AAV

Adeno-associated viruses (AAVs) have recently gained a lot of interest in gene therapy research for delivering modified genes into target cells to treat or prevent a disease by replacing a mutated gene with a healthy copy of the gene. Recombinant adeno-associated viruses (rAAV) are being investigated intensively in the development of gene therapies. However, in order to develop efficient rAAV therapies, we must address multiple challenges, such as optimal design, process and formulation conditions and comprehensive quality control (Figure 1).

Figure 1. Key activities and quality attributes required for development of stable and efficacious viral vectors ( DSP – downstream processing).

The importance of orthogonal techniques

Malvern Panalytical’s range of technologies can deliver important information for the rapid development of gene therapies. Table 1 summarizes the key parameters researchers must consider, and which Malvern Panalytical’s techniques can provide this information.

Key Sample ParametersMalvern Panalytical Techniques
Capsid size   DLS, NTA
Absolute Mw of viral capsids and transgeneSEC-MALS
Capsid titer or particle countMADLS®, SEC-MALS, NTA
Percentage of genome-containing virus particles/% full analysisSEC–MALS
Aggregate formationDLS, MADLS, SEC–MALS, NTA
FragmentationSEC–MALS
Thermal stabilityDLS, DSC
Higher-order structure analysisDSC
Serotype identificationDSC
Capsid uncoating and genome ejectionDLS, DSC
Binding to receptorITC
ChargeELS
DLS dynamic light scattering, SEC-MALS size exclusion chromatography multi-angle light scattering, NTA nanoparticle tracking analysis, MADLS multi-angle dynamic light scattering, DSC differential scanning calorimetry, ITC isothermal calorimetry, ELS electrophoretic light scattering.

DLS, MADLS, SEC-MALS, NTA, ITC, and DSC are label-free biophysical techniques that require minimal assay development and can be readily applied at all stages, strengthening the analytical workflow for gene therapy development. We have recently used multiple, orthogonal techniques to characterize rAAVs and this work has been published in the Pharmaceutics Journal.

Full and empty capsid analysis

SEC, together with compositional analysis, can help determine the concentration and molecular weight of two distinct components within a sample. For example, a multi-detection chromatogram for empty rAAV5 is shown in Figure 2. The refractive index (RI) signal is represented by the red channel, the ultra-violet (UV) at 260nm by the purple channel, and the right-angle light scattering (RALS) detector by the green channel. A compositional analysis method was used to generate the data in Table 2 for the empty rAAVs.

Table 2: Quantitative parameters for rAAV5 (Empty)

ParameterMonomerDimerAggregatesFragments
Mw (g/mol)3.84 x 1066.98 x 1061.77 x 107821,849
Mw/Mn1.0011.0101.1751.695
Fraction of Sample (%)84.77.22.75.4
Fraction of Protein (%)99.8
Total titer for sample (monomer and aggregate) = 5.91 x 1013 capsids/mL (2.6% RSD)
Figure 2. Triple detection chromatogram of rAAV5 (Empty). Red = RI, Purple = UV 260 nm, Green = RALS.

DLS and MADLS can determine the size of full and empty rAAV5 (27 ± 0.3 and 33 ± 0.4 nm respectively) (Figure 3). Linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 x 1011 to 8 x 1013 capsids/mL for the nominally full rAAV5 samples and 3.4 x 1011 to 7 x 1013 capsids/mL for the nominally empty rAAV5 samples.

Figure 3: Intensity particle size distributions obtained for empty and full rAAV samples using MADLS.

We can infer the structural stability and viral load release from a combination of DLS, SEC-MALS, and Differential Scanning Calorimetry (DSC). The structural characteristics of the rAAV5 start to change from 40°C onwards, with increasing aggregation observed.

In this study, we explored and demonstrated the applicability and value of the orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profile of rAAV5 samples. Importantly, the characterization can be conducted without a need for AAV calibration standards, extensive method development or dedicated reagents.

The findings from this study are also presented in a Malvern Panalytical webinar Delving Deeper Into AAV Attributes: Enhanced Characterization Using Multiple Technologies.