Last time we discussed Adeno-Associated Viruses (AAVs) as tools for gene therapy as well as the first steps in AAV preparation via the determination of the full to empty capsid ratio. Today, we will dive deeper into the process of AAV purification.

Once the ratio of full to empty capsids is established, the next step is the filtration of defective capsids to ensure accurate dosing and effective treatments. In general, to refine the capsid mixture into an Active Pharmaceutical Ingredient (API) containing a highly purified solution of full capsids, three molecular characteristics are leveraged in downstream bioprocessing: size, charge, and hydrophobicity. Most companies utilize a combination of chromatographic skids to take advantage of these properties in a strategic and efficient manner3. However, each purification step reduces the API yield; thus a careful balance between achieving the necessary purity and retaining the API needs to be achieved. Since gene therapy products generally suffer from a relatively low yield in large-scale manufacturing, it is not uncommon to employ fewer chromatographic steps to preserve yield1,2.

Unfortunately, there is limited difference between the full and empty AAV capsids when assessing two of the three properties listed above – size and hydrophobicity – since they are constructed based on a plasmid’s genetic code4. The only difference between them is the presence of DNA in the full capsid, which produces a slight negative charge. The purification process must be designed such that it can leverage this charge variablity2,4,5. To accomplish that, impurities are first removed via chromatography in order to allow for a more accurate resolution between full and empty capsids. Since the difference in charges is small between the full and empty capsids, AAV purification requires more precision than a typical chromatographic purification. Once the impurities have been removed, the resulting solution contains both empty and full capsids. An ion exchange chromatography step must be used to separate the two capsids. Removing the empty capsids is important because their presence can affect the functional capsid concentration and therapy effectiveness, since empty capsids can result in immune system reactions6 (more on this next time).

The next and final step in the process is the full separation of full and empty capsids. Stay tuned for a future post on this topic and more helpful information on AAVs!


  1. “Ion Exchange Chromatography Principles and Methods.” GE Healthcare”,
  2. Rieser, R., Koch, J., Faccioli, G., Richter, K., Menzen, T., Biel, M., … & Michalakis, S. (2021). Comparison of different liquid chromatography-based purification strategies for adeno-associated virus vectors. Pharmaceutics13(5), 748.
  3. Dickerson, R., Argento, C., Pieracci, J., & Bakhshayeshi, M. (2021). Separating empty and full recombinant adeno‐associated virus particles using isocratic anion exchange chromatography. Biotechnology Journal16(1), 2000015.
  4. Gonçalves, M. A., Pau, M. G., de Vries, A. A., & Valerio, D. (2001). Generation of a high-capacity hybrid vector: packaging of recombinant adenoassociated virus replicative intermediates in adenovirus capsids overcomes the limited cloning capacity of adenoassociated virus vectors. Virology288(2), 236-246.
  5. “DNA Is a Structure That Encodes Biological Information.” Scitable by Nature Education,
  6. “Clearing the hurdles of gene therapy manufacturing.” Pharma Manufacturing,

Written by Evan Riley