Rapid, accurate isolation and quantitation of viruses and virus-like particles

Isolate highly pure samples of viruses and virus-like particles for research, diagnostics, monitoring therapeutic response, vaccine development, and gene therapy. Carry out multi-parameter measurements with single-particle resolution and unmatched precision. Evaluate viral titre or viremia with high accuracy in a matter of minutes.

Izon’s unique system comprising qEV isolation columns and Tunable Resistive Pulse Sensing (TRPS) analysis provides the only standardised method of isolating and quantifying virions and virus-like particles and offers considerable advantages over other techniques in terms of speed, reproducibility, and simplicity. Measurement requires only 35 µL of sample and real-time analysis enables the assessment of sample aggregation over time.

Although viruses can have a general common structural backbone, indeed they are biological nanoparticles with an incredible diversity: sizes ranging from ~20 to ~1000 nm, DNA or RNA-based genomes, protein subunit-based capsids with different geometries and complexities, and presence or absence of cell-derived lipid envelope, to name a few.

Historically, viruses have played central roles in human wellbeing: as pathogens deteriorating health or as biomedical tools improving it. Hundreds of different pathogenic viruses can cause infectious diseases and death directly in humans, crops, and livestock. Quick and accurate diagnosis of these pathogens is critical for appropriate, timely and favourable treatment of subjects as well as to prevent further transmission in population leading to outbreaks or pandemics.

In turn, viruses’ cell-dependent life cycle and self-assembly capacity has converted them in versatile, bioengineer-able platforms with multiple utilities in medicine. Virus-like particles (VLPs) are synthetic, non-infective whole virus particles that do not contain any genetic material and can be effective stimulants of innate and adaptive immune responses. Altogether, viruses and VLPs have enabled the prevention and control of many viral and non-viral infectious diseases through the highly advanced field of vaccinology.

Research

Characterisation of virus structure and molecular composition, investigation of viral infection mechanisms, immunogenicity and transmission pathways.
Izon offers: qEV isolation for variable sample volumes, precise analytical tools, detailed support materials, training and guidance.

Diagnostics

Isolation, enrichment and identification of viruses in different type of biofluids.
Izon offers: qEV automated isolation for small samples, quick and reproducible analytical tools.

Therapeutics

Viruses offer efficient gene therapy for treatment of conditions and diseases, bacteriophage therapy for treatment of bacterial infections.
Izon offers: qEV isolation for large volumes, no toxic chemicals, accurate and reliable analytical tools.

Vaccines

Virus/VLPs are versatile, bioengineer-able platforms, can present heterologous antigens, effective stimulants of innate and adaptive immune responses, require large scale manufacturing processes.
Izon offers: qEV isolation for large volumes, no toxic chemicals, accurate and reliable analytical tools.

Reliable virus/VLP isolation

Achieve standardised and reproducible separation of viruses/VLPs from extracellular vesicles, protein aggregates, and other contaminants.

Accurate, direct virus/VLP quantification

Directly and accurately quantify viral particles without pre-analytical processing or highly skilled protocols, to optimise different stages of vaccine manufacture process.

Next-level characterisation of viruses/VLPs

Distinguish between viruses/VLPs of different sizes with nanometre precision and discriminate between populations with different surface properties with potential diagnostic applications.

Reliable Virus/VLP Isolation

To isolate viral nanoparticles, virologists face similar challenges as other fields, robust purification procedures ensuring virus/VLP separation from non-virus structures/molecules is essential. Viruses/VLPs have substantial similarities to other biological particles, such as Extracellular Vesicles (EVs and share some biophysical parameters (e.g. size, density), cell-derived membrane and abundant cell- originated release to the extracellular space (in the case of enveloped viruses/VLPs). Choosing the right virus/VLP purification method is critical to guarantee appropriate conclusions. SEC qEV columns enable fast, non-destructive, and reproducible isolation of virions from cell culture or biofluids, and remove >97% removal of contaminating proteins. The two types of qEV Isolation Columns, qEV/35 and qEV/70, are optimised for the isolation of particles between 35–350 nm and 70–1000 nm, respectively. Viruses/VLPs smaller than the isolation range (35 nm or 70 nm) are slowed because they enter the pores of the stationary phase resin within the column, while larger viruses/VLPs– which cannot enter the pores – flow around the resin and are eluted from the column more quickly.

Figure 1.

Separation of enveloped Ebola virus VLPs from EVs by size with qEV columns. Positive control VLP marker (VP40, NP, GP) profiles are shown in lane 1, typical profile of qEV collection volumes from unfiltered (top panel) or 0.22 µm filtered (bottom panel) supernatants from VLP-expressing cells are shown in lane 2-6. Markers for EVs are CD63, CD9, Actin. qEV collection volumes where VLPs are known to elute are indicated by pink box and fractions containing only EVs and no viral particles are indicated by blue box. Adapted from Pleet et al, 2019.

Figure 2.

Biochemical characterisation of enveloped lentiviral virions purified by qEV columns. Aliquots from each qEV collection volumes were analysed by PERT(Product-enhanced reverse transcriptase) assay (pink columns), immunoblot forp24 HIV CA protein (purple columns), total protein (beige columns) and silver staining (blue columns). This shows virus-specific markers (PERT and p24) are most prominent in early qEV collection volumes (7–10) yet clustered around volume8. Adapted from Heider et al, 2017.

VLPs attractiveness as vaccines resides on the facts that they can be bioengineered to present heterologous antigens, produced in numerous cellular expression systems (e.g. bacteria, yeasts, insect or mammalian cells), and have more easily up-scalable manufacturing processes. Virus/VLP SEC qEV purification has many advantages: minimal equilibration and calibration, no harsh buffers are required for binding or eluting of VLPs, ensuring VLP integrity is maintained, and their antigen/epitope exposure is not modified. Combining qEV Isolation Columns with Izon’s Automated Fraction Collector (AFC) creates a streamlined workflow and reduces inter- and intra-operator variability, improving reproducibility, and allows significant automation in the purification protocols.

Modern virus diagnostics in humans heavily depends on quick results, high through-put analysis, and close collaboration with clinicians. Detection and quantification of viral genomes is often how virus particles are identified with prior research providing knowledge of the virus structure and composition as well as characterisation of virion load isolated from different biofluids. qEVs have shown reliable and efficient separation from bulk proteins and cellular debris normally contaminating biofluids providing up to 60-foldenrichment of isolated particles.

Viruses are also useful therapeutics, as they can be specifically recognised, taken up and processed by host/target cells. These features allow efficient delivery of genetic material (i.e. DNA or RNA), causing a genetic modification that may confer some benefit to a disease, procedure known as gene therapy. Virus-based gene therapy candidates must be manufactured in an efficient and scalable manner, as ultra-high viral counts are necessary for administration in humans. qEV Isolation Columns provide many opportunities to optimise virus separation and purification techniques for required high yields and purity.

Accurate, Direct Virus/VLP Quantification

TRPS analysis enables viral particles to be counted directly, avoiding the need for the multiple steps or the destruction of virus particles. TRPS is a single-particle method which enables fast measurements, not requiring pre-analytical processing, fluorescent labelling, or highly skilled protocols, highlighting the shift in the field toward direct measurement techniques. TRPS offers accurate and reproducible analysis and the semi-automated Exoid system enables measurements to be carried out with minimal intra-/inter-operator variability. Alternative methods to actual virus particle quantification (e.g. plaque forming, PCR, ELISA) can require extensive sample preparation, be time consuming and complex, and are not always sufficiently sensitive or compatible with downstream analyses.

Figure 3.

Particle size resolving power between TRPS, NTA and MADLS. Averaged (3 runs) measurements of quadrimodal sample (CPN100/CPN150/CPN200/CPN240 at 1/1/1/1) showing that TRPS identifies all four sub populations clearly.

Figure 4.

Particle size distribution assessed by TRPS of Ebola virus VLP preparation after sonication and passage through a 0.45 μm (blue) or 0.8/0.2 μm (pink) filter. These results shows that double filtration retains smaller particles that are more amenable for manipulation of vaccine formulation. Adapted from Carra et al, 2015.

The small size of some viruses/VLPs is a challenge for other quantification techniques, however TRPS provides an accurate picture of the true particle size distribution and heterogeneity of a sample. Unlike some optical-based techniques which provide an average particle size, TRPS provides single-particle measurements and can precisely resolve multimodal or polydisperse samples.

In vaccine development and production, accurate quantification of the particles of interest is crucial as reliable measurement of yields is essential for quality control, and accurate concentration of the product must be known to ensure appropriate dosing or determining effects of storage conditions in viral particle’s stability. TRPS can be used to create both a better understanding of manufacturing processes and an accelerated purification optimisation when manufacturers are informed of accurate size distribution of virus particles.

Next-Level Characterisation of Viruses/VLPs

‍The ability of TRPS technology to measure particle-by-particle offers a more comprehensive characterisation of virion/VLP preparations, by simultaneously quantifying key physical parameters with high accuracy and precision. TRPS determines virus/VLP concentration, size, and zeta potential by measuring the characteristics of a blockade created when particles pass through the nanopore.

Figure 5.

TRPS is capable of successfully quantifying and identifying subpopulations of bimodal samples of low charged bare polystyrene particles and highly charged carboxylated polystyrene particles, without the need for multiple measurements or adjustment of settings.

Figure 6.

Analysis of zeta potential of three virus preparations: STAR and STAR-A-HV derived particles, and FHV-1particles. This shows no difference in zeta potential between two Lenti virus preparations from STAR and STAR-A-HV cells (full ellipsoid), however the Herpesvirus FHV-1 shows a different pattern, shifted to more negatively charged(broken ellipsoid). Adapted from Heider et al, 2017.

Many relevant biological interactions between viral particles or with a cellular recipient might depend on their charge or zeta potential. Zeta potential is a measure of effective charge of a nanoparticle in a certain medium and it represents the colloidal stability of particle−particle and particle− medium interactions. The tendency of virus/VLP preparations to aggregate or remain in suspension depends on the zeta potential and may be subject to different short or long-term formulation stability studies and subsequent manufacturing optimisation approaches. The charge of viruses/VLPs can influence their cellular uptake efficiencies and cytotoxicity effects in recipient host cells. Thus, opportunities arise for charge-based smart design and modifications of virus/VLP epitopes to optimise infectivity rates in the context of therapeutics or vaccines.

The ability of TRPS to analyse heterogenous populations with high resolving power enables discriminating subpopulations of viruses within a sample when species exhibit even slight differences in diameters or zeta potentials. Very small changes in zeta potential or particle size of viruses/VLPs in response to the binding of an antibody or aptamer can be evaluated using TRPS, allowing identification and specific quantification of subpopulations effectively labelled, leading to a reliable assessment at different stages of any assay optimisation process, especially important in diagnostics development.