A bivalent recombinant vaccine for human hookworm disease is under development. One of the lead candidate antigens in the vaccine is a glutathione S-transferase cloned from the hookworm Necator americanus (Na-GST-1) which is expressed in the yeast Pichia pastoris. Based on preliminary studies demonstrating that the recombinant protein was not stable in an acetate buffer at pH 6, we undertook an extensive stability analysis of the molecule. To improve and optimize stability we complemented traditional methods employed for macromolecule and vaccine stabilization with biophysical techniques that were incorporated into a systematic process based on an eigenvector approach. Large data sets, obtained from a variety of experimental methods were used to establish a color map ("empirical phase diagram") of the physical stability of the vaccine antigen over a wide range of temperature and pH. The resulting map defined "apparent phase boundaries" that were used to develop high throughput screening assays. These assays were then employed to identify excipients that stabilized the antigen against physical degradation that could otherwise result in losses of physicochemical integrity, immunogenicity, and potency of the vaccine. Based on these evaluations, the recombinant Na-GST-1 antigen was reformulated and ultimately produced under Good Manufacturing Practices and with an acceptable stability profile.