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.

Analysis of a common cold virus and its subviral particles by gas-phase electrophoretic mobility molecular analysis and native mass spectrometry.

Gas-phase electrophoretic mobility molecular analysis (GEMMA) separates nanometer-sized, single-charged particles according to their electrophoretic mobility (EM) diameter after transition to the gas-phase via a nano electrospray process. Electrospraying as a soft desorption/ionization technique preserves noncovalent biospecific interactions. GEMMA is therefore well suited for the analysis of intact viruses and subviral particles targeting questions related to particle size, bioaffinity, and purity of preparations. By correlating the EM diameter to the molecular mass (Mr) of standards, the Mr of analytes can be determined. Here, we demonstrate (i) the use of GEMMA in purity assessment of a preparation of a common cold virus (human rhinovirus serotype 2, HRV-A2) and (ii) the analysis of subviral HRV-A2 particles derived from such a preparation. (iii) Likewise, native mass spectrometry was employed to obtain spectra of intact HRV-A2 virions and empty viral capsids (B-particles). Charge state resolution for the latter allowed its Mr determination. (iv) Cumulatively, the data measured and published earlier were used to establish a correlation between the Mr and EM diameter for a range of globular proteins and the intact virions. Although a good correlation resulted from this analysis, we noticed a discrepancy especially for the empty and subviral particles. This demonstrates the influence of genome encapsulation (preventing analytes from shrinking upon transition into the gas-phase) on the measured analyte EM diameter. To conclude, GEMMA is useful for the determination of the Mr of intact viruses but needs to be employed with caution when subviral particles or even empty viral capsids are targeted. The latter could be analyzed by native MS.

Development of a bio-analytical strategy for characterization of vaccine particles combining SEC and nanoES GEMMA.

Commonly used methods for size and shape analysis of bionanoparticles found in vaccines like X-ray crystallography and cryo-electron microscopy are very time-consuming and cost-intensive. The nano-electrospray (nanoES) gas-phase electrophoretic mobility macromolecular analyzer (GEMMA), belonging to the group of ion mobility spectrometers, was used for size determination of vaccine virus particles because it requires less analysis time and investment (no vacuum system). Size exclusion chromatography (SEC) of viral vaccines and production intermediates turned out to be a good purification/isolation method prior to GEMMA, TEM (transmission electron microscopy) and AFM (atomic force microscopy) investigations, as well as providing a GEMMA analysis-compatible buffer. Column materials and different elution buffers were tested for optimal vaccine particle yield. We used a Superdex 200 column with a 50 mM ammonium acetate buffer. In addition, SEC allowed the removal of process-related impurities from the virions of interest. A sample concentrating step or a detergent addition step was also investigated. As a final step of our strategy SEC-purified or untreated vaccine-nanoparticles were further analyzed: (a) by immunological detection with a specific polyclonal antibody (dot blot) to verify the biological functionality, (b) by GEMMA to provide the size of the particles at atmospheric pressure and (c) by AFM and (d) TEM to obtain both size and shape information. The mean diameter of inactivated tick-borne encephalitis virions (i.e. vaccine particles) determined by GEMMA measurement was 46.6 ± 0.5 nm, in contrast to AFM and TEM images providing diameters of about 58 ± 4 and 52 ± 5 nm, respectively.

Sizing up large protein complexes by electrospray ionisation-based electrophoretic mobility and native mass spectrometry: morphology selective binding of Fabs to hepatitis B virus capsids.

The capsid of hepatitis B virus (HBV) is a major viral antigen and important diagnostic indicator. HBV capsids have prominent protrusions ('spikes') on their surface and are unique in having either T = 3 or T = 4 icosahedral symmetry. Mouse monoclonal and also human polyclonal antibodies bind either near the spike apices (historically the 'α-determinant') or in the 'floor' regions between them (the 'ß-determinant'). Native mass spectrometry (MS) and gas-phase electrophoretic mobility molecular analysis (GEMMA) were used to monitor the titration of HBV capsids with the antigen-binding domain (Fab) of mAb 3120, which has long defined the ß-determinant. Both methods readily distinguished Fab binding to the two capsid morphologies and could provide accurate masses and dimensions for these large immune complexes, which range up to ~8 MDa. As such, native MS and GEMMA provide valuable alternatives to a more time-consuming cryo-electron microscopy analysis for preliminary characterisation of virus-antibody complexes.