Systematic Development and Validation of a Bradford-Based Protein Quantification Method for Novel Multi-Dose R21...

Area of Science:

• Vaccinology
• Protein Chemistry
• Analytical Chemistry

Background:

• The R21 malaria vaccine, a next-generation tool, requires precise protein quantification for quality control, especially in complex formulations.
• Accurate quantification is challenging due to low antigen concentrations and the presence of adjuvants and preservatives.
• Multi-dose vaccine presentations are preferred for large-scale immunization programs like the Expanded Programme on Immunization (EPI).

Purpose of the Study:

• To evaluate and select a suitable protein estimation method for quantifying the R21 malaria vaccine antigen.
• To optimize and validate the chosen protein quantification method according to international guidelines.
• To ensure the reliability of quality control for R21 vaccine manufacturing and lot release.

Main Methods:

• Comparative evaluation of Micro-BCA, BCA, and Bradford assays for R21 antigen quantification.
• Selection of the Bradford assay based on precision, accuracy, and linearity.
• Method optimization, validation per ICH Q2(R2) guidelines, and assessment of specificity, accuracy, and precision.

Main Results:

• The optimized Bradford assay demonstrated high specificity, accuracy, and precision, with a limit of quantitation (LOQ) of 2 µg/mL.
• The assay showed sensitivity to detect antigen concentration changes and comparable performance to ELISA.
• Quantified protein content correlated with robust in vivo immunogenicity in release and stability studies.

Conclusions:

• The validated Bradford assay provides a rapid, cost-effective, and high-throughput solution for R21 vaccine quality control.
• This method sets a new benchmark for quality control of complex virus-like particle (VLP)-based vaccines.
• The assay's robustness supports the precision and reliability needed for global malaria prevention initiatives.