IMMOBILIZED COBALT AFFINITY PURIFICATION FOR HSV-1 BASED GENE THERAPY VECTORS

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

Study on the XPS-ESCA of Aluminum Phosphide Products

XPS-ESCA analysis showed small signal for phosphorus in fresh specimens of aluminum phosphide (AlP). After removal of a layer of about 0.5 – 1.0 μm by argon ion sputtering, it was observed that signal intensities from oxygen and aluminum increased. The oxygen signal decreased as a function of sputtering time, synchronously with the increase of the phosphorous signal from the AlP nucleus. The aluminum signal, which was considered to be mainly due to AlP and Al(OH)3, remained constant. Other impurity elements including N, Mg, etc., were identified in the technical 85% AlP and AlP tablet formulated products

Octopamine Levels in Blattella Germanica L. Tissues by Capillary Gas Chromatography with Electron Capture Detection

Distribution and levels of octopamine (OA), one of the biogenic amines in the invertebrate nervous system, may have significant effects on insect physiological processes including growth, feeding and reproduction. In this paper capillary gas chromatography with electron capture detection (GC-ECD) and mass selective detection (GC-MS) were used to determine the content of OA in Blattella germanica L. central nervous system (CNS), and that of OA in cockroach stressed by kinds of insecticides, known octopaminergic agonists and some essential oils. A derivatization method for organic extracts via reaction with pentafluoropropionic anhydride (PFPA) was developed. The resulting OA derivatives were confirmed by GC-MS to be tris-pentafluoropropionyl-OA. The method was used to quantify the amount of OA in insect issues by capillary GC-ECD through an extraction-derivatization-liquid/liquid partition procedure. Average OA content in normal cockroaches was determined to be 68.49 ± 7.31 ng/g tissue (N=5 determinations). It was shown that insecticides including chlordimeform, methomyl, permethrin, chlorfluazuron, malathion, trichlorfon and some oxazolidine agonists, essential oils including eugenol, cinnamic alcohol, phenyl ethyl alcohol could led to significant increase of OA levels in the cockroach CNS comparing with which in insect treated by 1-butanone. Malathion, trichlorfon, chlorfluazuron and cinnamic alcohol were shown to be able to cause a 20- fold increase in OA levels

Determination of Multiresidues in Rapeseed, Rapeseed Oil, and Rapeseed Meal by Acetonitrile Extraction, Low-Temperature Cleanup, and Detection by Liquid Chromatography with Tandem Mass Spectrometry

A multiresidue method for determining pesticides in rapeseed, rapeseed oil, and rapeseed meal by use of liquid chromatography–tandem mass spectrometry is developed. Samples were extracted with acetonitrile or acidified acetonitrile and cleaned up by a 12 h freezing step. The recovery data were obtained by spiking blank samples at three concentration levels. The recoveries of 27 selected pesticides in rapeseed, rapeseed oil, and rapeseed meal were in the range of 70–118%, at the concentration level of 10 μg kg^–1, with intraday and interday precisions of lower than 22 and 27%, respectively. Linearity was studied between 2 and 500 μg L^–1 with determination coefficients (<i>R</i>²) of higher than 0.98 for all compounds in the three matrices. The limits of quantitation (LOQs) of pesticides in rapeseed, rapeseed oil, and rapeseed meal ranged from 0.3 to 18 μg kg^–1. The <i>n</i>-octanol–water partition coefficient showed more influence than water solubility in extracting pesticides by acetonitrile from matrices of high fat content. This method was successfully applied for routine analysis in commercial products

Immobilized Cobalt Affinity Chromatography Provides a Novel, Efficient Method for Herpes Simplex Virus Type 1 Gene Vector Purification

Herpes simplex virus type 1 (HSV-1) is a promising vector for gene therapy applications, particularly at peripheral nerves, the natural site of virus latency. Many gene vectors require large particle numbers for even early-phase clinical trials, emphasizing the need for high-yield, scalable manufacturing processes that result in virus preparations that are nearly free of cellular DNA and protein contaminants. HSV-1 is an enveloped virus that requires the development of gentle purification methods. Ideally, such methods should avoid centrifugation and may employ selective purification processes that rely on the recognition of a unique envelope surface chemistry. Here we describe a novel method that fulfills these criteria. An immobilized metal affinity chromatography (IMAC) method was developed for the selective purification of vectors engineered to display a high-affinity binding peptide. Feasibility studies involving various transition metal ions (Cu(2+), Zn(2+), Ni(2+), and Co(2+)) showed that cobalt had the most desirable features, which include a low level of interaction with either the normal virus envelope or contaminating DNA and proteins. The introduction of a cobalt-specific recognition element into the virus envelope may provide a suitable target for cobalt-dependent purification. To test this possibility, we engineered a peptide with affinity for immobilized cobalt in frame in the heparan sulfate binding domain of HSV-1 glycoprotein B, which is known to be exposed on the surface of the virion particle and recombined into the viral genome. By optimizing the IMAC loading conditions and reducing cobalt ion leakage, we recovered 78% of the tagged HSV-1 recombinant virus, with a >96% reduction in contaminating proteins and DNA