Comprehensive Size-Determination of Whole Virus Vaccine Particles Using Gas-Phase Electrophoretic Mobility Macromolecular Analyzer, Atomic Force Microscopy, and Transmission Electron Microscopy

Abstract

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.

nES GEMMA Analysis of Lectins and Their Interactions with Glycoproteins – Separation, Detection, and Sampling of Noncovalent Biospecific Complexes

“…We found that also the sampling of the SNA–A1AT complex onto the NC membrane showed a noticeable staining comparable to A1AT sample. Interestingly, no distinct spot in the size of the ENAS electrode (9.5 mm diameter) was found, as observed previously after collecting significantly larger particles [16]. In our case, the applied NC membrane was evenly stained, probably due to the fact that the ENAS voltage was not high enough to deviate the particles from their trajectory imposed by the high nDMA sheath flow and to focus them on a distinct area.…”

“…nES GEMMA also allows a size-selective collection of analytes after gas-phase separation for consecutive investigations like microscopic measurements (transmission electron microscopy, TEM; atomic-force microscopy, AFM) or a biological test as an immunologic assay [14–16]. For this purpose, the CPC is replaced by an electrostatic nanoparticle sampler (ENAS).…”

Nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA) of liposomes: applicability of the technique for nano vesicle batch control

“…100 nm). We concluded already in a previous study 30 that this is most likely caused by force application of the AFM tip or interaction of particles with the wafer surface, or a combination of both.…”

Virus-like particle size and molecular weight/mass determination applying gas-phase electrophoresis (native nES GEMMA)

“…A collapse of VLP particles during gas-phase electrophoresis upon stripping of solvent molecules from their interior seems highly unlikely as AFM and dot-blot analyses of a vaccine VLP demonstrated particle integrity after gas-phase electrophoresis [44]. Instead, due to protein analyte inherent M W limitations, especially a direct comparison of a protein correlation with an EMD/M W correlation for VLPs is to date not feasible.…”

“…Besides yielding information on analyte surface-dry particle size and size distribution, particle number concentration detection is possible in accordance with recommendations of the European Commission for characterization of nanoparticle material (2011/696/EU from October 18 th , 2011). Furthermore, analytes can be size-selected for further analyses employing orthogonal methods for instance electron microscopy [43], atomic force microscopy [44], spectroscopic techniques [45, 46], or antibody-based nanoparticle recognition [44, 47]. It is of note that LiquiScan ES, MacroIMS, and SMPS are synonyms of the same instrument found in literature.…”