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 . 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.