Inactivation of herpes simplex type 1 gene vector on immobilized metal affinity chromatography: Oxidative damage by hydroxyl free radicals and its prevention
Inactivation of herpes simplex type 1 gene vector on immobilized metal affinity chromatography: Oxidative damage by hydroxyl free radicals and its prevention
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DOI：
10.1002/bit.20943
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发表时间：
2006-09-05
影响因子：
3.8
通讯作者：
Glorioso, Joseph C.
中科院分区：
文献类型：
--
作者：
Jiang, Canping;Ataai, Mohammad;Glorioso, Joseph C.
金属催化氧化（MCO）是导致溶液中许多生物分子降解的重要途径，通常涉及氧自由基的产生。MCO在固定化金属亲和色谱（IMAC）系统中的发生及其灭活生物制品的潜力尚未得到很好的认识。本文报道了用固定化钴亲和层析法灭活单纯疱疹病毒1型（HSV-1）基因治疗载体。我们观察到，使用粗上清液作为起始材料在IDA-CO2+柱上纯化KgBHAT（一种在表面上带有钴亲和标签（HAT）的HSV-1突变体）导致病毒感染性的显著损失（< 5%回收率）。电子自旋共振（ESR）表明，病毒灭活是由细胞杂质与柱上金属离子相互作用产生的羟基自由基引起的。在色谱移动的相中加入20 mM抗坏血酸（一种自由基清除剂）可有效清除羟基自由基，并将感染性恢复率显著提高至70%。这一发现是在实际的IMAC纯化中氧自由基介导的生物失活的第一个证明，以及如何有效地防止它的方法。
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. (c) 2006 Wiley Periodicals, Inc.