Key result
Inclusion of 20 mM ascorbate in the chromatography mobile phase effectively scavenged hydroxyl radicals and augmented HSV-1 infectivity recovery from <5% to 70% during IMAC purification.
Why the study?
Does the inclusion of ascorbate prevent the inactivation of HSV-1 gene therapy vectors during immobilized metal affinity chromatography?
Does the inclusion of ascorbate prevent the inactivation of HSV-1 gene therapy vectors during immobilized metal affinity chromatography?
Ascorbate prevents oxygen free radical-mediated biological inactivation of HSV-1 vectors during IMAC purification.
Answers are grounded in this paper and cite their sources.
May improve HSV-1 vector yields in IMAC; hypothesis-generating for clinical gene therapy manufacturing.
Metal catalyzed oxidation (MCO), which typically involves oxygen free radical generation, is an important pathway that leads to the deterioration of many biological molecules in solution. The occurrence of MCO in immobilized metal affinity chromatography (IMAC) systems and its potential for inactivating biological products has not been well recognized. In this study, we report the inactivation of herpes simplex virus type 1 (HSV-1) gene therapy vector on immobilized cobalt affinity chromatography. We observed that purification of KgBHAT, an HSV-1 mutant bearing cobalt affinity tags (HAT) on the surface, on an IDA-Co2+ column using crude supernatant as starting material resulted in signification loss in virus infectivity (<5% recovery). Electron spin resonance (ESR) revealed that the virus inactivation was caused by hydroxyl free radicals generated from the interactions between cellular impurities and the metal ions on the column. Inclusion of 20 mM ascorbate, a free radical scavenger, in the chromatography mobile phase effectively scavenged the hydroxyl radicals and dramatically augmented the infectivity recovery to 70%. This finding is the first demonstration of oxygen free radical-mediated biological inactivation in an actual IMAC purification and the way on how to effectively prevent it.
No takes yet. Share an insight, caveat, or question.
Jiang et al. (2006) studied HSV-1 gene therapy vector purification. Ascorbate vs. No ascorbate was evaluated on Virus infectivity recovery. Inclusion of 20 mM ascorbate in the chromatography mobile phase effectively scavenged hydroxyl radicals and augmented HSV-1 infectivity recovery from <5% to 70% during IMAC purification.
Synapse has enriched 10 related papers. Consider them for comparative context: