Adeno-associated virus (AAV), a prominently used vector in human gene therapy is a small, non-enveloped parvovirus consisting of a single-stranded DNA genome enclosed in a protein capsid.
AAV vector is used to transfer therapeutic genes to humans for the treatment of monogenic diseases, ocular diseases, and clinical trials of deficiencies like hemophilia and thalassemia. Its non-pathogenic nature, low immunogenicity, ability to retain long-term gene expression, and low likelihood to induce an inflammatory response make it very promising gene therapy vector.
This blog post provides a brief outlook on the importance of quantitation in AAV process development and how the innovative solutions from Gator Bio fill this gap in AAV characterization.
Importance of quantitation
Accurate quantitation of the AAV genome and capsid titer is an important step in AAV process development to ensure safe and efficacious dosing. High vector doses of AAV due to inaccurate quantitation can lead to an inflammatory response that is dangerous for sensitive organs such as the eyes.
Conventional AAV quantitation include PCR-based methods (qPCR). The major drawback of PCR-based quantitation is that it does not provide any information on the loss of integrity in viral particles. Moreover, PCR assays are prone to contamination by salts, proteins, or plasmid DNAs, and thus pose a risk of inaccurate quantitation.
Other established immunoassays such as ELISA, and Dot blot for capsid titer quantitation are time-consuming, labor-intensive, and thus significantly impede the AAV process development cycle and its manufacturing.
Gator Bio’s solutions for accurate quantitation
Gator Bio has developed Gator® Plus, unique plug-n-play, cost-effective high-throughput bioanalytical tool for accurate, high-sensitivity serotype quantitation in a highly parallelized, automated manner. It is based on Biolayer Interferometry (BLI) technology and uses CaptureSelect AAVX nanobody probes to bind and quantitate AAV capsid serotypes including AAV 1-8 and 10 with a dynamic range of 5×106 – 1×1013 vp/mL. The probes are compatible with various matrices used in process development The assay developed using these probes can be used for quantitation of AAVs from harvesting stage to final product QC Eight channels are operational in parallel and can process up to 96 samples within less than 30 minutes, resulting in high throughput quantification. The probes render an impressive regeneration performance and are reusable up to 10 times without any loss in binding, thus saving a lot of process time and operating costs.
Integration with GatorOne® software allows easy data analysis and sample AAV concentration measurement. It allows saving multiple standard curves needed to quantify different AAV serotypes, thereby saving the time to generate standard curves repeatedly, thus enhancing the process efficiency.
Drop us a line if you would like to know more about our quantitation solutions.
- Martinez-Fernandez de la Camara, C. et al. Genes 2021, 12, 601. https://doi.org/10.3390/genes12040601
- Shmidt, A.A. et al. Pharmaceuticals 2022, 15, 23. https://doi.org/10.3390/ph15010023
- Cui, M. et al. Molecules 2019, 24, 3973; doi:10.3390/molecules24213973