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The Importance of AAV Characterization in Ensuring Safe Gene Therapy Development

January 4, 2024

3d rendering of an adeno associated virus

Adeno-associated virus (AAV) capsids have emerged as frontrunners for in vivo gene delivery thanks to their unique safety profile and ability to provide long-term gene expression. With multiple serotypes of recombinant AAV vectors being utilized across various gene therapy programs, the field is witnessing unprecedented growth.

As of April 2020, there are 244 active worldwide gene therapy trials involving AAVs, including 24 in Phase III clinical trials.1 Even still, the path to developing comprehensive AAV analytics for process development and final drug product assessment is not without its obstacles.

Accurate AAV characterization is crucial to ensure the efficacy of gene therapies.1 Visible and subvisible particle (SVP) analysis plays a key role in this process. However, when applied to AAV-based gene therapies, it presents a unique challenge due to limited material availability for testing. Current AAV characterization methods like dynamic light scattering (DLS), size exclusion chromatography (SEC), light obscuration (LO), and flow imaging (FI), require large volumes of material, can be costly as well as time-consuming, creating bottlenecks in gene therapy development and manufacturing.1

Furthermore, these techniques do not provide a complete picture, as they focus primarily on soluble and stable AAVs. It’s equally crucial to study insoluble and unstable AAVs to identify any potential vulnerabilities or irregularities in the viruses and ensure their safe and effective use.

Thus, there is a need for new solutions that can overcome these challenges and help speed up the development and advancement of gene therapies.2

Traditional vs. Novel Analytical Approaches to Characterize AAV

Ensuring the consistent quality of AAV gene therapies is contingent on the ability to quickly characterize critical quality attributes (CQAs). This includes the measurement of virus titer, capsid content, and aggregation, all of which have an impact on the potency, purity, and safety of AAVs.3 Unfortunately, existing AAV characterization methods for measuring these attributes often take a long time or don’t work well for process development, but new rapid, high-throughput AAV analytical methods are being developed.3

In a recently published study in Science Direct, researchers examined both conventional and emerging techniques for measuring recombinant AAV (rAAV) quality attributes.

rAAVs are a promising gene therapy vector, but their quality attributes must be carefully analyzed to ensure safety and efficacy. Traditional AAV analytics and quality control assays are time-intensive and may not be suitable for rapid product development timelines. Rapid, high-throughput AAV analytical methods can yield actionable results swiftly, but they can be challenging to develop and implement due to limited sample quantities and matrix effects.

Despite these challenges, there has been significant progress in developing rapid, high-throughput analytical methods for rAAV characterization, especially for three potential CQAs: capsid titer, content ratio, and aggregate content. Chromatography and light-scattering-based methods can now be used to characterize these CQAs directly at or online, enabling real-time process monitoring and control.

According to the paper’s authors, some of the key benefits of rapid, high-throughput analytical methods for rAAV characterization include:

  • Faster product development: Rapid analytical methods can help accelerate product development by providing timely feedback on process performance and product quality.
  • Reduced costs: Rapid analytical methods can help to reduce costs by reducing the time and resources required for quality control testing.
  • Improved product quality: Rapid analytical methods can help to improve product quality by enabling real-time process monitoring and control.

Yet challenges do remain:

  • Limited sample quantities: rAAV production typically yields only small volumes of purified product, which can limit the amount of sample available for analysis.
  • Matrix effects: The presence of other molecules in the rAAV sample, such as buffers, residual DNA or protein, or ionic salts, can interfere with analytical measurements.
  • Method development: Developing rapid, high-throughput analytical methods that are accurate, reliable, and reproducible can be challenging.

The authors concluded that the development of AAV analytical methods that can support short timelines and high throughput of process development, while also maintaining reproducibility and specificity, will be critical.3

Beyond Traditional AAV Characterization Methods: Gaining Deeper Understanding of Samples with Aura

Characterizing AAV aggregation and stability at ultra-low volumes is crucial for achieving product purity and preventing DNA leakage or contamination. Technological advancements have opened up new possibilities in this area so it’s now feasible to rapidly screen for instability and AAV characterization for aggregation in the ultra-low volumes that make sense for gene therapy development. With Aura’s Side Illumination Membrane Imaging (SIMI) and Fluorescence Membrane Microscopy (FMM), technology combined with BMI’s refractive index capabilities, you can gain a deeper understanding of samples to identify protein or non-protein contents along with intrinsic or extrinsic elements—something that traditional flow-based particle analyzers could not provide. This allows for greater protection against costly recalls due to unseen particulate contamination.

Unlike flow imaging, which requires milliliters of sample, Aura systems can quickly and easily characterize viral vectors and other types of gene therapies with just 5 µL of sample. This is due to analyzing samples via membrane microscopy, which requires meniscal amounts while maintaining 100% sampling efficiency.

Aura GT technology also allows for AAV characterization with high throughput (<1 min/sample), quickly revealing insights into their stability. The system helps to ensure that a gene therapy’s most important stability characteristic — its SVP concentration — can be monitored throughout process development and through quality control testing.

Evaluate AAV stabilty under a variety of perturbations quickly and obtain quantitative results.
AAV2 vector was tested for aggregation under a variety of stress conditions—(a) the concentration of particles with an equivalent circular diameter (ECD) ≥ 2 μm and (b) the percentage of the membrane covered by particles.

Employ Multiple AAV Analytical Methods

By implementing a rapid analytical method for AAV characterization, you can begin candidate screening and selection much earlier in development, providing the confidence needed when commercializing your gene therapy.

Related
References
  1. McIntosh, N.L., Berguig, G.Y., Karim, O.A. et al. (2021). Comprehensive characterization and quantification of adeno associated vectors by size exclusion chromatography and multi angle light scattering. Scientific Reports, 11, 3012. https://doi.org/10.1038/s41598-021-82599-1
  2. Cole, L., Fernandes, D., Hussain, M.T., Kaszuba, M., Stenson, J., & Markova, N. (2021). Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques. Pharmaceutics, 13(4), 586. https://doi.org/10.3390/pharmaceutics13040586
  3. Gimpel, A.L., Katsikis, G., Sha, S., Maloney, A.J., Hong, M.S., Nguyen, T.N.T., Wolfrum, J., Springs, S.L., Sinskey, A.J., Manalis, S.R., Barone, P.W., & Braatz, R.D. (2021). Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies. Molecular Therapy – Methods & Clinical Development, 20, 740-754. https://doi.org/10.1016/j.omtm.2021.02.010