About adeno-associated virus

Adeno-associated virus (AAV) is a common viral vector for in vivo gene therapy because AAV induces low immunogenicity and pathogenicity and doesn’t integrate into the host genome. In addition, AAV is associated with stable and high in vivo expression levels. Several serotypes of AAV vectors are in use, with specificity for particular target tissues. In addition to the natural serotypes, several synthetic variants have been developed with the goal of improving the targeting and efficacy of the AAV-based gene therapy treatments.

AAV is a nonenveloped single-stranded DNA virus that’s smaller than other viral vectors (e.g., lentivirus and adenovirus) used to deliver genetic information. Despite its relative small size in the virus world, an AAV particle is ~ 5-fold larger and has a dramatically different structure than a monoclonal antibody (mAb; Fig 1). Keep these differences in mind when developing a manufacturing process for AAV, which will borrow from standard bioprocessing technology.

Relative size of an AAV particle vs a mAb.

Fig 1. Relative size of an AAV particle vs a mAb.

Workflow for AAV production

AAV is relatively stable compared with lentivirus, and it’s less sensitive to pH and salt fluctuations. Figure 2 shows a generic workflow for AAV production, which includes many of the same steps as a mAb workflow. Functional AAV vector is the product at the end of the process.

Generic workflow for adeno-associated virus (AAV) vector.

Fig 2. Generic workflow for adeno-associated virus (AAV) vector.

AAV cell culture – what to consider

When developing an upstream AAV process, you will optimize the conditions for AAV production. Consider how you will produce the AAV vector, as well as which equipment and reagents you will use. Throughout the workflow, single-use, closed systems help to manage biosafety concerns and minimize the risk of contamination. They also reduce steps and manual handling.

There are several options for an AAV production platform. The pros and cons of each are summarized in Table 1.

Table 1.
AAV production platforms



Plasmid transfection – HEK293 with or without T antigen • Scalable
• Flexible
• Quick
• High cost – multiple plasmids, large quantity needed as you scale up
• Low to moderate titers
• High percentage of empty capsids (serotype dependent)
Baculovirus expression in insect cells (Sf9) • Scalable with high titers • Requires removal of baculoviruses downstream
• Requires extensive lysis to release AAV
Cell line induced by virus infection – dependent on helper virus • Can achieve high viral titers • Need to remove helper virus during purification
Stable, inducible producer cells • Scalable and reproducible
• Can be more economical than plasmid-based process
• High titers
• Challenging and time consuming to prepare from scratch
• Is less flexible for AAV design modifications
• Available cell lines may be subject to intellectual property (IP)

Tip: Regardless of the production platform that you choose, it's very important to consider certain things, such as the cell line documentation, testing, and safety track record. This will help you with your regulatory submission.

Also, think about whether you will use adherent or suspension culture. Adherent cultures can give you higher productivity per cell. But on the other hand, they’re more difficult to scale up. One option for scaling adherent cells is to use microcarriers to produce AAV vectors production. This lets you grow adherent cells in bioreactors. If you choose to adapt cells for suspension culture, you will need to decide whether to do this directly or sequentially. If you do a direct adaptation as we did at Cytiva, it’s important to control viability, robust cell growth, and aggregation. When it comes to scalability, a suspension cell line is preferred.

And finally, consider whether you will supplement your culture with serum, which can boost titers.

Tip: We strongly recommend using a chemically defined cell culture medium without any animal-derived components. This is clearly a regulatory advantage.

AAV purification – what to consider

After the AAV vector is produced, it needs to be released from the cells, even in serotypes that can secrete it. A scalable lysis uses a detergent, such as Tween™ 20. Also, host cell DNA (hcDNA) must be fragmented to reduce viscosity. This will improve the capacity of the filtration steps and ease hcDNA removal in the downstream steps. Optimizing the lysis/fragmentation step, including the amount of DNA nuclease, will reduce cost and improve the performance of the clarification step.

Tip: Check to ensure that the detergent or other additives are suitable for a manufacturing environment. For example, instead of Triton™ X-100, which is on the authorization list (Annex XIV) of REACH, we use Tween™ 20.

After lysis/fragmentation, the cell debris is removed during a clarification step. Evaluate the need for depth filters to efficiently remove particulates.

Tip: Evaluate using uncharged or charged depth filters to efficiently clarify the harvest before the final normal flow filtration (NFF) step. For challenging feeds, consider a low pH precipitation step prior to NFF.

Next is a buffer exchange step to prepare for the AAV vector capture step. Consider whether you need to concentrate the AAV vector to reduce the loading time or to improve results in the capture step. Choose an appropriately sized filter and nominal molecular weight cutoff (NMWCO) and depth filters (e.g., PDP8), if needed, that will separate the virus from debris without premature filter clogging and excess loss of virus.

Tip: We recommend using hollow fibers with a NMWCO of 300 kDa to maximize the removal of impurities into the permeate while retaining the AAV. It may be possible to omit this step for other chromatography formats like membranes or fibers.

When designing a purification scheme, consider the goals of each step and the available options. Keep scalability in mind. Then optimize the conditions to maximize recovery and purity.

The objectives of the capture step are to isolate, stabilize, and concentrate the virus. Some of the options for this step are summarized in Table 2.

Table 2.
Some options for the capture step in AAV purification



Affinity chromatography resin • High impurity reduction ― efficient
• Suitable for large-scale processes
• May allow for platform solution for several serotypes
• High capacity
• May need to concentrate material prior to loading
• Harsh elution conditions (e.g., low pH)
• Affinity can vary for different AAV serotypes and recombinant capsids
• Aggregation with some serotypes
• Does not separate empty and full capsids
Ion exchange resin • Flexible – not serotype dependent
• Possible to separate empty and full capsids
• Scalable
• Compatible with single-use technology
• Will need more extensive process development than affinity resins
• Lower capacity – conditions may need to be optimized depending on serotype
• Lower cost
• Lower impurity reduction
• Lower recovery

The next purification step, the polishing step, aims to remove both process and product impurities. The major challenge is to purify full capsids from empty capsids. Even when upstream conditions have been optimized to maximize the percentage of full capsids, there will still be a substantial percentage of empties.

Because the two populations vary in density, centrifugation is a popular method to separate them. However, a process developed using centrifugation does not scale up well.

Ion exchange is frequently used in the polishing step for AAV. The separation is mainly based on small differences in capsid pI. The exact numbers vary by serotype but average about 5.9 for full capsids vs 6.3 for empty capsids. Anion exchange will elute the empty capsids at lower conductivity compared to the full capsids. Cation exchange will elute in the opposite order.

Tip: Additives such as sucrose and poloxamer 188 have a positive effect on the separation, possibly by reducing the capsid-capsid and/or capsid-impurity interactions. Also, MgCl2 improves the separation. It’s believed that its differential binding to full and empty capsids affects the binding to the ion exchange ligand.

Another option to remove impurities is multimodal core resins, such as Capto™ Core 400. It is comprised of a bead with an inner surface that contains a ligand. AAV vectors flow through, while smaller impurities (such as host proteins, small DNA fragments, enzymes, and detergents) bind to the ligand in the pores. Use of Capto™ Core 400 in flow-through mode allows large load volumes (> 30 CV).

And don’t overlook the importance of analytics

Analytics are critical to support success in AAV process development and production. Different analytical assays measure the titer, purity, and identity of the AAV preparation. Virus titer is measured with respect to viral particle/capsid, viral genome, and infectious virus titer. Host cell impurities that must be removed to sufficient levels include host cell protein and DNA. Characterization assays evaluate virus size and shape or integrity, as well as ratio of full to empty capsids. Viral protein composition is also analyzed to confirm the ratio of viral proteins VP1, VP2, and VP3.

When developing an assay, keep these things in mind:

  1. The limit of detection (LOD) of your assay might not be low enough to get accurate results. For example, it’s difficult to accurately determine the titer of low-concentration material in harvest and clarified material.
  2. Detergents or buffer components (e.g., high salt) could affect your assay(s).
  3. Accuracy may depend on sample impurity level, especially in upstream samples where there are a lot of impurities.
  4. Be aware of the variation in your assays. Variation can be particularly high for manual assays.

Learn more about AAV production.