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.

Upstream process optimization and micro- and macrocarrier screening for large-scale production of the oncolytic H-1 protoparvovirus

“…In our process, the majority of infective virus particles are cell-associated at the time of harvest. To harvest H-1PV, a freeze–thaw cell lysis in Tris–EDTA buffer (VTE) (Leuchs et al, 2016 ) or Tris–HCl buffer (VT) (Leuchs et al, 2017 ) was previously reported for stationary cultures. For large-scale production with adherent cells on carriers, a scalable cell lysis is required.…”

White paper on microbial anti-cancer therapy and prevention

“…The rodent protoparvovirus H-1 is currently in Phase I/IIA clinical trials against glioblastoma [ 214 , 215 ] and pancreatic cancer [ 216 ] in Germany. Viral capsids are non-enveloped and extremely rugged, simplifying their production, purification and storage [ 217 – 219 ], as well as enhancing their tissue penetration properties. The protoparvoviruses belong to the same virus family as the adeno-associated viruses (AAV), which have already found extensive clinical use as gene therapy vectors.…”

Untitled

“…Selectivity is described frequently as correlating with protein isoelectric (pI) point [12][13][14][15][16] . Differences in pI have been documented among full and empty capsids for several parvoviruses, including AAV [17][18][19][20][21] but descending pH gradient elution has not proven effective for separating their empty and full capsids. A comprehensive study with structurally-similar minute virus of mice (MVM) showed that selectivity was dominated by the distribution of charges on capsid surfaces rather than their net charge [20] .…”