Quantification of AAV Capsid Loading Fractions: A Comparative Study

Akash Bhattacharya, Shawn M Sternisha, Ross VerHeul, Kyle Curry, Jian Zhang, Sean Kelly, Mingjuan Liu, Jeffrey Hung
Beckman Coulter Life Sciences, Indianapolis, IN
Vigene Biosciences, Rockville, MD


The Therapeutic Context:

Gene therapy has the potential to treat a wide range of diseases from cancer to cardiovascular disorders. Disease caused by mutated or defective genes can be treated by therapeutic genes which
have to be packaged and delivered to the target cells. Recombinant Adeno Associated Virus (rAAV) vectors are one of the most promising mechanisms for therapeutic gene delivery.


The Viral Vector Production / Quality Control Challenge:

The production of large quantities of bioactive rAAV particles is fraught with production challenges such as maintaining integrity and maximizing the packaging efficiency of the delivery vehicle. A typical rAAV prep will include bioactive particles/capsids which are fully loaded with the therapeutic gene, as well as undesirable but partially loaded and empty capsids. Identifying and quantifying these different species is vital from a quality control standpoint, since improperly loaded viral capsids do not produce the desired therapeutic effect, but still elicit an immune response.

Solving the Challenge with AUC:

Analytical Ultracentrifugation (AUC) is a native state, solution phase, first-principle technique used to quantitate the amounts of empty, full and partially loaded AAV capsids. Here we present the results of a panel of blind tests on AAVs which were conducted to compare and contrast the empty/full ratio readout from AUC vs. that from Transmission Electron Microscopy (TEM). The AAV samples used are the AAV reference standard materials from Vigene Biosciences. We observe that AUC can potentially have more resolution to distinguish partially vs. fully packaged capsids.

Adeno Associated Virus as a Gene Therapy Vector 



AAV Packing Fraction


AUC Workflow


AUC can quantitate AAV Empty/Partial/Full Ratios


TEM can quantitate AAV Empty/Full Ratios but cannot detect partially loaded capsids



AUC experiments provided a native-state, serotypeindependent quantification of Empty/Full ratios for the Vigene AAV Reference Standards in a single blind test.

  • Analyzed data from AUC is in agreement with orthogonal data from TEM.
  • Data from AUC experiments can be analyzed to quantify partially loaded capsids, which TEM cannot discriminate.

Disclaimer: Several third party software vendors supply programs for the analyses of the raw data generated by the Beckman Coulter Inc. (“Beckman”) Analytical Ultracentrifuge. Third party analysis software has not been validated by Beckman for use with the Beckman Analytical Ultracentrifuge. Training conducted by Beckman on the third party software, does not imply a recommendation for use or the suitability of the third party software use by the customer. Beckman does not endorse any third party analyses software. Beckman warranty and/or performance guarantee that may be applicable or are provided by Beckman for Beckman Analytical Ultracentrifuge do not apply to any third party software. Copyright, License and Terms of Use Disclaimers are documented for the below software on their respective pages: Sedfit, https://sedfitsedphat.nibib.nih.gov/software/default.aspx Beckman Coulter makes no warranties of any kind whatsoever express or implied, with respect to this protocol, including but not limited to warranties of fitness for a particular purpose or merchantability or that the protocol is non-infringing. All warranties are expressly disclaimed. Your use of the method is solely at your own risk, without recourse to Beckman Coulter. Not intended or validated for use in the diagnosis of disease or other conditions. This protocol is for demonstration only, and is not validated by Beckman Coulter.

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