Presence of imidazole in loading buffer prevents formation of free radical in immobilized metal affinity chromatography and dramatically improves the recovery of herpes simplex virus type 1 gene therapy vectors.

We have recently shown that immobilized metal affinity chromatography (IMAC) is an effective technique for purification of herpes simplex virus type 1 (HSV-1) gene vector engineered to display cobalt affinity tag on the envelope. However, the tagged HSV-1 viruses were severely inactivated by oxidative hydroxyl free radicals when crude HSV-1 supernatant was applied on an immobilized cobalt column and eluted by a low pH buffer. Furthermore, we have reported that virus inactivation could be prevented by inclusion of high concentration of ascorbate in chromatographic mobile phase. In this paper we report that when elution of bound virus was attempted by inclusion of imidazole in elution buffer, rather than lowering the pH of elution buffer, similar inactivation was also observed. The results also demonstrated that virus inactivation was dramatically reduced by inclusion of 20mM imidazole in the loading buffer. Electron spin resonance (ESR) experiments suggest that imidazole prevents hydroxyl free radical generation from the cobalt complexes. This is the first report describing the role of imidazole in preventing free radical formation in an IMAC column. From a practical stand point, our results imply that inclusion of appropriate amount of imidazole in the loading buffer is an effective strategy for improving the recovery yield of active products and for enhancing product quality during IMAC purification.