title: A novel scalable, robust downstream process for oncolytic rat parvovirus: isoelectric point-based elimination of empty particles.
abstract: The rodent protoparvovirus H-1PV, with its oncolytic and oncosuppressive properties, is a promising anticancer agent currently under testing in clinical trials. This explains the current demand for a scalable, good manufacturing practice-compatible virus purification process yielding high-grade pure infectious particles and overcoming the limitations of the current system based on density gradient centrifugation. We describe here a scalable process offering high purity and recovery. Taking advantage of the isoelectric point difference between full and empty particles, it eliminates most empty particles. Full particles have a significantly higher cationic charge than empty ones, with an isoelectric point of 5.8-6.2 versus 6.3 (as determined by isoelectric focusing and chromatofocusing). Thanks to this difference, infectious full particles can be separated from empty particles and most protein impurities by Convective interaction media®diethylaminoethyl (DEAE) anion exchange chromatography: applying unpurified H-1PV to the column in 0.15 M NaCl leaves, the former on the column and the latter in the flow through. The full particles are then recovered by elution with 0.25 M NaCl. The whole large-scale purification process involves filtration, single-step DEAE anion exchange chromatography, buffer exchange by cross-flow filtration, and final formulation in Visipaque/Ringer solution. It results in 98% contaminating protein removal and 96% empty particle elimination. The final infectious particle concentration reaches 3.5E10 plaque forming units (PFU)/ml, with a specific activity of 6.8E11 PFU/mg protein. Overall recovery is over 40%. The newly established method is suitable for use in commercial production.
authors.affiliation: German Cancer Research Center Tumor Virology F010, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany. B.Leuchs@dkfz.de.
authors.forename: Barbara
authors.identifier: http://orcid.org/0000-0001-6955-8393
authors.initials: B
authors.lastname: Leuchs
authors.affiliation: German Cancer Research Center Tumor Virology F010, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
authors.forename: Veronika
authors.initials: V
authors.lastname: Frehtman
authors.affiliation: German Cancer Research Center Tumor Virology F010, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Riese
authors.affiliation: German Cancer Research Center Tumor Virology F010, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Müller
authors.affiliation: German Cancer Research Center Tumor Virology F010, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
authors.forename: Jean
authors.initials: J
authors.lastname: Rommelaere
mesh_terms: D000818:Animals; D002213:Capsid / Q000737:chemistry* / Q000821:virology; D002412:Cations; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005374:Filtration / Q000379:methods; D052661:H-1 parvovirus / Q000737:chemistry* / Q000302:isolation & purification* / Q000648:ultrastructure; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D008854:Microscopy, Electron; D051381:Rats
chemical_list: D002412:Cations
keywords: CIM® column; Chromatography; Empty capsid elimination; H-1 parvovirus; Upscaling; pI