Virus Titre Determination
Virus Titer Determination
Viral vectors used in gene therapy applications offer many advantages and gene and cell therapy based medicines are experiencing a resurgence due to the introduction of next generation transfer vectors including adenovirus, adeno-associated virus and lentivirus. Vector quality and purity/potency are of course of paramount importance, and as part of the testing package for vectors, determination of viral titre is a critical measurement.
Multiple methods can be used for quantification of virus preparations. The plaque assay is a classical biological assay used to measure the infectivity of a virus in a test article. It is based on the assumption that a single infectious virus particle gives rise to a single macroscopic area of cytopathology called a plaque on an otherwise healthy monolayer of cultured cells. If a single cell in a monolayer is infected with a single virus particle, new viruses resulting from the initial infection can infect surrounding cells, hence producing a plaque that is visible by microscopy. Shortly after inoculation, the cell monolayer is overlaid with a semisolid agar medium to prevent virus progeny spreading to other parts of the culture. At the end of the assay. the monolayer is attained with a vital dye and the plaques in the cell monolayer are visualized.
The TCID50 assay (Tissue Culture Infectious Dose) is a system for determining titration of virus. The procedure is performed to determine the infectious titre of any virus which can cause cytopathic effects (CPE) in tissue culture and is carried out over a period of between 5 to 20 days while cells in culture remain viable. This procedure is performed to quantify how much infectious virus is in a preparation. It should be noted that not all virus types cause CPE in tissue culture, and the cell line and virus are aligned in order to see a cytopathic effect.
This is a quantal assay in which replicate wells are infected with serial dilutions of the virus challenged samples. Each well is scored as either positive or negative based on the presence of virus induced CPE. The virus titre is calculated according to the Spearman Kärber formula as described in the Federal Gazette No.84 4 May 1994 and by Schmidt, N.J and Emmons, R.W (1989) in Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infection, 6th Edition.
The assay systems are fully validated assays which have been evaluated for accuracy, repeatability, reproducibility, linearity, limit of detection and limit of quantitation. The statistical treatment of the data is considered as entirely appropriate and was performed in line with guidance within the ICH Q5A(R1) guideline.
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