:10.1016/j.xphs.2017.07.019

Abstract

A novel protein adjuvant double-mutant Escherichia coli heat-labile toxin, LT (R192G/L211A) or dmLT, is in preclinical and early clinical development with various vaccine candidates. Structural characterization and formulation development of dmLT will play a key role in its successful process development, scale-up/transfer, and commercial manufacturing. This work describes extensive analytical characterization of structural integrity and physicochemical stability profile of dmLT from a lyophilized clinical formulation. Reconstituted dmLT contained a heterogeneous mixture of intact holotoxin (AB5, ∼75%) and free B5 subunit (∼25%) as assessed by analytical ultracentrifugation and hydrophobic interaction chromatography. Intact mass spectrometry (MS) analysis revealed presence of Lys84 glycation near the native sugar-binding site in dmLT, and forced degradation studies using liquid chromatography-MS peptide mapping demonstrated specific Asn deamidation and Met oxidation sites. Using multiple biophysical measurements, dmLT was found most stable between pH 6.5 and 7.5 and at temperatures ≤50°C. In addition, soluble aggregates and particle formation were observed upon shaking stress. By identifying the physicochemical degradation pathways of dmLT using newly developed stability-indicating analytical methods from this study, we aim at developing more stable candidate formulations of dmLT that will minimize the formation of degradants and improve storage stability, as both a frozen bulk substance and eventually as a liquid final dosage form.

Highlights

There is a clinical need for new adjuvants to enhance and broaden mucosal immune responses for various vaccine candidates
Escherichia coli labile toxin (LT) exists as heterohexamer (AB5) complex consisting of a single enzymatically active A subunit which is noncovalently linked to a pentameric B subunit that is responsible for binding to the host's intestinal epithelial cells.[1,8]
Results of intact mass spectrometry (MS) analysis of dmLT samples exposed to 2 concentrations of hydrogen peroxide (0% and 1% v/v) produced 3 peaks corresponding to the dmLT A chain (27,645.5 Da), B chain (11,719.5 Da), and modified B chain (12,043.0 Da; Fig. 6a)

Summary

Introduction

There is a clinical need for new adjuvants to enhance and broaden mucosal immune responses for various vaccine candidates. Vaccines administered by the mucosal route have the potential to provide both mucosal and systemic immunity and result in logistical and regulatory advantages over traditional vaccines given by injection.[1,2,3,4] One of the reasons for the current limited availability of vaccines that activate mucosal immunity is the lack of effective mucosal adjuvants. Bacterial enterotoxins such as cholera toxin (CT) and heat-labile toxin (LT). Two mutations (R192G/L211A) were introduced into the A subunit of LT (dmLT) that minimize the molecule toxicity while retaining its ability to stimulate immunogenicity.[8,9]

Methods

Results

Conclusion