ABSTRACT
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.
Subject(s)
Bioreactors , Cell Culture Techniques/methods , Kidney/virology , Mechanotransduction, Cellular/physiology , Physical Stimulation/methods , Simplexvirus/growth & development , Simplexvirus/isolation & purification , Animals , Cell Culture Techniques/instrumentation , Cells, Cultured , Cricetinae , Motion , Physical Stimulation/instrumentation , Pilot Projects , Shear Strength , Stress, Mechanical , Virus InactivationABSTRACT
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.
