A method for quantifying the proportion of supercoiled circular (SC) forms in DNA solutions is described. The method (SCFluo) takes advantage of the reversible denaturation property of SC forms and the high specificity of the PicoGreen fluorochrome for double-stranded (ds)DNA. Fluorescence values of forms capable of reversible denaturation after a 5 min heating, 2 min cooling step are normalised to fluorescence values of total dsDNA present in the preparation. For samples with a SC content >20-30%, good regression fits were obtained when values derived from densitometric scanning of an agarose gel and those derived from the SCFluo method were compared. The method represents an attractive alternative to currently established methods because it is simple, rapid and quantitative. During large-scale processing and long-term storage, enzymatic, chemical and shear degradation may substantially decrease the SC content of plasmid DNA preparations. Regulations for pharmaceutical grade products for use in gene therapy and DNA vaccination may require >90% of the plasmid to be in the SC form. In the present study the SC content of 6.9, 13 and 20 kb plasmid preparations that had been subjected to chemical and shear degradation was successfully quantified using the new method.
Despite continuous improvements in culturing and recovery techniques, high-titer stocks of purified disabled herpes simplex virus type-1 (HSV-1 DIS) vector for drug discovery and use in preclinical and clinical trials are currently difficult to achieve. Efforts to improve their centrifugal recovery have been addressed in this paper. The operation of a swing-out centrifuge rotor was assessed, and its operational conditions were defined for the recovery of viable HSV-1 DIS. 80% virus recovery was achieved after 90 min at 26000g. The 20% loss of virus was attributed to damage to the viral envelope by overcompaction of the pellet and impaction with the base of the centrifuge tube. Virus recovery was increased by a further 10% by using a fixed-angle centrifuge rotor operating at 26000g. Plaque assays of recovered HSV-1 DIS gave values on the order of 10(6) pfu/mL, compared to values typically above 10(9) pfu/mL obtained for the replication-competent HSV-1 viron.
A new process route is proposed to increase the production yield of disabled herpes simplex virus type 1 (HSV-1 DIS). Infected baby-hamster kidney (BHK) cells were subjected to a range of shear rates between 3.69 x 10(3) s(-1) and 51.3 x 10(3) s(-1) in the gap between a pair of co-axial cylinders. Analysis of the supernatant fractions of sheared material established that optimal virus release was achieved by exposing the infected cells to a shear rate of 42.7 x 10(3) s(-1) for a period of 1 min. Compared with the current laboratory process, the titre of HSV-1 DIS was increased over 30-fold, from about 1 x 10(6) to 30 x 10(6) pfu (plaque-forming units)/ml. Evaluation of the supernatant fractions by flow cytometry, total protein assay, PAGE and dot-blot assays showed no evidence of cell disruption, supporting the hypothesis that shear-induced release of the cell-membrane-bound virus was achieved without compromising downstream purification. The proposed method is scalable, and since no additional chemicals are required, it provides an attractive option for enhanced recovery of virus particles for therapeutic applications.
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