Herpes simplex virus type 1 (HSV-1) is a promising vector for gene therapy applications. To be used as therapeutic agents, HSV-1 vectors must meet the stringent criteria of high titer and purity. Thu, development of scalable, efficient HSV-1 vector purification strategies is essential to advance HSV-1 vectors into clinic.
In this dissertation, a novel, efficient HSV-1 vector purification method, based on immobilized metal affinity chromatography (IMAC), was developed.
I first evaluated the feasibility of using various transition metal ions (Cu2+, Zn2+, Ni2+, and Co2+) for the purification of HSV-1 vectors. Results show that none of the metals investigated provided a means of separating the virus from impurities. However, of interest is the finding that neither the virus nor the impurities bound to immobilized Co2+, suggesting that this metal could be useful for HSV-1 vector purification if the vector could be endowed with the affinity toward cobalt.
Accordingly, I constructed an HSV-1 recombinant bearing a cobalt affinity tag (HAT) in the heparan sulfate binding domain of the virion envelope glycoprotein B (gB). It was found that the productivity and infectivity of the tagged HSV-1 mutant (KgBHAT) was not adversely affected by the mutation; while the binding and elution of KgBHAT on cobalt charged iminodiacetate (IDA-Co2+) columns confirmed that efficient purification was possible. By reducing cobalt ion leakage and optimizing the loading conditions, flow rate, and chromatographic substrate, efficient purification of KgBHAT from crude supernatant was achieved with over 70% virus infectivity recovery and over 95% reduction in protein and DNA impurities.
Finally, I found that purification of KgBHAT on IDA-Co2+ columns using crude supernatant as starting material resulted in significant loss in virus infectivity. Electron spinning resonance revealed that the virus inactivation was caused by hydroxyl free radicals generated from the interaction between cobalt ions and components in crude virus supernatant. Appropriate amounts of free radical scavenger, a free radical scavenger, or imidazole in the loading material was able to protect HSV-1 from inactivation, and led to high virus infectivity recovery from IMAC purification. This finding is the first report of free radical mediated biological inactivation in an actual IMAC purification.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-03012006-140224 |
| Date | 02 June 2006 |
| Creators | Jiang, Canping |
| Contributors | William Federspiel, Richard Koepsel, Joseph C Glorioso, Mohammad Ataai |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-03012006-140224/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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