Viral Vector Characterization

Cell and gene therapy involves leveraging a gene delivery vehicle to introduce a therapeutic into the body. Many gene therapy companies are working with Adeno-Associated Virus (AAV) as their gene delivery vehicle of choice. AAVs are ideal because the viral capsids can be modified to carry a therapeutic cargo. Researchers first need to determine what percent of their viral capsids are intact, they then need to understand how many intact viral capsids are full. This transgenic cargo is critical for the success of the therapy.

empty capsid, partial capsid, full capsid the ultimate goal viral vector characterization with auc

It’s particularly important for researchers to understand the viral load of these particles because viral particles that do not contain the full target therapeutic gene are unlikely to produce therapeutic activity, instead these partial capsids might produce adverse reactions, such as an immunogenic response. Researchers need to understand the quality and purity of a given viral prep, and many are turning to analytical centrifugation for this insight.

The solution to measuring rAAV vector homogeneity, purity (and more): analyze viral particles in solution.

Recombinant adeno-associated viral vectors (rAAV) could hold great promise for new, life-saving gene therapies.

But while classic techniques such as dynamic light scattering (DLS) or high performance liquid chromatography (HPLC) can characterize rAAV heterogeneity and aggregation, they simply don’t provide sufficient resolution for quantifying homogeneity and viral particle load. When it comes to producing clinical-grade rAAV vector preparations, one obstacle has always been achieving high-resolution measurements.

As gene therapy researchers know, with so much at stake in early product development, failing fast is fundamental. Adoption of analytical ultracentrifugation makes it possible to produce clinical-grade rAAV vectors, unlike the other usual technologies.

The main obstacle? Lack of a high-resolution, quantitative technique for monitoring therapeutic quality with regard to:

  • Homogeneity
  • Purity
  • Product consistency
  • Viral fullness

The ideal solution: in-solution analysis with analytical ultracentrifugation (AUC).

As scientists such as those at Genethon have discovered, by enabling matrix-free analysis of rAAV vector preparations—independent of serotype and transgene—AUC helps them:

  • Determine viral assembly state or homogeneity
  • Empirically quantify aggregation
  • Determine subparticle contamination
  • Quantify mass to accurately measure genetic payload

View this webinar to learn how Genethon’s Christine LE BEC and her team have used AUC to successfully characterize scAAV and ssAAV vectors—including homogeneity and viral particle load.

Analytical Methods to Measure Empty and Full AAV Particles


As gene therapy approaches usually require large amounts of AAV vectors for clinical use, few manufacturing processes have been reported to provide high titer and potent quantities of products. The dose control of AAV vectors is commonly established on titration methods relying either on the quantitation of DNA (viral genome) or transgene expression following cell transduction (infectious genome). However, AAV vector lots are generally a heterogeneous mixture of empty particles (i.e. do not contain DNA) and full particles (i.e. contain DNA). Therefore, it could be considered that empty particles are product related impurities, which can impact on the immunogenicity profile of the product when high doses are administered to patients. Different indirect methods can be used to establish the ratio between full and empty AAV particles. The quantification of full capsids is performed by using qPCR based technology. The total particles can be evaluated by an ELISA assay, spectrometric analysis, ion-exchange chromatography or SDS-PAGE. However, these methods have limitations and are not applicable to all serotypes without performing a new development. Using analytical ultracentrifugation approach, we have developed a method to quantify simultaneously empty and full AAV particles as well as intermediate species containing fragmented or incomplete vector genome. We have applied this technique to AAV lots of different serotypes, several sizes of transgene and different process of production. Several examples of AAV vectors analysis will be presented showing that AUC could be implemented as a routine test to monitor AAV product quality and manufacturing consistency.

Product for Characterizing Viral Vectors

Optima AUC

The next generation analytical ultracentrifuge frequently used in viral vector QC to quantify the percent load in AAV capsids 

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