title: Downstream processing of virus-like particles: single-stage and multi-stage aqueous two-phase extraction.
abstract: The demand for vaccines against untreated diseases has enforced the research and development of virus-like particle (VLP) based vaccine candidates in recent years. Significant progress has been made in increasing VLP titres during upstream processing in bacteria, yeast and insect cells. Considering downstream processing, the separation of host cell impurities is predominantly achieved by time-intensive ultracentrifugation processes or numerous chromatography and filtration steps. In this work, we evaluate the potential of an alternative separation technology for VLPs: aqueous two-phase extraction (ATPE). The benefits of ATPE have been demonstrated for various biomolecules, but capacity and separation efficiency were observed to be low for large biomolecules such as VLPs or viruses. Both performance parameters were examined in detail in a case study on human B19 parvovirus-like particles derived from Spodoptera frugiperda Sf9 insect cells. A solubility-guided approach enabled the design of polyethylene (PEG) salt aqueous two-phase systems with a high capacity of up to 4.1mg/mL VLPs. Unique separation efficiencies were obtained by varying the molecular weight of PEG, the pH value and by using neutral salt additives. Further improvement of the separation of host cell impurities was achieved by multi-stage ATPE on a centrifugal partition chromatography (CPC) device in 500mL scale. While single-stage ATPE enabled a DNA clearance of 99.6%, multi-stage ATPE improved the separation of host cell proteins (HCPs). The HPLC purity ranged from 16.8% (100% VLP recovery) for the single-stage ATPE to 69.1% (40.1% VLP recovery) for the multi-stage ATPE. An alternative two-step downstream process is presented removing the ATPS forming polymer, cell debris and 99.77% DNA with a HPLC purity of 90.6% and a VLP recovery of 63.9%.
authors: Karlsruhe Institute of Technology, Institute of Process Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe, Germany.
authors: Christopher
authors: C
authors: Ladd Effio
authors: Karlsruhe Institute of Technology, Institute of Process Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe, Germany.
authors: Lukas
authors: L
authors: Wenger
authors: Karlsruhe Institute of Technology, Institute of Process Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe, Germany.
authors: Ozan
authors: O
authors: Ötes
authors: Karlsruhe Institute of Technology, Institute of Process Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe, Germany; Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany.
authors: Stefan A
authors: SA
authors: Oelmeier
authors: Diarect AG, Freiburg, Germany.
authors: Richard
authors: R
authors: Kneusel
authors: Karlsruhe Institute of Technology, Institute of Process Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe, Germany. Electronic address: juergen.hubbuch@kit.edu.
authors: Jürgen
authors: J
authors: Hubbuch
mesh_terms: D000818:Animals; D036022:Capsid Proteins / Q000032:analysis; D002498:Centrifugation; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000302:isolation & purification; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016732:Parvovirus B19, Human / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D061987:Sf9 Cells / Q000166:cytology / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry; D012995:Solubility; D018411:Spodoptera; D058425:Vaccines, Virus-Like Particle / Q000032:analysis* / Q000302:isolation & purification* / Q000378:metabolism; D014773:Virology / Q000379:methods*
chemical_list: D036022:Capsid Proteins; D058425:Vaccines, Virus-Like Particle; C095548:capsid protein VP2, parvovirus B19; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D004247:DNA
keywords: Aqueous two-phase systems; Centrifugal partition chromatography; DNA removal; High-throughput screening; Parvovirus; VLP