the number of viral particles in this assay by targeting the conditions where 50% of

the wells are infected. A conversion factor based on the Poisson law distribution

(TCDI50 = 0.69 × PFU) has been proposed to compare the results obtained for the

two infectivity assays.

Both assays, as other cell-based assays, are highly dependent on the quality of the

cells used. Operator effect and methodologies to maintain the cell lines could highly

impact the level and variability of the assay. Thus, it is quite common to obtain a

variability between 0.5 and 1 log viral particles/ml for such assays. Additionally,

one should remember that infectious particles are present in a very limited amount

compared to total viral particles in the viral samples. Thus, tissue culture might react

differently to samples carrying a high level of viral particles and develop a strong

innate cell defense mechanism that will impact the quantification assay.

On the other hand, in some processes, the target analyte is not a full “infectious

particle.” For virus-like particles (VLPs) or defective viral vectors, the infectivity as-

says will be limited to viral particles entering the target cells without further replicating

and eventually expression of the transgene in the case of defective viral vectors. Here,

the virus can get inside a cell, and just express the transgene without replication. This is

referred to as transduction with the additional notion of transducing units.

Transducing units correspond to the number of viral particles capable to

transfer a gene of interest into a cell. This is commonly evaluated using a reporter

gene as fluorescent GFP or RFP proteins. Transducing particles could either be

quantified with an assay adapted from the TCID50 assay presented previously, or

with flow cytometry. Indeed, the principle of applying the suspension of virus onto

a cell culture is maintained and the read-out is no longer the cytopathic effect of the

suspension but the expression of the reporter gene. Positive wells are then numbered

based on the presence or absence of fluorescent gene expression.

Additional protocols based on flow cytometry were developed to achieve more

statistically significant results and to develop high throughput assays. After sufficient

cultivation duration following cells contact with the virus suspensions at different

dilutions, cells are resuspended to be analyzed by flow cytometry.

8.3.2

TOTAL VIRAL PARTICLES QUANTIFICATION

As described in the first section, the number of total particles including defective

particles is of interest for qualification of some process steps. Several tools were

proposed to capture such information. In the 1960s, early analysis was based on

microscopic observation using electron microscopy [5]. In this case, the method has

been used satisfactorily with poliovirus, bacteriophage, and vaccinia virus, three

viruses differing greatly in size and morphology.

Negative staining transmission electron microscopy (NSTEM) is the only assay

allowing for direct observation of the viruses. Such a tool is fully adapted to observe

viruses that have a range of sizes between 30 nm to 300 nm. It gives the possibility to

describe viral particles morphology, structure, and composition. To confirm the

presence of viral proteins, it is possible to label specific antibodies with gold particles

(see Figure 8.2). The negative staining technic principle holds on viral suspension

staining with electron-dense stain prepared from heavy metals after its fixation. The

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Bioprocessing of Viral Vaccines