heavy metals then coat the surface of the virus particles to reveal their internal and

external structures [6]. The stained samples are then deposited and dried on dedicated

grids. The quantitative evaluation of viral particles is feasible but with high variability

as the analysis is performed by visual counting on NSTEM grids.

Such tools also permit to qualify the viral suspension quality as it will be pos-

sible to observe other nanoparticles like exosomes or micro-vesicles. However,

such assays are very complex to implement as a quantification assay. NSTEM is

very long and costly. Consequently it can not be used for screening. It is most of

the time applied on highly purified samples as protein and cell debris will also have

a strong response to electron-dense stain.

For process optimization and development, electron microscopy is not the

appropriate tool as it will be necessary to screen production conditions with impure

material. Thus, other types of assays allowing the quantification of parts of the virus

(genome, viral protein) are preferred for high-throughput analyses. In such a case,

the evaluation of the number of total viral particles is performed by calculating the

theoretical amount of genome or protein within a viral particle. Such indirect

quantification methods imply that an external standard is used to calibrate the

amount of total viral particles. Thus, biochemical or molecular biology assays are

qualified with electron microscopy viral particle counting.

Viral genome quantification by quantitative qPCR and RT-qPCR was in the

last 10 years the method of choice to determine total viral particles amount. Indeed,

the assumption is here that each viral particle carries a single copy of its genome.

Thus, quantifying the number of viral genomes within a sample gives access to the

number of particles. Such tools based on molecular biology techniques allow for the

design of probes that could have nucleotide sequences highly specific to a virus or a

viral strain. It was particularly exploited for the presence of viral adventitious agents

within pharmaceutical products as it could allow screening for several viruses at the

same time. As preliminary steps, this assay necessitates extracting the viral genome

from the particles and eventually convert RNA in cDNA if the virus strain is an RNA

virus. These two steps include purification and sample handling steps, which might

affect again the variability of the assay. The second step consists of the incubation of

the viral genomes with a specific probe carrying both a fluorophore and its quencher to

allow for fixation of the probes on the nucleic acid sequences. Release of the fluor-

ophore then happens while the DNA polymerase degrades the specific probe.

Correlation between the number of fluorophores released and the number of viral

particles then must be established upfront. This means that a reference material with

its associate reference qualification assay is then necessary. In the last decade,

high‐throughput droplet‐based digital PCR (ddPCR) has been developed as an im-

provement of the conventional polymerase chain reaction (PCR) methods. In ddPCR,

DNA/RNA is encapsulated inside reaction chambers formed of microdroplets. This

was render possible thanks to the improvement reached in microfluidic science

working on mixture between immiscible fluid (i.e., the so-called dispersed fluid) and a

continuous fluid to generate submicroliter droplets at kilohertz rates [7]. Thus, the

reaction chamber contains one or fewer copies of the DNA or RNA. Some of the main

features of ddPCR include high sensitivity and specificity, absolute quantification

without a standard curve, high reproducibility, good tolerance to PCR inhibitor, and

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