media and GMP-compliant cell lines is encouraged to prevent the introduction of

adventitious agents [17]. HIV-1 Gag VLPs, as well as lentivirus and other viruses,

have a mean particle diameter between 100 and 200 nm. Because of that, when

sterile filtration is applied (<0.2 um), a substantial titer reduction might occur [128].

Therefore, this step could be omitted when the whole process is demonstrated to be

performed under aseptic conditions (FDA 1997) [17].

10.7

CHARACTERIZATION AND QUANTIFICATION OF VLPs

The quantification and characterization of VLPs is a challenge. As VLPs are not

infective viruses, the use of classical techniques like infectivity assays (TCID50) or

the quantification of the viral genomes by qPCR is not possible.

Compared to simple protein-based bioproducts, the characterization of VLPs be-

comes more difficult, since not only protein composition but also their three-

dimensional structure should be assessed. Biochemical, biological and biophysical

methods have been used in VLP characterization [3]. Biochemical protein gels and

biological enzyme-linked immunosorbent assay (ELISA) or immunoblot are normally

used to assess product purity and VLP quantification, respectively [26,47,129,130].

However, these assays cannot distinguish assembled from non-assembled structures

[131] and may require biophysical methods to study the structural integrity of the

obtained VLPs.

Analytical ultracentrifugation, dynamic light scattering (DLS), and transmission

electron microscopy (TEM) were primarily used to assess VLP physical properties [3].

Recently, technical progress in the field of microscopy, as well as the application of

nanotechnology to virology, have given rise to several single-particle analytical

technologies. Nanoparticle tracking analysis (NTA), tunable-resistive pulse sensor

(TRPS), flow virometry, cryogenic electron microscopy (cryo-EM), or atomic force

microscopy (AFM) represent very advanced methods to evaluate VLP size, poly-

dispersity, purity, and even nanoparticle composition [131].

The specific detection and quantification of VLPs entails several difficulties,

especially for enveloped VLPs, which are composed of a protein capsid surrounded

by the host-cell lipid membrane. These structures must be distinguished from other

similar nanovesicle structures like adventitious viruses, baculoviruses, [132] and

extracellular vesicles (EVs), including exosomes and microvesicles [133].

When using transmission electron microscopy, the classical method is to use

negative staining sample preparation, in which VLPs are mixed with a very dense

salt concentration, normally uranyl acetate. The stain interacts with the particles

that are present in the sample, so the presence of Gag polyprotein inside

the particles and the difference in density of the lipid membrane can be observed.

The drawback of this method is that different unwanted backgrounds can appear

depending on the production platform, which can alter the final visualization of

the samples. Also, the interaction with the staining reagents can deform the

membrane, hence the visualization of the native structure of the particle in the

sample is no longer possible.

The use of Cryo-TEM allows the visualization of the samples without any sample

pre-treatment, allowing the observation of the native structure of the particles

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Bioprocessing of Viral Vaccines