Assessing stability of protein drug substance & formulated drug product

Oct 21. 2013

After protein purification, an important issue is the stability of a protein drug substance and formulated drug product. 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.

Alphalyse performed a stability assay on a protein vaccine:

  • Vaccine vials were incubated at the normal storage temperature at 4 °C and at 37 °C in an accelerated stability study.
  • Samples were taken at different storage intervals for stability measurements.
  • For analysis of protein degradation products, the protein was eluted from the alum hydroxide by treatment with SDS-PAGE sample buffer.
  • The samples were analyzed by electrophoresis, and the gels were stained with a sensitive silver staining method compatible with MS analysis.

The figure below shows a silver-stained gel of the drug product stored for nine months at 37 °C in the accelerated stability study. Degradation protein fragment bands were cut out from the silver-stained 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 fragments. Those 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. Thus, the combination of SDS-PAGE and MS peptide mapping again provided very valuable information (about protein degradation patterns, in this case) that is not easily obtained by other analytical techniques.

Stability analysis of alum formulated protein stored at 37oC for 9 months.

Stability analysis of alum formulated protein stored at 37oC for 9 months.

Figure: Stability analysis of alum-formulated protein stored at 37 °C for nine months. The protein was eluted from the alum, and 10 μg 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 (, and the results are shown in the protein sequence coverage map.

More about: Protein Stability Assays.

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