processes’ efficiency. In the last 10 years, the scientific community has developed

more and more quantification tools to describe and better understand the viral re-

plication mechanisms associated with these observations. Nevertheless, this concept

is not yet fully understood, as fundamental virology has for long not been suffi-

ciently quantitative with regards to incomplete processing of the viral particles

leading to DIPs. As an example, it is not rare that the ratio between the infectious

particles and the total particles generated by an identical production process

changes with the viral strain as it was demonstrated for influenza viruses [3].

Regarding the selection of assays, further guidelines will be discussed depending

on the information that must be provided to the regulatory agencies. Besides, several

critical quality attributes will have to be evaluated at several steps of the process. In

general, for viral production processes, three main attributes of the product are tar-

geted (i) the infectious viral particles (IVP), (ii) the total viral particle (VP), and (iii)

the total antigen content.

8.3

VIRAL QUANTIFICATION METHODS

As described earlier, several biochemical, cell-based, or molecular biology methods

are applied for the quantification of viral samples. Such methods can be categorized

into four main sections: infectivity assays, protein content or nucleic acid content

(genome) assessment, and particle counts. Assays will be further presented in the

following sections.

The following section describes the main assays implemented for viral pro-

duction process monitoring. Assays presently in use have several limiting draw-

backs for both the qualification of virus lots and the monitoring of in-process

analysis of product quality. Indeed, most of these viral quantification methods are

time consuming and costly experiments. They also present a lot of variabilities,

either operator-dependent, standard-dependent, or matrix-dependent. This limits the

use of a unique assay throughout the entire process. They are not extremely specific,

and the limits of detection and quantification are high. Thus, for now, the scientific

community does not have access to highly satisfactory assays for the evaluation of

their processes. Therefore, different orthogonal assays are combined to determine

the consistency of the process and its productivity.

8.3.1

INFECTIOUS PARTICLE QUANTIFICATION

For the evaluation of infectious viral particles, methods test the capacity of the virus

to infect, to replicate, and kill plated cultivated cells (Figure 8.1). Such cell-based

assays have high variability and could take from days up to weeks to provide results.

They could fall in two categories: plaque assays and tissue culture infectious doses

(TCID50).

Plaque assays are one of the oldest assays for detecting viruses and quantifying

the number of infectious viral particles. Plaque assay is based on putting in contact

different dilutions of virus suspension on confluent cells for a short period (com-

monly below 30 min). Plated cells are then covered with a hydrogel commonly

composed of soft agarose prepared within a culture medium (see Figure 8.1). This

Analytics and virus production processes

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