Comparison of sample preparation techniques for the physicochemical characterization of Orf virus particles

• Published in: Journal of virological methods Vol. 310; p. 114614
• Main Authors: Eilts, Friederike, Steger, Marleen, Pagallies, Felix, Rziha, Hanns-Joachim, Hardt, Martin, Amann, Ralf, Wolff, Michael W.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-12-2022
• Subjects: Isoelectric point; Orf virus; Sucrose; Surface charge; Transmission electron microscopy; Ultracentrifugation; Viral vector vaccine; Virion; Viruses; Zeta potential; PBS; DF; Surface charge; Viral vector vaccine; SXC-DF; DLS; Orf virus; AM; IU; Elu; SC; MWCO; PEG; Transmission electron microscopy; CPB; PDI; Isoelectric point; Zeta potential; ORFV; UAc; pI; TEM; SXC
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2022.114614

The determination of the electrostatic charge of biological nanoparticles requires a purified, mono-disperse, and concentrated sample. Previous studies proofed an impact of the preparation protocol on the stability and electro-hydrodynamics of viruses, whereas commonly used methods are often complex and do not allow the required sample throughput. In the present study, the application of the (I) steric exclusion chromatography (SXC) for the Orf virus (ORFV) purification and subsequent physicochemical characterization was evaluated and compared to (II) SXC followed by centrifugal diafiltration and (III) sucrose cushion ultracentrifugation. The three methods were characterized in terms of protein removal, size distribution, infectious virus recovery, visual appearance, and electrophoretic mobility as a function of pH. All preparation techniques achieved a protein removal of more than 99 %, and (I) an infectious ORFV recovery of more than 85 %. Monodisperse samples were realized by (I) and (III). In summary, ORFV samples prepared by (I) and (III) displayed comparable quality. Additionally, (I) offered the shortest operation time and easy application. Based on the obtained data, the three procedures were ranked according to eight criteria of possible practical relevance, which delineate the potential of SXC as virus preparation method for physicochemical analysis.
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•Evaluation of three rapid virus sample preparation methods.•Virus preparation methods impact charge and size measurements.•Diafiltration introduces Orf virus aggregates and reduces virus recovery.•Ultracentrifugation is best suited for high virus titers.•Virus charge characterization after steric exclusion chromatography possible.