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Separation and characterization of viruses using multi-detector field-flow fractionation

by Dr Robert Reed and Dr Soheyl Tadjiki

Viruses are increasingly used as gene therapy vectors in modern medicine. In order to minimize unwanted side effects such as immunogenicity, accurate determination of viral aggregation status is important. Field-flow fractionation is emerging as one of the most robust techniques for the separation and size measurement of viruses, their fragments, and their aggregates.


The global coronavirus pandemic is the biggest story of 2020, thrusting viruses to the forefront of almost every person’s consciousness around the world. Characterization of viruses and the development of vaccines have become the most important scientific pursuits for many labs.

Vaccines can be complex and may contain many molecular species that span a large size range, from antibodies, virus fragments such as proteins and nucleic acids, polysaccharide–protein complexes and up to large aggregates that are often over 100 nm in diameter. This means these samples are challenging to separate and characterize by column-based chromatography techniques such as size exclusion chromatography.

‘Asymmetrical flow field-flow fractionation’ (AF4) can help researchers in this field as it is a stationary phase-free separation technique for characterization of polydisperse samples from approximately 1 nm to 1 μm in diameter [1, 2]. AF4 has already shown merit in the characterization of viruses and virus-like particles [3, 4], as well as antibodies and antigens and their aggregates and agglomerates [5, 6].

A schematic for the AF4 channel is shown in Figure 1. The combination of cross flow and channel flow causes size separation over the course of the analysis, with smaller particles eluting to connected detectors before larger particles, including aggregates.

For characterization of the separated particles or molecules, a variety of detectors can be connected to the AF4, including dynamic light scattering and multi-angle light scattering (MALS) for size measurement, with the latter also providing data on molecular weight when combined with a concentration detector such as UV/Vis or refractive index.

In this article, AF4 coupled with multiple detectors was used to separate and characterize:

  • adeno-associated virus (AAV) monomers and their fragments/ aggregates; and
  • a mixture of two AAV serotypes after exposure to heat stress.

Separation and characterization of stressed- AAV fragments and aggregates with a single method using AF4-MALS

AAVs are increasingly used for gene therapy owing to their versatility and safety. They can be loaded with DNA or RNA and delivered to a specific cell type, with the goal being to treat or cure disease [7]. One of the biggest concerns for producing a uniform AAV sample is the purity of the virus monomer in solution, as both fragments and aggregates can be present in significant amounts.

An AAV sample was divided into two aliquots, one of which was stressed using a proprietary method to induce fragmentation and aggregation. Two samples were analysed, ‘non-stressed’ AAVs (Fig. 2) and ‘stressed’ AAVs (Fig. 3). To separate by size and characterize the AAVs and their fragments/aggregates, an AF4 system (Postnova AF2000) was used with two detectors monitoring the eluent. Firstly, a 21-angle MALS detector (PN3621) was used for measuring the radius of gyration (Rg); secondly a UV/Vis detector (PN3211), which is sensitive to virus concentration, was used to calculate the relative amount of fragment/aggregate present compared to AAV monomer.

In Figure 2, the UV/Vis response for the non-stressed sample is plotted as the red trace, with Rg calculated at each time point from MALS data (not shown) and plotted as red dots. The void peak (analogous to the solvent peak in chromatography) elutes first, around 2 minutes. The UV/Vis detector is sensitive to concentration and the relative amount of each species is calculated by integrating the area under the peaks, excluding the void peak. The monomer elutes starting around 5 minutes, and has a measured Rg consistent with most AAVs, approx. 12–15 nm in radius. The large aggregates in the non-stressed AAV sample are monodisperse, with an Rg of approx. 80 nm.

The UV response and Rg measurements for the stressed AAV sample are shown in Figure 3. Fragments generated during stressing the sample comprised 15.0% of peak area. These fragments were too small for their Rg to be measured by MALS (<8 nm radius.) The peak between 5 and 10 minutes contains the AAV monomer, which was also observed in this retention time range in the non-stressed sample. However, in the stressed sample, a population of larger Rg values is observed eluting later in this peak, likely due to dimers and trimers having formed during the stress protocol. For the non-stressed sample, the aggregate was present at 6.1% (Fig. 2), and for the stressed sample the aggregate was present at 23.7% (Fig. 3). For the stressed AAV, there is substantial polydispersity in the aggregate size, ranging from about 40 nm to 120 nm in radius. In addition to quantification of a wide size range from small virus fragments to large virus aggregates, fractions can be collected at any point in the fractogram. This could be done to enable further off-line analysis of nucleic acids or proteins contained in the fragments.

Studying stability of two AAV serotypes under heat stress using AF4-MALS

One of the critical quality attributes that needs to be quantified for a safe and effective AAV product is its stability. In this application, AF4-MALS with a UV/Vis diode array detector was used to study the aggregation of two AAV serotypes when they are stressed by heat.

Two AAV serotypes, AAV1 and AAV5, were used for this study. The samples were stressed at 60 °C and 75 °C for 35 minutes. An AF4 system (Postnova AF2000) interfaced with a MALS detector (PN3621) and a diode array UV/Vis detector (PN3241) were used to analyse the AAV samples using the formulation buffer as the AF4 carrier solution.

Figures 4a and 4b show plots of the UV signals obtained at 219 nm absorbance wavelength versus retention time for non-stressed and stressed AAV1 and AAV5 serotypes. The numbered peaks in the graphs indicate the subpopulations of molecular species present in the samples that have been separated by AF4 based on their hydrodynamic sizes.

Eluting first are the smaller viral fragments in peaks 1 and 2, followed by the virus monomer and oligomers in peaks 3 and 4 respectively. Peak 5 contains the larger virus aggregates that elute last in the separation (at 35 to 60 minutes), which are detected only for the stressed AAV1 sample at 75 °C. For non-stressed AAV1, monomeric and oligomeric viral particles constitute about 26% and 7% of the sample, respectively.

The non-stressed AAV5 sample contained more monomer than AAV1, with 43% in the monomeric and 5% in the oligomeric forms.

These data indicate that both serotypes are reasonably stable up to 60 °C. However, they exhibited a significant change in their size distributions when they were stressed at 75 °C. In both stressed samples, the monomer (peak 3) vanished completely, and the oligomer peak (peak 4) grew substantially (to 66% for AAV1 and 26% for AAV5). This suggests that the virus particles form oligomers as they undergo heat stress.

The Rg of the AAVs in non-stressed and stressed samples was obtained from evaluation of the MALS signal intensities at different angles. The Rg data points (black circles) of non-stressed and stressed AAV1 and AAV5 samples are shown in Figures 5a and 5b, respectively. Each point represents a size measurement for a volume of sample passing through the MALS flow cell. On average, virus monomers (peak 3) are in the order of 8–10 nm in radius. The oligomer peak varies from 10–30 nm in radius. The large aggregates (peak 5), only present in the stressed AAV1 sample, are approx. 100 nm in radius and represent approx. 2% of the population.


As virus research continues to push forward, it is clear that the separation techniques need to keep pace with the more demanding analytical requirements that emerge. Current AF4 technology provides a solid leap forward but further developments will enhance the capabilities in speed, resolution and detection. The open channel design of AF4 is considerably advantageous over chromatographic techniques, as the absence of a stationary phase results in less sample or aggregate loss due to interaction or filtration in the column packing. From the data presented here, we see that AF4 can already separate a wide range of sizes within a single method, from virus fragments less than 10 nm in radius, to large aggregates above 100 nm in radius. It can be equally applied to any or all of the components in virus and vaccine research from small antibodies of only a few nanometres in size, up to large protein–polysaccharide complexes or virus particles and aggregates of well over 100 nm. In addition to other commonly used characterization methods for virus and antibody analysis, AF4 provides a powerful orthogonal approach that can help ensure safe and effective vaccine production.

The authors
Robert Reed PhD*, Soheyl Tadjiki PhD
Postnova Analytics Inc. Salt Lake City, UT, USA

*Corresponding author


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