The grids were imaged in a TEM under cryogenic conditions and at 120kV. Sample grids were prepared by pipetting a few µL of sample solution onto a holey carbon copper grid and plunge-freezing in liquid ethane. All samples were column purified after primary Ab treatment and again after secondary Ab treatment to remove unbound Ab’s. Secondary Ab’s recognizing the spike protein or the endogenous protein and conjugated to 6 nm or 10 nm gold nanoparticles were used to detect bound primary Ab’s. For the controls, the wildtype (WT) virus was incubated with the anti-S or the anti-HA primary Ab. Vaccine material B (VM-B) with a spike (S) protein gene inserted into the viral genome was incubated with a primary antibody (Ab) that binds the spike protein (anti-S), or two primary Ab’s that bind the spike protein and an endogenous surface protein (anti-HA) of the virus. Methods: Vaccine material A (VM-A) harvested from a cell line was digested with an enzyme to remove cellular materials, column purified (CP), ultrafiltered, and concentrated with a default pump (UFP-1) or a low shear pump (UFP-2). In a second scenario, cryoTEM combined with immunogold staining helped to verify if a surface glycoprotein inserted into the viral genome was present on the surfaces of viral particles.
In one scenario, cryoTEM results were used to guide vaccine process development and improvement. Results are reported for two vaccine materials. We have applied cryo transmission electron microscopy (cryoTEM) combined with immunogold staining 1 to answer these questions. However, these methodologies do not offer information on protein distribution on or within viral particles.
The identity and relative amounts of different proteins in a vaccine material can be determined by Western blotting and mass spectrometry. While information about the size of the viral particles can be obtained from flow cytometry, dynamic light scattering, and microflow imaging, these techniques do not provide structural understanding at the nanometer scale of critical features of viral particles such as surface proteins affecting biodistribution, replication in vivo, and overall immune responses. Purpose: The increasing complexity of vaccine materials necessitates characterization using a multitude of analytical methodologies to obtain sufficient product understanding.
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