Virus titration / TCID50 assay
Virus titration for samples up to biosafety level S2
Virus titration is an integral part of the development, manufacturing and quality control of virus products, including advanced therapy medicinal products (ATMPs). Coriolis offers a variety of methods to obtain virus titers. One option is to determine how many virus particles per volume are inside a sample (i.e. the physical titer). This is done by using particle characterization techniques - you can read more about that here. But such techniques may not tell the entire story, because the infectious or functional titer (i.e. how much virus can infect the target cell) is usually more important and meaningful and may not directly correlate to the number of virus particles in a sample.
TCID50 is a common assay type
The number of infectious virus particles is frequently quantified by using the Median Tissue Culture Infectious Dose (TCID50) assay. The assay works by adding a serial dilution of the virus sample to cells in a 96 well plate format. The type of cell is specifically selected to show a cytopathic effect (CPE), i.e. morphological changes upon infection with the virus or cell death. After an incubation period, the cells are inspected for CPE or cell death and each well is classified as infected or not infected. Colorimetric or fluorometric readouts are also possible, which can increase assay sensitivity. The dilution, at which 50% of the wells show a CPE, is used to calculate the TCID50 of the virus sample. This calculation can generally be done by a variety of mathematical approaches, e.g., the Spearman-Karber method or the Reed-Muench method. Virus titer is expressed as TCID50 / ml.
Other biological assays are available
Depending on the virus, the type of cells and the readout parameter indicating an infection, a variety of other virus titration assays are possible. For viruses that lyse the infected cell, for example, a plaque forming assay is commonly employed for quantification. The virus sample is added (in a suitable dilution) to a monolayer-forming cell culture and incubated over several days. Areas with infected cells will be visible as holes or plaques either by the microscopy, or by colorimetric or fluorometric staining. Virus quantity is expressed as infectious units (IFU) / ml or plaque forming units (PFU) / ml.
Coriolis offers following services regarding virus quantification up to biosafety level S2:
- Transfer, implementation and application of existing TCID50 assays
- Transfer, implementation and application of existing plaque forming assays
- Development of tailor-made virus titration / quantification assays
We perform virus titration / quantification as a standalone service or as part of a formulation development program for viruses, ATMPs and drug substances up to biosafety level S2.