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A novel scalable, robust downstream process for oncolytic rat parvovirus: isoelectric point-based elimination of
Barbara Leuchs1, Veronika Frehtman2, Markus Riese2
1German Cancer Research Center Tumor Virology F010, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany. B.Leuchs@dkfz.de.
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.
Insights
A new scalable purification method effectively separates infectious H-1PV particles from empty ones using anion exchange chromatography. This process yields high-purity oncolytic virus suitable for clinical applications and commercial production.
Area of Science:
- Biotechnology
- Virology
- Oncology
Background:
- Rodent protoparvovirus H-1PV exhibits oncolytic and oncosuppressive properties, making it a promising anticancer agent.
- Current purification methods for H-1PV are limited, necessitating a scalable, GMP-compatible process for clinical applications.
Purpose of the Study:
- To develop a scalable, high-purity virus purification process for H-1PV.
- To overcome the limitations of density gradient centrifugation for H-1PV purification.
Main Methods:
- Utilized the isoelectric point difference between full and empty H-1PV particles.
- Employed Convective interaction media® diethylaminoethyl (DEAE) anion exchange chromatography for separation.
- Integrated filtration, DEAE chromatography, buffer exchange, and final formulation into a large-scale process.
Main Results:
- Achieved 98% removal of contaminating proteins and 96% elimination of empty particles.
- Obtained a final infectious particle concentration of 3.5E10 plaque forming units (PFU)/ml.
- Reported an overall recovery rate exceeding 40%.
Conclusions:
- The developed DEAE anion exchange chromatography method is scalable and efficient for H-1PV purification.
- This process yields high-grade pure infectious particles suitable for commercial production.
- The method offers a significant improvement over existing purification techniques for oncolytic viruses.
