Using LC-MS to Identify Clipping in Self-Assembled Nanoparticles During Vaccine Development

Area of Science:

• Vaccine development
• Biopharmaceutical analysis
• Protein chemistry

Background:

• A hemagglutinin stabilized stem nanoparticle (HA-SS-np) vaccine candidate was developed for broad influenza protection.
• Early formulation studies revealed conflicting stability data between reducing gel electrophoresis (rCGE) and size exclusion chromatography (SEC) assays.

Purpose of the Study:

• To investigate the conflicting stability results observed for the large HA-SS-np vaccine molecule.
• To elucidate the nature of the observed product degradation using advanced analytical techniques.

Main Methods:

• Utilized liquid chromatography-mass spectrometry (LC-MS) to analyze the integrity of the HA-SS-np vaccine.
• Compared LC-MS findings with results from rCGE and SEC assays.

Main Results:

• LC-MS identified unexpected sequence clipping within the HA-SS-np subunits, explaining the rCGE results.
• An intact disulfide bond was found to hold the clipped subunits together, explaining the unchanged SEC profile.
• Conflicting data from rCGE and SEC were rationalized by the subunit clipping and intact disulfide bond.

Conclusions:

• LC-MS is critical for analyzing large nanoparticle vaccine integrity and resolving conflicting assay results.
• The identified degradation pathway (subunit clipping) informed a successful redesign of the formulation buffer.
• This analytical approach enabled mitigation of the stability issue, supporting vaccine development.