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).
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 Parameters||Malvern Panalytical Techniques|
|Capsid size||DLS, NTA|
|Absolute Mw of viral capsids and transgene||SEC-MALS|
|Capsid titer or particle count||MADLS®, SEC-MALS, NTA|
|Percentage of genome-containing virus particles/% full analysis||SEC–MALS|
|Aggregate formation||DLS, MADLS, SEC–MALS, NTA|
|Thermal stability||DLS, DSC|
|Higher-order structure analysis||DSC|
|Capsid uncoating and genome ejection||DLS, DSC|
|Binding to receptor||ITC|
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)
|Mw (g/mol)||3.84 x 106||6.98 x 106||1.77 x 107||821,849|
|Fraction of Sample (%)||84.7||7.2||2.7||5.4|
|Fraction of Protein (%)||99.8||–||–||–|
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.
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.