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.
Institute of Chemical Technologies and Analytics, Vienna University of Technology , A-1060 Vienna, Austria.
Marlene
M
Havlik
Institute of Chemical Technologies and Analytics, Vienna University of Technology , A-1060 Vienna, Austria.
Martina
M
Marchetti-Deschmann
Institute of Chemical Technologies and Analytics, Vienna University of Technology , A-1060 Vienna, Austria.
Gernot
G
Friedbacher
Institute of Chemical Technologies and Analytics, Vienna University of Technology , A-1060 Vienna, Austria.
Wolfgang
W
Winkler
Department of Nanobiotechnology, University of Natural Resources and Life Sciences , A-1180 Vienna, Austria.
Paul
P
Messner
Baxalta Innovations, A-2304 Orth/Donau, Austria.
Laura
L
Perez-Burgos
Baxalta Innovations, A-2304 Orth/Donau, Austria.
Christa
C
Tauer
Institute of Chemical Technologies and Analytics, Vienna University of Technology , A-1060 Vienna, Austria.
Günter
G
Allmaier
D002850:Chromatography, Gel; D004586:Electrophoresis; D004669:Encephalitis Viruses, Tick-Borne / Q000648:ultrastructure*; D046911:Macromolecular Substances / Q000737:chemistry; D018625:Microscopy, Atomic Force; D010316:Particle Size; D014771:Virion / Q000648:ultrastructure*; D014773:Virology / Q000379:methods*
D046911:Macromolecular Substances