Sneak a peek at your AAV aggregation and titer with Stunner

Quantifying adeno-associated virus (AAV) titer and checking for aggregates are major challenges for developing and manufacturing these gene therapy vectors. The classic AAV tools include chromatography, AUC, TEM, ELISA, and PCR-based methods, but none of these deliver results quickly, easily, and from low sample volumes. When it comes to optimizing processes or running extensive DoEs, faster and easier titers enable evaluation of all the constructs and conditions you want. Since AAV aggregates can mess up the results from many of the classic tools, spotting aggregation before it causes issues can save lots of time and sample.

Stunner gives researchers a full read-out on AAV aggregation and titer on any serotype using only 2 μL of sample. By combining high-speed UV/Vis spectroscopy with static and dynamic light scattering (SLS & DLS), Stunner measures titers down to 1e12?vg/mL and checks size and aggregation of 96 samples in just 1 hour.

AAV aggregation and titer, all at once

AAV samples often contain a mix of intact viruses, free protein, free DNA, and aggregated viruses. Identifying and quantifying these impurities is vital to optimizing any manufacturing process. AAV aggregation in particular complicates quantification since it negatively impacts the accuracy of many titer methods. Only with UV/Vis , DLS and SLS combined can you gain insight into all these impurities at the same time.

Stunner found that an example stock of empty AAV5 contained mostly intact capsids with a small amount of aggregated protein impurities. The total capsid titer of this empty AAV5 was 1.73e13 cp/mL. However, a purchased stock of refined "full" AAV5 with a total capsid titer of 1.56e13 cp/mL contained significantly more aggregation – and contained 70% full capsids. The aggregation and degradation of the AAV could have happened during storage or transportation. Regardless of how clean a sample is or how many full capsids it has, Stunner helps you figure out exactly what you’ve got.

AAV serotypes have different aggregation behavior

Assessing AAV storage conditions is a slow process, made even slower when the only analytical tools you have are bioassays. Functional assays might show the loss of infectious particles but won’t tell you why. Stunner’s DLS detects AAV aggregation in less than a minute so you can tell when a sample has gone bad without wasting time on cell-based assays.

The DLS intensity distributions of AAV2 and AAV9 stored at 4 °C for 2 weeks show AAV2 had peaks at much larger hydrodynamic diameters than AAV9, indicating significant aggregation. Quickly rejecting samples you know won’t work in a cell-based assay saves time and means you can focus on the samples you know will work.

TL;DR

Quantifying AAV aggregation and titers is an arduous task. Other methods are time-consuming and require significant sample amounts. Stunner speeds things up with high-throughput determination of capsid titer, ssDNA titer, and AAV aggregation from tiny sample volumes. Stunner’s combo of UV/Vis and DLS makes it easy to look deeper into AAV quality – identifying aggregates, problem samples, or testing stress conditions in just minutes. Regardless of whether a vector is in research, development or manufacturing, Stunner gives you the data you need and helps you know your capsid inside out.