Hydrodynamic characterization of a vesicular stomatitis virus-based oncolytic virus using analytical ultracentrifugation

Abstract

Determination of the size, density, and mass of viral particles can provide valuable information to support process and formulation studies in clinical development. Analytical ultracentrifugation (AUC), as a first principal method, has been shown to be a beneficial tool for the characterization of the non-enveloped adeno associated virus (AAV). Here, we demonstrate the suitability of AUC for the challenging characterization of a representative for enveloped viruses, which usually are expected to exhibit higher dispersity than non-enveloped viruses. Specifically, the vesicular stomatitis virus (VSV)-based oncolytic virus VSV-GP was used to evaluate potential occurrence of non-ideal sedimentation by testing different rotor speeds and loading concentrations. The partial specific volume was determined via density gradients and density contrast experiments. Additionally, nanoparticle tracking analysis (NTA) was used to determine the hydrodynamic diameter of VSV-GP particles to calculate their molecular weight via the Svedberg equation. Overall, this study demonstrates the applicability of AUC and NTA for the characterization of size, density, and molar mass of an enveloped virus, namely VSV-GP.

BASIS: BioAnalysis SEDFIT integrated software for cGMP analysis of SV-AUC data

“…With such a wide range of applications, it's easy to imagine how useful AUC can be for characterization of biopharmaceuticals. Whereas antibodies have long been examined by sedimentation velocity AUC (SV-AUC) for aggregate quantitation 4,5,10,[12][13][14][15][16] , of particular interest recently is the use of SV-AUC for determination of loading states among adeno-associated virus (AAV) and other viral vectors 8,[17][18][19][20][21][22][23][24] .…”

An automated interface for sedimentation velocity analysis in SEDFIT

“…Specifically, the combination of efficient Lamm equation modeling [5], novel size-distribution techniques for direct data fitting with and without diffusional deconvolution [6][7][8], and systematic noise analysis [9,10] as combined in the ls-g*(s) and the high-resolution c(s) approach implemented in the software SEDFIT has garnered widespread applications. These include analyses of size-distributions, hydrodynamic properties, and interactions of particles across the entire size-range accessible to SV-AUC from below 1 kDa to above 10 GDa, ranging from small carbohydrates and peptides [11][12][13] to proteins and protein complexes [14][15][16][17], carbohydrates [18], synthetic polymers [19], small and large nanoparticles [20,21], multi-protein complexes and interacting systems [15,[22][23][24][25], lipid vesicles and emulsions [26,27], viral particles [28][29][30][31][32][33][34], and entire cellular organisms [35], at concentrations from picomolar to millimolar [3,36,37]. Due to the high resolution and sensitivity, and the measurement of particle sedimentation free in solution in the absence of surfaces or labels, this approach has also proven to be advantageous in biotechnology for the characterization of therapeutic and vaccine products, for example including antibody and other protein therapeutics [38][39][40]…”

An automated interface for sedimentation velocity analysis in SEDFIT

“…Specifically, the combination of efficient Lamm equation modeling [5], novel size-distribution techniques for direct data fitting with and without diffusional deconvolution [6][7][8], and systematic noise analysis [9,10] as combined in the ls-g*(s) and the high-resolution c(s) approach implemented in the software SEDFIT has garnered widespread applications. These include analyses of size-distributions, hydrodynamic properties, and interactions of particles across the entire size-range accessible to SV-AUC from below 1 kDa to above 10 GDa, ranging from small carbohydrates and peptides [11][12][13] to proteins and protein complexes [14][15][16][17], carbohydrates [18], synthetic polymers [19], small and large nanoparticles [20,21], multi-protein complexes and interacting systems [15,[22][23][24], lipid vesicles and emulsions [25,26], viral particles [27][28][29][30][31][32], and entire cellular organisms [33], at concentrations from picomolar to millimolar [3,34,35]. Due to the high resolution and sensitivity, and the measurement of particle sedimentation free in solution in the absence of surfaces or labels, this approach has also proven to be advantageous in biotechnology for the characterization of therapeutic and vaccine products, for example including antibody and other protein therapeutics [36][37][38][39]…”