Summary
Stability assays of protein vaccines formulated with alum adjuvants are not straightforward to perform because most standard analytical methods for protein characterization cannot be easily applied to proteins immobilized on alum.

In this stability assay, vaccine protein degradation products were eluted from the alum hydroxide adjuvant with SDS-PAGE sample buffer and separated by 1D SDS PAGE. The gels were stained with a sensitive silver staining method compatible with MS analysis. Degradation protein fragment bands were cut out from the gel and analyzed by in-gel trypsin digestion and MS peptide mapping. The protein sequence coverage map shows peptide maps obtained for the individual protein fragment bands in the gel. The peptide maps confirm that the proteins are drug substance degradation products. The sequence coverage maps show that bands 1 and 2 are protein fragments missing the C-terminal region, band 3 and 4 are also missing the N-terminal region, and band 5 and 6 found around 6 kDa in the gel contain only the middle part of the sequence.

Figure 4: Stability analysis of alum-formulated protein stored at 37 °C for nine months. The protein was eluted from the alum, and 10 ìg were analyzed by 1D SDS-PAGE. The break-down products (Bands 1–6) were cut out from the silver-stained gel together with intact protein (Band 0) and analyzed by MALDI MS peptide mapping. Peptide masses obtained from the degradation products were correlated to the protein sequence using GPMAW software from Lighthouse Data (www.gpmaw.com), and the results are shown in the protein sequence coverage map.

Reference:
Mortz, E. et al. “Proteomics technology applied to upstream and downstream process development of a protein vaccine”, Bioprocess International 6, p36-43, 2008.