Adeno-associated Virus

Improving Efficiency of rAAV Production and Purification with Chromatography

Since the discovery of the adeno-associated virus (AAV) in 1965, its use in gene therapy as a transfer vector has increased along with demand for characterization, production, and purification. Recombinant adeno-associated virus (rAAV), the smallest particulate gene delivery vector at 20 nm in diameter, is derived from a small, nonenveloped icosahedral virus with a linear single-stranded DNA genome of 4.7–6 kilobases (kb). The small size of the virion permits the packaging of only 5 kb of exogenous DNA, including the promoter. More recent work has exploited the unique ability of AAV to link together in doublets or strings to bypass this size limitation [1,2].

More than twelve naturally occurring serotypes of AAV have been characterized, each with unique surface proteins and glycosylation patterns [3,4]. Novel serotypes have also been developed. Advantages of rAAV include non-pathogenicity, low immunogenicity, and the ability to transduce specific target tissues (i.e., tissue tropism) and establish long-term gene expression. Understanding these differences in tropism is critical when choosing a specific gene delivery vector to achieve a successful therapeutic outcome [5,6]. With a better understanding of AAV structure and biology, new vectors with modifications to the genome or capsids may be designed to facilitate more specific transduction of cells for gene therapy. Also, structural differences in surface proteins and aminoglycans may affect efficient approaches for vector purification.

Production of rAAV typically follows a streamlined workflow of plasmid production, cell expansion and plasmid transfection, viral vector production, and viral recovery and purification before the final fill-finish (Figure 1). Each step in the workflow influences the quality of material produced and the efficiency of the process. Downstream purification depends on the clearance of impurities such as host cell proteins, debris, media components, exogenous DNA, and empty vector particles. Designing an efficient and effective downstream purification system involves using advantages of different chromatography types with the least amount of processing steps to minimize viral vector loss. Approaches to vector purification exploit the viral particle physical characteristics such as size, surface charge, and hydrophobicity. Viral vectors vary greatly in physical characteristics depending on the type of virus and for the same virus, can vary depending on serotype and transgene inserts. Greater purity of the final vector can reduce immunogenic responses and increase the efficiency of gene transfer. However, if purification is not ideal, rAAV may lose essential structural or genetic components [7,8].

Overview of AAV production workflow for gene therapy

Figure 1. Typical workflow for rAAV production.

One primary consideration when moving to clinical manufacturing for gene therapy is how to increase production scale. At larger scales, the purification methods need to accommodate large volumes with high efficiency and speed. Historical purification methods involved tedious and unscalable ultracentrifugation or lengthy density gradient steps, which were a bottleneck to rAAV production. Recent advances in production methods now focus on scalable chromatography [7,9]. AAV virions are relatively stable to heat, nonionic detergents, and proteolytic enzymes, making them good candidates for chromatographic purification [10]. Two standard modes of chromatography applicable to downstream purification include affinity and ion exchange. As with all chromatography, results are sensitive to buffer and mobile phase composition. The importance of precise buffer systems will be discussed in relation to these different types of chromatography.

Affinity chromatography

Affinity chromatography relies on the interaction of the external capsid structure of a viral particle with a ligand. While AAV serotypes share a common capsid morphology, the presence of specific amino acids in certain regions determines whether antibodies are able to bind to them [11]. Different types of affinity resins interact with unique capsid structures present in different serotypes, offering options for AAV vector–specific purification. Novel AAV variants that are mosaics of capsid subunits from different serotypes are being developed to help evade preexisting neutralizing antibodies generated as a result of the humoral immune response to natural infection or prior treatment with AAV-based vectors [8]. Affinity resins may be used to efficiently purify these new types of AAV.

However, during purification of both naturally occurring serotypes and in mosaic AAV, non-specific binding of impurities can occur, decreasing the efficiency of product production. Another limitation of affinity chromatography is that it cannot easily separate full vectors from empty capsids. This process requires additional purification steps that may involve ion-exchange chromatography or ultracentrifugation. Centrifugation purification has limited utility because it is difficult to scale-up and is relatively harsh on the virus capsid.

Affinity chromatography combined with ion-exchange chromatography is commonly implemented to maximize AAV purification efficiency [9,12,13].

Ion exchange chromatography

Ion exchange chromatography involves the separation of ionizable molecules based on their total charge. Ion exchange resins consist of two main types: cation exchange resins that exchange positively charged ions and anion exchange resins that exchange negatively charged ions. The charge on the molecule of interest may be manipulated by changing buffer pH or salt concentration, allowing separation of biological molecules such as proteins, peptides, and rAAV.

Surface proteins on the capsids of different AAV serotypes have charges that allow binding to ion-exchange chromatographic resins [6]. It is important to note that when working outside of their optimal pH range, some resins may rapidly lose capacity and resolution. When the pH is higher than the isoelectric point (pI) of the AAV capsid, the net charge on the protein is negative, allowing binding to an anion exchanger. Conversely, when the pH is lower than the pI, the net charge on a protein is positive, and it can bind to a cation exchanger [8,14].

Ion exchange resins work well when used with low-salt buffers. Higher salt concentrations may impede binding to the resins and cause osmotic shock, resulting in ruptured viral capsids [9]. The inclusion of buffer-exchange steps before ion-exchange chromatography may prevent the osmotic shock issue. Other methods require the use of salt gradients that are optimized to separate viruses from impurities.

The principles of ion exchange chromatography can be used in either a column or membrane format. Membranes characteristically have a binding ligand immobilized on their absorbing surface that allows protein binding. Although membranes generally have a lower binding capacity, they have a larger surface area, which allows the use of faster flow rates. In some cases, the use of membranes may result in greater productivity [15,16].


Precise buffer composition is critical for reliable and efficient rAAV purification. Due to specific differences in capsid morphology, complete buffer systems designed for each AAV serotype are of great importance. Capsid sequences of different AAV serotypes show electrostatic differences that are reflected in their pI. Optimizing the affinity of the viral particles for the resin will allow better recovery rates of purified material.

Multiple buffers are used throughout the purification process, and the composition of each will affect the final result. A buffer containing the wrong counterion can prevent binding of the protein of interest to the column resin. The charged species in buffers used for ion-exchange chromatography should generally have the same charge as the resin. For example, although phosphate buffers are commonly used for protein purification, they are not appropriate for anion exchange chromatography because the phosphate ion interacts strongly with positively charged anion exchange resins [8,14].

Ion exchange resins and membranes used in the purification process may lose binding capacity and resolution outside their optimal pH range [15]. Anionic and cationic beads and membranes must be matched with a buffer or mobile phase of appropriate pH. Depending on their environment, proteins may carry a net positive charge, a net negative charge, or no charge. In addition to monitoring pH, measuring the conductivity of solutions is another way to ensure consistency in buffer composition and performance.

Looking beyond the purification process, the effectiveness of the final rAAV product is influenced by the same buffer variables discussed earlier. Some AAV serotypes have functional proteases, enzymes that catalyze autolytic cleavage of the capsid as well as external substrates [17]. Outside of their ideal pH range, AAV proteases also show reduced function. These proteases induced significant autocleavage of capsid proteins at pH 5.5, while no autocleavage was seen at pH 7.5. Precise pH ranges have been shown to be essential for productive AAV infections [17].


Gene therapy products, as with many biologics, are quite complex and difficult to characterize. Current FDA guidance for gene therapy notes that for purity, the main concerns are impurities that can be process-related and include residual DNA, protein, or culture reagents. Consistency in the manufacturing process is critical to producing a consistent product. Having reliable sources for all production components, especially buffers, will allow greater control of the process and permit focus on the more variable aspects of rAAV production.


Learn more about Teknova Resources for Gene Therapy. To consult with our rAAV specialists about your project, email us at


  1. Chamberlain K, Riyad JM, Weber T. (2016) Expressing transgenes that exceed the packaging capacity of adeno-associated virus capsids. Hum Gene Ther Methods, 27(1):1–12. doi: 1089/hgtb.2015.140
  2. Wang D, Tai PWL, Gao G. (2019) Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov, 18(5):358–378. doi: 1038/s41573-019-0012-9
  3. Hammond SL, Leek AN, et al. (2017) Cellular selectivity of AAV serotypes for gene delivery in neurons and astrocytes by neonatal intracerebroventricular injection. PLOS ONE, 12(12):e0188830. doi: 1371/journal.pone.0188830
  4. Viney L, Bürckstümmer T, et al. (2021) Adeno-associated Virus (AAV) capsid chimeras with enhanced infectivity reveal a core element in the AAV genome critical for both cell transduction and capsid assembly. J Virol, 95(7): e02023-20. doi: 10.1128/JVI.02023-20
  5. Balakrishnan B and Jayandharan GR. (2014) Basic biology of adeno-associated virus (AAV) vectors used in gene therapy. Curr Gene Ther, 14(2):86–100. doi: 10.2174/1566523214666140302193709.
  6. Srivastava A. (2016) In vivo tissue-tropism of adeno-associated viral vectors. Curr Opin Virol, 21:75–80. doi: 10.1016/j.coviro.2016.08.003
  7. Clément N and Grieger JC. (2016) Manufacturing of recombinant adeno-associated viral vectors for clinical trials. Mol Ther Methods Clin Dev, 3:16002. doi: 1038/mtm.2016.2
  8. Naso MF, Tomkowicz B, et al. (2017) Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs, 31(4):317–334. doi: 10.1007/s40259-017-0234-5
  9. Tomono T, Hirai Y, et al. (2018) Highly efficient ultracentrifugation-free chromatographic purification of recombinant AAV serotype 9. Mol Ther Methods Clin Dev, 11:180–190. doi: 1016/j.omtm.2018.10.015
  10. Burova E and Ioffe E. (2005) Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications. Gene Ther, 12(S1):S5–S17. doi: 10.1038/
  11. Adachi K, Dissen GA, et al. ( 2020) Adeno-associated virus-binding antibodies detected in cats living in the Northeastern United States lack neutralizing activity. Sci Rep, 10(1):10073. doi: 10.1038/s41598-020-66596-4
  12. Nass SA, Mattingly MA, et al. (2018) Universal method for the purification of recombinant AAV vectors of differing serotypes. Mol Ther Methods Clin Dev, 9:33–46. doi: 1016/j.omtm.2017.12.004
  13. Guidance for industry: chemistry, manufacturing, and control (CMC) information for human gene therapy investigational new drug applications (INDs). [PDF] US Food and Drug Administration [Accessed April 13, 2021]
  14. Ion exchange chromatography. Bio-Rad Laboratories [Accessed April 13, 2021].
  15. Wang C, Mulagapati SHR, et al. (2019) Developing an anion exchange chromatography assay for determining empty and full capsid contents in AAV6.2. Mol Ther Methods Clin Dev, 15:257–263. doi: 1016/j.omtm.2019.09.006
  16. Boi C, Malavasi A, et al. (2020) A direct comparison between membrane adsorber and packed column chromatography performance. J Chromatogr A, 1612:460629. doi: 10.1016/j.chroma.2019.460629.
  17. Kurian JJ, Lakshmanan R, et al. (2019) Adeno-associated virus VP1u exhibits protease activity. Viruses, 11(5):399. doi: 10.3390/v11050399