Real-Time Single-Aggregate Analysis of Vaccine Antigen Stability via Stochastic Electrochemical Blocking

Protein aggregation remains a major challenge in vaccine stability, particularly for protein antigens that undergo structural perturbation during storage, reconstitution, and transport. Here, we apply stochastic electrochemical blocking to observe aggregation of the glycoprotein E (gE) antigen from the SHINGRIX™ vaccine under different formulation and temperature conditions. This orthogonal technique to more traditional light-based methods enables rapid, single-particle, qualitative measurements in solution without introducing shear forces or nonnative interfaces. We examined gE aggregates after reconstitution over a two-week period via additions of MilliQ water, the buffer matrix of the liposome-based adjuvant AS01_B (sans the liposome itself), and the complete AS01_B liposome adjuvant system. Day 0 measurements revealed formulation-dependent aggregation, with the AS01_B Buffer addition promoting the largest aggregates, water addition producing intermediate aggregation, and AS01_B itself minimizing aggregation. Time trial experiments showed that refrigeration (4 °C) consistently reduced aggregate size, whereas 37 °C accelerated aggregation until particles became too large to reach the electrode. Overall, stochastic electrochemical blocking provides a rapid, tangential method for monitoring protein aggregation in vaccine formulations and offers actionable insights into optimizing storage and reconstitution conditions.