Journal: Applied Microbiology and Biotechnology

Article Title: A novel scalable, robust downstream process for oncolytic rat parvovirus: isoelectric point-based elimination of empty particles

doi: 10.1007/s00253-016-8071-x

Figure Lengend Snippet: Optimal buffer and salt conditions for DEAE chromatography. a Pre-purified empty and full H-1PV particles in 50 mM Tris-HCl pH 8.7 were applied to the DEAE column and eluted with a continuous 0–0.5 M NaCl gradient. The elution profile shows the elution of empty capsid particles in fraction 8 (first UV absorbance peak) with 0.15 M NaCl (conductivity 22 mS). The second UV absorbance peak, starting at fraction 11, shows desorption of full capsid particles at 0.2–0.25 M NaCl (conductivity 29–32 mS). Physical particles ( PP ) and genome-containing particles ( GP ) were quantitated. b Clarified cell lysate was diluted in 50 mM Tris-HCl pH 8.7 containing 0.15 M NaCl and applied to the DEAE column. Empty particles were recovered in the flow through and in the 0.15 M wash (first UV absorbance peak). Upon elution with a 0.15–0.4 M NaCl gradient, full particles were eluted near 0.25 M NaCl (second UV absorbance peak). Physical particles ( PP ) and genome-containing particles ( GP ) were quantitated

Article Snippet: All chromatographic studies were performed with a 0.34-ml CIM ® DEAE monolith disk or an 8-ml CIM ® DEAE monolith column (1.3 μm pore size; Bia Separations, Ajdovscina, Slovenia) and ÄKTAprime (GE Healthcare Europe GmbH, Freiburg, Germany) at room temperature (RT) under laminar flow.

Techniques: Chromatography, Purification

Journal: Applied Microbiology and Biotechnology

Article Title: A novel scalable, robust downstream process for oncolytic rat parvovirus: isoelectric point-based elimination of empty particles

doi: 10.1007/s00253-016-8071-x

Figure Lengend Snippet: Large-scale chromatography of a 2.3E8 cell harvest (corresponding to one 10-layer CellSTACK®, reproduced five times). When clarified cell lysate diluted in 50 mM Tris-HCl pH 8.7 containing 0.15 M NaCl was loaded onto the 8-ml DEAE column, empty capsids was eluted in the flow through and wash (first UV peak). Full capsids was eluted in fractions 1–5 at NaCl concentration 0.25 M during elution with a continuous 0.15–0.3 M NaCl gradient (second UV peak, PP/GP ratio ~1)

Article Snippet: All chromatographic studies were performed with a 0.34-ml CIM ® DEAE monolith disk or an 8-ml CIM ® DEAE monolith column (1.3 μm pore size; Bia Separations, Ajdovscina, Slovenia) and ÄKTAprime (GE Healthcare Europe GmbH, Freiburg, Germany) at room temperature (RT) under laminar flow.

Techniques: Chromatography, Concentration Assay

Journal: Applied Microbiology and Biotechnology

Article Title: A novel scalable, robust downstream process for oncolytic rat parvovirus: isoelectric point-based elimination of empty particles

doi: 10.1007/s00253-016-8071-x

Figure Lengend Snippet: Comparison of H-1PV recovery and depletion of empty particles as well as protein impurities by chromatography- and density gradient ultracentrifugation-based downstream process from the same starting material

Article Snippet: All chromatographic studies were performed with a 0.34-ml CIM ® DEAE monolith disk or an 8-ml CIM ® DEAE monolith column (1.3 μm pore size; Bia Separations, Ajdovscina, Slovenia) and ÄKTAprime (GE Healthcare Europe GmbH, Freiburg, Germany) at room temperature (RT) under laminar flow.

Techniques: Comparison, Chromatography, Purification, Formulation

Journal: Applied Microbiology and Biotechnology

Article Title: A novel scalable, robust downstream process for oncolytic rat parvovirus: isoelectric point-based elimination of empty particles

doi: 10.1007/s00253-016-8071-x

Figure Lengend Snippet: Protein composition and electron microscope images of virus batches before and after the downstream process. a , b Protein extracts of virus samples (1.0E10 PP) were analyzed by SDS-PAGE and revealed by a silver staining or b immunoblotting with αVP antibodies. M indicates markers constituting the BenchMark™ Protein Ladder, lane 1 indicates virus harvest, lane 2 indicates IOD-PBS-VIS-Ringer, lane 3 indicates DEAE-VivaspinTM-final formulation, and lane 4 indicates DEAE-HiTrap-final formulation. c , d , e Electron micrographs showing clarified cell lysate ( c ), IOD-PBS-VIS-Ringer ( d ), DEAE-final formulation ( e ). Scale bar is 100 nm

Article Snippet: All chromatographic studies were performed with a 0.34-ml CIM ® DEAE monolith disk or an 8-ml CIM ® DEAE monolith column (1.3 μm pore size; Bia Separations, Ajdovscina, Slovenia) and ÄKTAprime (GE Healthcare Europe GmbH, Freiburg, Germany) at room temperature (RT) under laminar flow.

Techniques: Microscopy, Virus, SDS Page, Silver Staining, Western Blot, Formulation

Journal: Applied Microbiology and Biotechnology

Article Title: A novel scalable, robust downstream process for oncolytic rat parvovirus: isoelectric point-based elimination of empty particles

doi: 10.1007/s00253-016-8071-x

Figure Lengend Snippet: Flow diagram of H-1PV downstream purification methods, comparing IOD-PBS-VIS-Ringer gradient purification with DEAE-final formulation methods

Article Snippet: All chromatographic studies were performed with a 0.34-ml CIM ® DEAE monolith disk or an 8-ml CIM ® DEAE monolith column (1.3 μm pore size; Bia Separations, Ajdovscina, Slovenia) and ÄKTAprime (GE Healthcare Europe GmbH, Freiburg, Germany) at room temperature (RT) under laminar flow.

Techniques: Purification, Formulation