In 2020, Cytiva scientists hosted several webinars on developing a scalable adeno-associated virus (AAV) production process, with a focus on AAV serotype 2 (AAV2). They detailed the AAV production workflow, including transient transfection and cultivation in single-use bioreactors, AAV purification, and analytics. And they also previewed a new purification technology. Here they answer questions from attendees.

How long does it take to adapt adherent cells to suspension for the HEK 293T cells?

It took about 2 months using direct adaptation. We made 10 cell passages to control cell growth before creating a working cell bank. One tip is to control cell aggregates during adaptation and not exceed 2.5 × 10⁶ cells/mL, because aggregates will increase when viable cell density (VCD) is too high.

Do you expect your PEI-based transient transfection method for AAV will be scalable to 500 L?

Yes, this is what we expect. But so far for AAV2 we have only done the scaling up from 20 mL to 20 L. We obtained similar titers for several serotypes. A customer has a scalable transfection procedure in the Xcellerex XDR bioreactor up to 500 L.

How did you select your lysis conditions for AAV2?

We have been doing a study comparing different detergents for lysis of HEK293 cells in an adenovirus process that we published and compared with freeze-thawing. Tween™ 20 showed good performance. It is not on the REACH list (i.e., it is health and environmentally friendly and can be used for large-scale production).

Do you have any recommendations to maximize recovery on Capto AVB?

Yes, a few things. Make sure you do not underload the column. Capacity is very high, approx. 10¹⁴/mL resin. If the titer is very low in the harvest, try to concentrate it. And try different elution procedures (flows and buffers). Also, the titer in the sample that is loaded can affect recovery (higher titer is an advantage).

The AAV recovery from the concentration and buffer exchange step was up to 80%. Where was the loss of 20%?

We did analyze the permeate, and no virus was detected there for the 300 MWCO hollow fiber filter. It could be trapped to some degree in the system tubing or filter, or it could also be analysis error. Sometimes we have achieved 90% to 100% recovery. So, we generally get very good recovery over this step.

How does Capto Core technology work for AAV purification?

This is a flow-through procedure. The virus will pass through due to the size, and the impurities will go into the pores and bind the ligand (octylamine) inside. We have Capto Core 400 and Capto Core 700. Capto Core 400 is suitable for AAV. The 400 and 700 reflect the MWCO of the outer shell of the bead.

How do people manage viral clearance studies in gene therapy products and processes (e.g., AAV)?

It’s important that the raw materials are not contaminated. But depending on the virus to be purified and what it can withstand, you can also introduce steps with low pH or detergent, so that more sensitive viruses will be cleared.

What is the best way to remove empty AAV capsids?

By ion exchange (IEX) chromatography if you want it to be scalable. We are still working on the full/empty separation, but we believe Capto Q ImpRes is suitable for most serotypes. We also suggest you maximize the % full capsids in your preparation by optimizing upstream production.

What method do you use for measuring AAV titer?

We are using two similar assays: an enzyme-linked immunosorbent assay (ELISA) and a Biacore assay for surface plasmon resonance (SPR). We get very similar values that compare to the ATCC reference standard. But we have lower variation (CV%) with the Biacore assay.

Did you check residual DNA impurities in your AAV preparation?

So far, we have done PicoGreen™ analysis for total DNA. After the affinity step we could not detect any DNA; it was below the detection limit (1 ng/mL). We plan to perform a qPCR for HEK293 DNA that we have already set up. In the AAV DNA qPCR, we always check background from plasmids and have carefully set up the assay to have full control of any background sígnals from plasmids. We see no or very low background in our qPCR assay.

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• Plasmid production
  • Xcellerex XDR microbial system – single-use, stirred-tank fermentor for growing plasmid-containing microbial cultures
  • Capto PlasmidSelect resin and columns – to purify supercoiled plasmid DNA
• Upstream AAV production
  • Xcellerex XDR platform – single-use, stirred-tank bioreactors for cell cultivation up to 2000 L
  • ReadyToProcess WAVE 25 bioreactor – single-use, rocking bioreactor for growing cells up to 25 L
  • Cell culture media development service – 3 levels of service to optimize media for your cell line
  • HyClone media and reagents – for cell growth, transfection, and buffer preparation
  • Cytodex microcarriers – for growing adherent cells in suspension culture
  • Single-use bags and connectors – to make aseptic connections between single-use components
• AAV purification
  • Xcellerex mixers – single-use mixers for media and buffer preparation
  • Single-use bags and connectors – to make aseptic connections between single-use components
  • ULTA NFF filters – single-use capsules for harvest, clarification, and final filtration
  • ReadyToProcess TFF filters – single-use cartridges for concentration
  • ÄKTA tangential flow filtration systems – range of single-use filtration systems from benchtop to GMP production
  • ÄKTA chromatography systems – range of automated systems from benchtop to GMP production
  • Anion exchange resins and columns – for virus purification
  • ReadyToProcess Adsorber Q – single-use anion exchange membranes for virus purification
  • Capto Core 400 resin and columns – for intermediate purification and polishing
  • Capto AVB resin and columns – affinity purification of AAV, with ligand derived from single-chain antibody
  • Size exclusion resins and columns – for virus purification and analysis
• AAV analytical assays
  • Biacore T200 system – for surface plasmon resonance (SPR)
• Manufacturing platforms and facilities – for viral vectors and plasmids
• CMO service: Research-grade AAV – for preclinical studies

• Case study: AAV production on microcarriers – OBiO Technology
• Case study: Efficient plasmid purification – Cobra Biologics
• Online learning – a bevy of self-paced digital courses
• Biotech Makers LinkedIn group – ask your AAV and plasmid questions here