title: Separation of HIV-1 gag virus-like particles from vesicular particles impurities by hydroxyl-functionalized monoliths.
abstract: 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 × 1011particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 109particles.
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Petra
authors.initials: P
authors.lastname: Steppert
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Daniel
authors.initials: D
authors.lastname: Burgstaller
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Miriam
authors.initials: M
authors.lastname: Klausberger
authors.affiliation: BIA Separations d.o.o., Ajdovščina, Slovenia.
authors.forename: Petra
authors.initials: P
authors.lastname: Kramberger
authors.affiliation: Icosagen AS, Tartumaa, Estonia.
authors.forename: Andres
authors.initials: A
authors.lastname: Tover
authors.affiliation: Austrian Centre of Industrial Biotechnology, Vienna, Austria.
authors.forename: Eva
authors.initials: E
authors.lastname: Berger
authors.affiliation: VetCore Facility for Research, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: Katharina
authors.initials: K
authors.lastname: Nöbauer
authors.affiliation: VetCore Facility for Research, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: Ebrahim
authors.initials: E
authors.lastname: Razzazi-Fazeli
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.|Austrian Centre of Industrial Biotechnology, Vienna, Austria.
authors.forename: Alois
authors.initials: A
authors.lastname: Jungbauer
mesh_terms: D002478:Cells, Cultured; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D015683:Gene Products, gag / Q000302:isolation & purification* / Q000378:metabolism; D015497:HIV-1 / Q000302:isolation & purification*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D017665:Hydroxyl Radical / Q000378:metabolism; D040901:Proteomics; D058425:Vaccines, Virus-Like Particle / Q000302:isolation & purification
chemical_list: D015683:Gene Products, gag; D058425:Vaccines, Virus-Like Particle; D017665:Hydroxyl Radical
keywords: hydrophobic interaction chromatography; mixed mode chromatography; monoliths; virus-like particles; viruses