Mastering AAV Titre Determination

Dana Holzinger, Head of Product Management at PROGEN gave a highly insightful overview of the challenges associated with AAV titre determination. Ensuring that you have highly precise quantification methods is key. Holzinger argued that the most crucial challenges are accuracy and comparability. Methods must be precise and sensitive to ensure reliable titre measurements. Variability among orthogonal methods can lead to inconsistencies across labs. While the ELISA method is the most commonly used assay, Holzinger proposed an alternative solution. She demonstrated that by automating their ELISAs, PROGEN could improve reproducibility, efficiency, and throughput. Furthermore, automating ELISAs is useful in a high throughput setting, Holzinger stated: “If you are in a high throughput screening setting, then you really are suffering maybe from the number of samples, but you would like to reduce the hands-on time, maintain at least reproducibility and accuracy of your test system and you would like to reduce the time pressure.” Holzinger compared various orthogonal methods, such as biolayer interferometry (BLI), capillary electrophoresis (CE), size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS), and dynamic light scattering (DLS). These methods offer varying advantages and challenges, often influenced by AAV serotype and assay design. For instance, while ELISA and BLI showed consistent results using shared standards, discrepancies emerged with CE. Holzinger discussed CE results briefly: “With regard to the titre recovery, we had some good correlation for AAV9. However, AAV8 was overestimated while AAV2 and AAV5 were underestimated,” she continued to explain, “And this discrepancy might be due to the to the use of different standard materials in both immunoassays.” Biophysical methods like SEC-MALS, demonstrated high precision but occasionally underestimated or overestimated titres depending on the serotype. The variability among methods showed that there is room for improvement particularly when investigating serotype-specific factors, such as the aggregation tendencies of AAV2. Holzinger stated: “However if you have a closer look again at the AAV2, you'll see that three out of the four orthogonal methods were out of the range. So when we know that AAV2 has a strong tendency to form aggregates, aggregations, and that might be a factor that could influence several methods here,” she added, “And of course, these serotype-specific and also method-dependent factors need to be analysed further if we again have a closer look to the immune assays.” Developing and characterising high-quality reference standards are key to improving method comparability. PROGEN’s standards undergo rigorous quality control, including genomic titre measurement, full/empty capsid ratio analysis, and purity assessment. These standards are tailored to commonly used serotypes, ensuring high purity, consistent viral genome loads, and low endotoxin levels. Although methods like ELISA remain indispensable, advancements in orthogonal techniques and robust standards are leading to more precise and scalable solutions to meet demands in gene therapy. Holzinger showed that interrogating traditional methods can go above and beyond to tackle AAV capsid titre determination.