The first particle counter used for viral particle counting in solution was

dynamic light scattering (DLS), allowing for both size and zeta potential mea-

surement of the particles.

Nanoparticle tracking analysis (NTA), developed by NanoSight Ltd., is based

on a laser-illuminated microscope technique. The technique relies on the detection of

the Brownian motion of nanoparticles in liquid which are visualized through an in-

tense illumination by a laser beam passing through an optical prism. NTA was already

successfully used for quantification of vaccinia virus, rabies, adenovirus, phage,

vesicular stomatitis virus G (VSV-G), human cytomegalovirus, respiratory syncytial

virus (RSV), and HIV [18,28,29]. An important limitation of the technique is the viral

particle range necessary for the analysis, between 10⁷ and 10⁹ particles per mL. The

dilution steps needed to reach such a narrow quantification range can result in long

sample preparation. It has recently been applied to sizing and quantifying RSV pre-

paration by coupling the detection principle with a fluorescent aptamer [28].

Flow cytometry principles based on light scattering were also exploited and

adapted to virus particle size. The scientific community also calls such technologies

flow virometry. Commercial solutions (Virocyt®– Sartorius) or in-house developed

protocols were proposed in the last 20 years with successful application with several

viruses. The only limitation is the power of the cytometer lasers to detect very small

particles. In this case, the viral particles are labeled with fluorescent dyes which

could be either specific antibodies, lipid bilayer dyes, or nucleic acid intercalators.

The Coulter effect was also used for the detection of a particle as they present

some charges at their surface. Indeed, coulter counter principle target counting and

sizing particles suspended in electrolytes. The commercial application of this

technology is referred to tunable resistive pulse sensing technique (TRPS).

Viruses covered with proteins have a specific charge that is commonly negative.

The reference commercial equipment for virus particle counting is the qNano®

equipment (IZON). Here the particles are pushed through pores of different sizes by

electric current between two electrodes. The number of charges present at the

surface of the particles will induce differences in the speeds rate of the particles

through the holes. This way, particles are discriminated into populations based on

their particle’s diameter but also their zeta-potential (charges). The Coulter effect is

also now referenced in literature for the characterization and quantification of

several virus types (adenovirus, VSV, influenza) [11,30].

Charges of viral particles were also exploited for virus separation and quantifi-

cation by liquid chromatography. Ion-exchange chromatography IEX-HPLC was

applied to quantify several viruses and viral vectors (influenza virus, baculovirus,

adenovirus, adeno-associated virus) [3], [31–33]. In this case, the charges of the

virus particles are used to create interaction with cationic support and to allow for

separation from low negatively charged free proteins and strongly negative DNA/

RNA molecules. Separation is commonly realized by saline buffer allowing to

conserve structural integrity of the viral particles. Detection of the viral particle

peaks is performed on native absorbance or fluorescence of the proteins with re-

spectively either UV detectors at 280 nm (protein main absorbance) or fluorescence

detectors excitation at 290 nm and emission at 335 nm (specific fluorescence of

tryptophan residues).

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Bioprocessing of Viral Vaccines