Adeno-associated viral vectors have quickly become the vector of choice for many gene therapy developers. With a strong safety profile in that AAV is non-pathogenic, is unable to replicate and does not integrate itself into the host genome, demand for this vector is increasing rapidly.

How will the AAV industry deal with the level of expansion it is currently experiencing and how will developers approach the challenges presented by AAV?

This is where alternative AAV packaging systems become of interest, including Porton Advanced’s insect cell lines for AAV research, development, and scaling up. But how do they compare when considering the cost, yield, scalability, and immunogenicity of current AAV systems?

We sat down with Ling-Jie Kong, CSO at Porton Advanced, to learn more about the area, and what the future may hold for the field.

AAV Production

Porton Advanced, Dr. Kong explained, offers two different cell packaging systems for AAV: HEK293, derived from human embryonic kidney cells, and SF9, derived from insect cells grown in tissue culture. He noted that HEK293 packaging systems are currently more popular, despite expensive, the SF9 system offers a significant reduction in cost while increases AVV production.

“There are advantages and disadvantages for using each system of cell lines. Some of our customers prefer HEK293 cell lines and some of the customers prefer the insect cell system. We work with our customers to accommodate their needs,” Dr. Kong commented.

Human Cell Lines

• AAV production is very costly, for example, a P&D batch can cost around $500,000.
• AAV yield is relatively low. For example, a 200L batch will only produce 10^15 to 10^16 AAV vectors depends on the products.
• HEK293 requires the use of 3 plasmids. Plasmids are very expensive and have limited transfection efficiency, creating barriers to scalability.

Insect Cell Lines

• SF9 insect cell lines don’t require the use of plasmids. Instead, they use baculovirus (BV) to infect cells, making them much easier to use and scale up compared to HEK293.
• AAV yield is much higher. A 200L batch can regularly produce 10^16 to 10^17 AAV vectors depends on the products
• However, there are also specific challenges involved with using the SF9 cell line. The production process requires baculovirus production and downstream baculovirus clearance.
• The other disadvantage is that the baculovirus is not stable. During the scaling up process, there is possibility of losing the gene of interest.

 “We have worked with our partners in Europe to make the system much more stable to support large volume expansion,” reassures Ling-Jie.

The Future of AAV

Technology

In the future, it is expected that the overall costs of AAV production will reduce significantly as greater funding, infrastructure and technological advances streamline and scale-up processes.

However, there are a lot of challenges that AAV production has to overcome.

This includes improving the upstream process to increase the total AAV titer and reduce the empty AAV, and down-stream purification process, to reduce costs – an issue that could be improved through the development and implementation of advanced chromatography technology.

“A key contributing factor to the future of AAV will be developments in technology. The earlier AAV packaging used viruses. Now the safer and more effective triple plasmids transfection system is commonly used in HEK293 system. In last a few years the much improved insect cell systems have been produced,” comments Ling-Jie.

He adds that:

“The technology is improving day by day. For example, at the beginning, customers expressed worry about insect cell infectivity, however, with technological improvements, the insect cell-packaged AAV is as effective as those from HEK293 cells. New technology advance has allowed us at each step to increase efficiency, production, and quality whilst reducing costs.”

Porton Advanced and AAV

As Porton Advanced prepares for the challenges that lie ahead it focuses on a high volume of in-house design of experiments in product development.

“At Porton we are improving the AAV production platforms. For example, we are currently working on a HEK293 cell line grows in higher densities and can produce a lot more viruses. We are also working on a more stable baculovirus system to support high volume process in the insect cell system. We also generated a rhabdovirus-free insect cell line to make the process easier and the final products safer.” said Ling-Jie.

They also note the ongoing work to their downstream purification process to try and improve recovery -currently, recovery for HEK293 can reach 60% and after purification, the full capsid can reach higher than 95%, with overall downstream purification recovery around 50% – comparatively high yields when considering direct competitors.

Global Reach

Porton Advanced is a company based in China, a location the team champions as greatly beneficial.

China has a lot of advantages for AAV production, most notably as one of the biggest global hubs for biotechs, home to many talented scientists including local talent, and attracted talent from around the world. Based in Suzhou, just half an hour from Shanghai, Porton Advanced headquarters are also located nearby many Universities providing access to talent and resources.

The Chinese market is very large with a substantial local customer base, reducing the need for global vendors and decreasing costs significantly. Costs are further reduced by the lower labor costs compared to Western countries.

While the local market has provided a great source of clientele for Porton, Ling-Jie highlighted plans to expand to reach a global customer base.

“In terms of infrastructure, we already have a 4000m2 PD lab and plan to finish the 16000m2 building of out GMP facility by end of 2022. This facility will provide us with more new bioreactors. We also plan to grow our team, recruiting people from both overseas and inside China. As far as our systems go, we continue to work with our well established processes for both the HEK293 and insect systems,” concluded Ling-Jie, as Porton Advanced continues to advance and develop their AAV packaging systems offerings, and reach.

This blog has been produced in partnership with Porton Advanced.

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