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.
Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria. guenter.allmaier@tuwien.ac.at.
Marlene
M
Havlik
Martina
M
Marchetti-Deschmann
Gernot
G
Friedbacher
Paul
P
Messner
Wolfgang
W
Winkler
Laura
L
Perez-Burgos
Christa
C
Tauer
Günter
G
Allmaier
D002850:Chromatography, Gel; D004586:Electrophoresis / Q000379:methods; D004669:Encephalitis Viruses, Tick-Borne / Q000737:chemistry / Q000276:immunology*; D004675:Encephalitis, Tick-Borne / Q000276:immunology / Q000517:prevention & control / Q000821:virology*; D006801:Humans; D018625:Microscopy, Atomic Force; D046529:Microscopy, Electron, Transmission; D010316:Particle Size; D015164:Vaccines, Inactivated / Q000737:chemistry* / Q000276:immunology; D014765:Viral Vaccines / Q000737:chemistry* / Q000276:immunology; D014771:Virion / Q000737:chemistry* / Q000276:immunology / Q000648:ultrastructure
D015164:Vaccines, Inactivated; D014765:Viral Vaccines