Next-Generation Light Scattering: Determining AAV Titer with Multi-Angle Dynamic Light Scattering


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Gene therapy is a rapidly growing area for therapeutic development, due to the potential to cure a wide range of genetic disorders. Although gene therapy has been around for many years, advances in gene editing and the discovery of CRISPR has allowed the potential of such therapies to be realized. As with all proteinaceous therapies, there is a need for detailed and thorough characterization of the therapeutic virus, including accurate determination of viral titer. The viral titer, the number of viral particles per unit volume, is a very important product parameter, as it is directly related to the efficacy of the product as well as allowing monitoring of processing efficiency.

Zetasizer UltraAdeno-Associated Virus (AAV) is a small virus with a diameter of approximately 25 nm and has become the viral vector of choice for most gene editing therapies due its attractive properties, including its low pathogenicity, ability to infect non-dividing cells, ability to integrate into a specific location on human chromosome 19 and its tunable tropism toward different tissue types. To date, there have been over 130 clinical trials worldwide utilizing AAV as a viral vector.

Despite the well-known structure of AAV, gene therapy researchers are still limited in their ability to rapidly measure AAV particle concentration. Due to its small size, AAVs are too small for titer determination from some existing rapid measurement technologies, such as Nanoparticle Tracking Analysis, and so industry has adopted techniques like quantitative polymerase chain reaction (qPCR) and colorimetric assays such as enzyme-linked immunosorbent assay (ELISA) to measure genome counts and capsid proteins, to estimate AAV titer. However, these methods require hours or even days to complete. As a result, there is high demand for a method that can rapidly and directly measure AAV particle concentration. With the development of Multi-Angle Dynamic Light Scattering (MADLS®), the Zetasizer Ultra from Malvern Panalytical provides both rapid, accurate viral titer measurements and aggregation profiles in minutes.

Concentration data from capsid ELISA assay and MADLS-based particle concentration
Figure. Concentration data from capsid ELISA assay and MADLS-based particle concentration results shown for three samples: ATCC AAV2 reference sample, Allergan sample 1, Allergan sample 2. All concentrations are given as particles/mL. n = in how many of the three repeat measurements the peak was identified.

A typical intensity-weighted distribution profile from traditional DLS allows rapid assessment of the aggregation profile and therefore insights into the stability of the AAV preparations. However, using an advanced correlation technique called Adaptive Correlation, the Zetasizer Ultra can generate rapid, high quality-data, even for samples that are typically challenging for conventional DLS instruments. This high-quality correlation data, when combined with data collected from three separate scattering angles, allows the measurement of the AAV concentration. The data below contain the MADLS-derived viral titer values, together with the associated ELISA data, for three AAV batches. The measurements were performed on a Zetasizer Ultra, using the low-volume quartz cuvette (ZEN2112) and the particle concentration measurement, which gives both the MADLS size distribution and the concentration per peak. The titer values show good agreement between the two technologies, even when an aggregate peak is present. Each repeat MADLS measurement takes less than 10 minutes and the sample can be kept enclosed inside the cuvette for the whole measurement to minimize the risk of contamination and operator exposure.

These data highlight the capability of Zetasizer Ultra’s MADLS-based particle concentration measurement in providing both the size distribution and accurate titer of AAV virus samples. The titer data are consistent with technologies that are currently used for this application but can be generated in a matter of minutes—a fraction of the time required for the existing techniques. Consequently, the Zetasizer Ultra can provide rapid, valuable data during the development and manufacturing of AAV-based gene therapeutic products.


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