ForteBio
Hands-on time reduced by 50%. Sample prep time reduced by 15-25%. Near real-time bioprocessing decisions.
Rapid and accurate methods for the quantitation of Adeno-associated virus (AAV) particles are an unmet need for advancing bioprocessing in gene therapy. Viral capsid titer is commonly measured by ELISA, empty vs. full capsid titer differentiation and ratio is obtained by Analytical Ultracentrifugation (AUC), and viral genome titer is measured increasingly by ddPCR1. These methods are generally time consuming and labor-intensive, and are hence not practical for at-line, rapid measurement of viral titer during bioprocessing and manufacturing.
ForteBio has developed a quick, high-throughput and robust AAV2 capsid quantification method, capable of virus titer determination in samples along the purification process. The method accelerates assay of multiple 96-well or 384-well plates in less than 1 hour, saving significant time and labor compared to ELISA and ddPCR assays which require approximately 5 and 8 hours respectively, and involve significant hands-on time at the bench.
Octet® instruments utilize the Bio-Layer Interferometry (BLI) technology in combination with 96- or 384-well sample plates and off-the-shelf biosensor probes to provide rapid and accurate analysis of biomolecular interactions in real time. The Octet platform has been used for both titer and kinetic properties determination of molecules ranging from recombinant proteins to monoclonal antibodies. In virus vaccine studies, Octet systems have been deployed for research on a diverse range of virus and virus-like particles including HIV, influenza virus and Ebola virus2.
The Octet AAV2 titer assay demonstrated excellent precision and reliability, with very minimal matrix effects relative to ELISA and ddPCR. The method can be extended as a generic viral titer assay for at-line testing of any AAV serotype in a bioprocess setting. The development of the generic method can be achieved by coating the appropriate specific capture ligand on a commercially available biosensor. We postulate that the Octet platform offers near real-time feedback on the bioprocess, saving significant time and resources, thereby enhancing efficiency and productivity for virus manufacturing. Some of the key highlights of the Octet AAV2 quantitation assay are listed in Table 1.
In our latest application note, we describe the development of an assay for quantifying AAV2 virus particles. The assay was constructed by capturing AAV2 virus using heparin immobilized onto Streptavidin biosensors. We show that the working assay can be used to quantify AAV2 particles from purified as well as complex bioprocess matrices with a dynamic range of 4.15x108 – 2.66x1010 gc/mL (genome copy/mL). Depending on the Octet instrument used, the quantitation assay can be completed in as fast as 30 minutes, significantly accelerating assay timelines compared to ELISA and ddPCR based methods. The Octet assay can be extended to any AAV serotype by using an appropriate capture molecule and following the assay development steps described herein. Download application note.
References
1 Dobnik D, Kogovšek P, Jakomin T, Košir N, Tušek Žnidaric M, Leskovec M, Kaminsky SM, Mostrom J, Lee H and Ravnikar M, Accurate Quantification and Characterization of Adeno-Associated Viral Vectors, Front. Microbiol., 2019, 10:1570. doi: 10.3389/fmicb.2019.01570.
2 Rejane Petersen, Strategies Using Bio-Layer Interferometry Biosensor Technology for Vaccine Research and Development, Biosensors (Basel), 201,7 Oct 31, 7(4), pii: E49, doi:10.3390/bios7040049.