high efficacy compared to conventional molecular methods. This tool is nowadays

deployed on a variety of viruses including influenza, cytomegalovirus, HIV, hepatitis,

and was extensively evaluated during the SARS‐CoV‐2 outbreak. Indeed, some re-

sults reveal that ddPCR was 500 times more sensitive to SARS‐CoV‐2 than RT‐PCR

in low‐viral throat swabs. Nevertheless, as the pandemic is still underway, much of

the findings concerning SARS‐CoV‐2 detection should be taken with causion [7]

(Table 8.2).

Viral protein quantifications could be performed using several types of assays.

Here diagnosis and biochemistry tools have reached years of development, allowing

one to choose between biochemical assays (protein activity or total protein quanti-

fication assays), immuno-based assays (ELISA, SRID), biosensors (SPR), chroma-

tography (UPLC, HPLC), or mass spectrometry following liquid chromatography

separation (LC-MS). Most of the quantification tools developed for viral proteins

often target the dominant viral antigens or external proteins. Indeed, their amount or

activity is one of the main quality attribute of the viral-based products. Thus, these

tools could either be exploited as the evaluation of critical quality attributes of a viral-

based product or to access the number of total virus particles for process optimization.

The first assays presented are historical virology assays allowing for viral activity

evaluation and description. Nevertheless, most of them remain reference methods to

evaluate the quality of the viral product for product lot release.

The hemagglutination assay is still in use in many labs for different types of

viruses. This includes the viral families of orthomyxoviridae, paramyxoviridae,

togaviridae, reoviridae, adenoviridae with for example influenza, measles, or ru-

bella viruses [21]. The hemagglutination assay detects the interaction between the

virus and red blood cells. Virus suspensions are incubated with red blood cells

(RBCs) to allow for attachment of viral antigens with RBC specific receptors. In

highly concentrated viral suspension, RBC and virus will then form a network

blocking the RBC sedimentation. When performed within a conical bottom well

plate, it is thus easy to visually distinguish a condition where the network has been

formed (no sedimentation) from a condition where not enough viruses were present

to form a network (sedimentation observed with a red dot). Hemagglutination assay

is highly dependent on the purity of the viral material tested and on the RBC quality

and origin. Mostly RBCs are used from chicken blood, but guinea pigs and other

types of poultry can also be used. RBCs are used fresh, ideally collected the day

before the assay which renders the analysis process complex to plan. Donor-to-

donor animal variability could strongly impact the results; therefore, standard re-

ference samples are required. The assay sensitivity is also quite poor compared to

further detailed immune-based assay. Nevertheless, this essay is simple to perform,

rapid, easy to read by visual evaluation,and allows for the comparison of many

conditions. Such assay has already been used to quantify total viral particles.

Indeed, in 1954, Donald and Issacs established a quantification method of viral

particles based on the hypothesis that there is approximately one influenza virus for

each red blood cell at the end point of agglutination [22]. Viruses preparations were

quantified by both electron microscopy and red blood cells assay to establish such

correlation. Nevertheless, because of its high degree of variability and dependency

on RBC origin or operator reading, hemagglutination assay should be used with

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Bioprocessing of Viral Vaccines