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

The Analyst (Impact Factor: 4.11). 01/2014; 139(1):1412-1419. DOI: 10.1039/c3an01962d
Source: PubMed

ABSTRACT 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.

Download full-text


Available from: Guenter Allmaier, Jul 25, 2014
42 Reads
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Nanoparticle characterization is gaining importance in food technology, biotechnology, medicine, and pharmaceutical industry. An instrument to determine particle electrophoretic mobility (EM) diameters in the single-digit to double-digit nanometer range receiving increased attention is the gas-phase electrophoretic mobility molecular analyzer (GEMMA) separating electrophoretically single charged analytes in the gas-phase at ambient pressure. A fused-silica capillary is used for analyte transfer to the gas-phase by means of a nano electrospray (ES) unit. The potential of this capillary to separate analytes electrophoretically in the liquid phase due to different mobilities is, at measurement conditions recommended by the manufacturer, eliminated due to elevated pressure applied for sample introduction. Measurements are carried out upon constant feeding of analytes to the system. Under these conditions, aggregate formation is observed for samples including high amounts of non-volatile components or complex samples. This makes the EM determination of individual species sometimes difficult, if not impossible. With the current study we demonstrate that liquid phase electrophoretic separation of proteins (as exemplary analytes) occurs in the capillary (capillary zone electrophoresis, CE) of the nano ES unit of the GEMMA. This finding was consecutively applied for on-line desalting allowing EM diameter determination of analytes despite a high salt concentration within samples. The present study is to our knowledge the first report on the use of the GEMMA to determine EM diameters of analytes solubilized in the ES incompatible electrolyte solutions by the intended use of electrophoresis (in the liquid phase) during sample delivery. Results demonstrate the proof of concept of such an approach and additionally illustrate the high potential of a future on-line coupling of a capillary electrophoresis to a GEMMA instrument.
    Analytica Chimica Acta 09/2014; accepted(100). DOI:10.1016/j.aca.2014.05.043 · 4.51 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 soft desorption/ionization technique preserves non-covalent 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, virus capsids 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.
    Analytical Chemistry 07/2015; 87(17). DOI:10.1021/acs.analchem.5b01450 · 5.64 Impact Factor