Type of publication:
Type of document:
Steppert, P; Burgstaller, D; Klausberger, M; Kramberger, P; Tover, A; Berger, E; Nöbauer, K; Razzazi-Fazeli, E; Jungbauer, A
Separation of HIV-1 gag virus-like particles from vesicular particles impurities by hydroxyl-functionalized monoliths.
J Sep Sci. 2017; 40(4):979-990
Authors Vetmeduni Vienna:
Vetmed Research Units
- The downstream processing of enveloped virus-like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl-functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV-1 gag virus-like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith. The binding capacity was 2 × 1012 /mL monolith and was only limited by the pressure drop. By applying either a linear or a step gradient elution, to decrease the ammonium sulfate concentration, the majority of double-stranded DNA (88-90%) and host cell protein impurities (39-61%) could be removed while the particles could be separated into two fractions. Proteomic analysis and evaluation of the p24 concentration showed that one fraction contained majority of the HIV-1 gag and the other fraction was less contaminated with proteins originated from intracellular compartments. We were able to process up to 92 bed volumes of conditioned loading material within 3 h and eluted in average 7.3 × 1011 particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 109 particles.© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Chemistry Techniques, Analyticalmethods
Gene Products, gagisolation & purificationmetabolism
HIV-1isolation & purification
Hydrophobic and Hydrophilic Interactions
Vaccines, Virus-Like Particleisolation & purification