1 research outputs found
Comprehensive Size-Determination of Whole Virus Vaccine Particles Using Gas-Phase Electrophoretic Mobility Macromolecular Analyzer, Atomic Force Microscopy, and Transmission Electron Microscopy
Biophysical properties including
particle size distribution, integrity,
and shape of whole virus vaccine particles at different stages in
tick-borne encephalitis (TBE) vaccines formulation were analyzed by
a new set of methods. Size-exclusion chromatography (SEC) was used
as a conservative sample preparation for vaccine particle fractionation
and gas-phase electrophoretic mobility macromolecular analyzer (GEMMA)
for analyzing electrophoretic mobility diameters of isolated TBE virions.
The derived particle diameter was then correlated with molecular weight.
The diameter of the TBE virions determined after SEC by GEMMA instrumentation
was 46.8 ± 1.1 nm. Atomic force microscopy (AFM) and transmission
electron microscopy (TEM) were implemented for comparison purposes
and to gain morphological information on the virion particle. Western
blotting (Dot Blot) as an immunological method confirmed biological
activity of the particles at various stages of the developed analytical
strategy. AFM and TEM measurements revealed higher diameters with
much higher SD for a limited number of virions, 60.4 ± 8.5 and
53.5 ± 5.3 nm, respectively. GEMMA instrumentation was also used
for fractionation of virions with specifically selected diameters
in the gas-phase, which were finally collected by means of an electrostatic
sampler. At that point (i.e., after particle collection), AFM and
TEM showed that the sampled virions were still intact, exhibiting
a narrow size distribution (i.e., 59.8 ± 7.8 nm for AFM and 47.5
± 5.2 nm for TEM images), and most importantly, dot blotting
confirmed immunological activity of the collected samples. Furthermore
dimers and virion artifacts were detected, too