Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques

Abstract

Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies produced through controlled and economical manufacturing processes, multiple challenges need to be addressed starting from viral capsid design through identification of optimal process and formulation conditions to comprehensive quality control. Addressing these challenges requires fit-for-purpose analytics for extensive characterization of rAAV samples including measurements of capsid or particle titer, percentage of full rAAV particles, particle size, aggregate formation, thermal stability, genome release, and capsid charge, all of which may impact critical quality attributes of the final product. Importantly, there is a need for rapid analytical solutions not relying on the use of dedicated reagents and costly reference standards. In this study, we evaluate the capabilities of dynamic light scattering, multiangle dynamic light scattering, and SEC-MALS for analyses of rAAV5 samples in a broad range of viral concentrations (titers) at different levels of genome loading, sample heterogeneity, and sample conditions. The study shows that DLS and MADLS^® can be used to determine the size of full and empty rAAV5 (27 ± 0.3 and 33 ± 0.4 nm, respectively). A linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 × 10¹¹-8.7 × 10¹³ cp/mL for the nominally full rAAV5 samples and 3.4 × 10¹¹-7 × 10¹³ cp/mL for the nominally empty rAAV5 samples with 3-8% and 10-37% CV for the full and empty rAAV5 samples, respectively. The structural stability and viral load release were also inferred from a combination of DLS, SEC-MALS, and DSC. The structural characteristics of the rAAV5 start to change from 40 °C onward, with increasing aggregation observed. With this study, we explored and demonstrated the applicability and value of orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples.

Keywords: SEC–MALS; differential scanning calorimetry (DSC); dynamic light scattering (DLS); high-throughput; multiangle dynamic light scattering (MADLS®); orthogonal techniques; recombinant adeno-associated viruses (rAAV).

Figure 1

Triple detection chromatogram of rAAV5 (Empty). Red = RI; purple = UV 260 nm; green = RALS.

Figure 2

Triple detection chromatogram of rAAV5 (Full). Red = RI; purple = UV 260 nm; green = RALS.

Figure 3

Comparison of full and empty rAAV titer (capsid particles/mL) using SEC–MALS and MADLS.

Figure 4

A plot of the expected and calculated percentage of full rAAV5 virus particles.

Figure 5

Thermal stress conditions for full and empty rAAV5 stability study with the Zetasizer Ultra. (A) Hydrodynamic diameter trend with temperature. (B) Overlay of intensity particle size distributions at 30 °C and 45 °C for full rAAV5 samples.

Figure 6

RI detection chromatograms of full rAAV5 samples incubated at 45 °C and aliquoted at 2, 5, 10, and 15 min for analysis by SEC–MALS.

Figure 7

A plot of the scattering intensities (in kilocounts per second (kcps)) as a function of temperature for full and empty rAAV5 samples.

Figure 8

Thermal ramps of full rAAV5 capsids using different buffer and experimental conditions. Sample 1 = PBS at 1 °C/min; Sample 2 = PBS at 3 °C/min; Sample 3 = PBS plus 20 mM EDTA at 1 °C/min.

Figure 9

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

Figure 10

Hydrodynamic sizes of full and empty rAAV5 samples measured at a range of dilutions expressed in volume-by-volume percentages using MADLS measurements.

Figure 11

Particle concentrations values obtained for empty and full rAAV5 samples across a range of dilutions expressed in volume-by-volume percentages.

Figure 12

Overlay of the DSC traces of nominally empty and nominally full rAAV5 samples. DSC data corrected for the instrumental blank and the baseline. Vertical lines mark regions with distinctly different regimes of thermally induced processes.