Conquering The Complexities Of Aav Manufacturing: Advances In Efficiency, Scalability, Compliance

Adeno-associated viruses (AAV) have emerged as pivotal delivery vehicles for gene therapy due to their overall safety (less immunogenic), efficiency (broad tropism), and long-term gene expression. These small non-pathogenic viruses carry a single-stranded DNA genome (~ 4.7 kb) encased in a protein capsid. Several AAV-mediated gene therapies have already received regulatory approval or are in late stages of clinical trials, and many more are on the horizon (a search of clinicaltrials.gov disclosed at least 114 studies employing AAV-mediated gene therapies for a host of diseases).

However, AAVs are replication-incompetent, lacking the appropriate genes to reproduce. This deficiency makes the manufacturing of therapeutic AAVs a complex, multi-step, and costly process that involves the preparation not only of the AAV plasmid but also helper plasmids or viruses that express the critical proteins necessary for AAV assembly and replication.

GEN talked with several contract development and manufacturing organizations (CDMOs) and/or contract research organizations (CROs) about new ways they are addressing AAV manufacturing challenges.

Novel capsid challenges

“One of the biggest challenges in AAV manufacturing today revolves around novel capsids,” remarks Ify Iwuchukwu, PhD, head of process and analytical development at OXB. “Many companies, including our own, have developed platform processes that perform very well across a variety of AAV serotypes, but may not prove to be ‘plug-and-play’ when it comes to novel capsids. A bit of tuning is required to achieve the optimum process outputs for both titer and product quality.”

Ify Iwuchukwu, PhD
Head of Process and Analytical Development, OXB

The company has built an extensive toolbox to deal with such novel capsids, which allows them to adapt client processes without the need for extensive development. For manufacturing, the company utilizes suspension cultures of HEK293 cells. Iwuchukwu elaborates, “These cells are transfected with either triple or dual plasmid transfection systems, producing the vector of choice. The HEK293 cells are then lysed, and the harvest material is clarified for purification. Purification includes a two-step chromatography to first isolate the vector produced from the rest of the cell culture materials, including host cell proteins, residual DNA, etc. This is followed by a full capsid enrichment process. Finally, the enriched vector product is buffer exchanged into the final formulation solution.”

The company’s proprietary dual transfection system contains an optimized arrangement of sequences that are critical for AAV production, leading to a significant increase in productivity. According to Iwuchukwu, “We consistently achieve high titers and employ a robust and scalable affinity capture and anion exchange chromatography process that efficiently removes host cell/process impurities. We also can achieve a high percentage of full capsid (>90%).”

OXB’s novel Dual-Plasmid system resolves some of the key manufacturing challenges faced by AAV developers.

Iwuchukwu notes that transfection scale-up is rather unique to the field of viral vector manufacturing. “Earlier during our development, we invested in gaining an understanding of the fundamental kinetics of transfection mixture generation, including complex size formation, mixing, equilibration time, and equipment required to scale up the transfection operation. This understanding has led us to successfully scale up our upstream process to 2000L.”

Increasing production efficiency

Another major challenge of the industry is high production costs, according to Xin Swanson, PhD, chief strategy officer at PackGene Biotech. “Traditional AAV manufacturing is expensive, making it difficult for therapies to be widely available,” she remarks.

Xin Swanson, PhD
Chief Strategy Officer
PackGene Biotech

According to Swanson, the company is addressing these challenges by creating more innovative and cost-saving approaches for manufacturing, including plasmids for AAV replication and capsid formation (RC plasmids). “Unlike traditional batch processes, PackGene’s proprietary Π-Alpha 293 AAV production platform includes suspension-based production, unique designs of modified RC and helper plasmids, and efficient column-based downstream processing steps to enable high-yield, cost-effective AAV production with flexible scaling options,” Swanson says. “As a result, we have successfully increased AAV production yields by up to tenfold. Further, through actively incorporating advanced analytics during development and optimizing critical process parameters, the platform improves product safety by greatly reducing unique process-related impurities, such as empty capsids and encapsulated DNA.”

Swanson adds that providing companies with end-to-end supporting services at every step also helps reduce costs. “Optimizing manufacturing workflows by leveraging platform technology allows both reduced production costs and flexible production scaling,” he indicates. “Further, by combining our high-yield P-Omega plasmid production platform and AAV production under one roof, we can effectively lower the overall AAV production costs by decreasing the cost of these key raw materials while simplifying supply chains and reducing overall program timelines. Additionally, we are razor-focused on quality and cGMP compliance to ensure production meets regulatory requirements, de-risking clinical development and avoiding costly setbacks for our clients.”

For companies considering in-house AAV manufacturing versus outsourcing to a CDMO, Swanson offers some advice. “Building an in-house facility requires significant investment in infrastructure, equipment, and personnel with deep technical expertise. You also need robust quality systems and manufacturing technology innovations. Such investments often compete with resources required to accelerate discovery research, pipeline development, and clinical progress. Outsourcing to a specialized CDMO for production activities can greatly accelerate timelines and reduce overall costs.”

Off-the-shelf advances

Production and full quality-controlled (QC) release of AAV plasmid DNA can require several months of painstaking work prior to and after manufacture. Charles River Laboratories (CRL) decided to premanufacture and QC a variety of AAV, as well as lentiviral vector, plasmids so that they are ready to use by clients. “We now provide the needed AAV plasmids as standard offerings since we decided to make them in bulk as off-the-shelf products,” reports Matthew Hewitt, PhD, Corporate VP and CTO Manufacturing at the company.

Matthew Hewitt, PhD
VP, CTO Manufacturing
Charles River Laboratories

Hewitt likens the process to ordering from Amazon for quick availability of critical quality items. “Utilizing off-the-shelf plasmids saves a lot of time and money that can be devoted to discovery and development work.”

As a CDMO, CRL has a “vertically integrated portfolio” and offers AAV manufacturing services for small to very large companies. Hewitt notes, “We control all aspects of the process from raw materials to finished products. We also continually identify promising new technologies to bring into our portfolio.”

Another important consideration for AAV manufacturing is the reliable regulatory compliance of finished products. Hewitt philosophizes, “This is an industry where no one wants to be first, but everyone wants to be second! However, we already have a considerable breadth of regulatory experience that allows a client to come into our facilities as, for example, a clinical customer and then progress all the way through product commercialization without the worry of whether we have done this before. We already have the infrastructure set up to deal with commercial regulatory scrutiny.”

Hewitt sees many future innovations for AAV manufacturing and the field in general. He projects, “If you look at science today, the case of innovation has sped up considerably. Technologies can evolve much quicker than in the past. So basically, technologies are catching up to our ideas. I feel that we are moving overall to be able to make viral vectors much more efficiently in the future. The intent is to bring therapies to more patients at a lower cost.”

Industrializing manufacturing

Lonza is aiming to industrialize AAV manufacturing for a variety of client demands. The company has created two Xcite^® AAV platforms that employ either transient transfection or stable producer cell-line (PCL) technologies.

Bingnan Gu, PhD
Director, R&D, Cell & Gene
Lonza

Bingnan Gu, PhD, director of R&D, cell & gene at the company elaborates. “The transient transfection platform, leveraging Lonza’s proprietary, suspension clonal HEK293 cell line and rationally designed Helper and RepCap plasmids, produces AAV with up to 1E+12 vg/mL (viral genome per milliliter) titer with 50% full capsid at harvest across multiple serotypes in 3L to 250L single-use bioreactors. The platform allows rapid manufacturing of recombinant AAV with minimal optimization of the established platform process.”

To help overcome some of the issues of transient transfections, Lonza has also developed a next-generation, helper virus-free stable PCL technology. Gu summarizes, “This offers innovative solutions to the challenges of transient AAV manufacturing, enabling simple, robust, large-scale AAV production for common indications, including neurological and metabolic diseases. The technology removes plasmid transfection by stably integrating DNA cassettes of Helper, RepCap, and therapeutic gene into the genome of HEK293 cells. The Lonza PCL platform consistently delivers over 1012vg/mL titer and 30% full capsid in crude harvest across 30 cell passages. The high productivity and scalability have the potential to reduce the cost of goods by over 80% compared to the transient transfection process.”

Lonza’s AAV manufacturing systems also have homed in on ensuring AAV product quality and safety by developing a suite of assays that accurately measure genome titer, packaging, and integrity. Gu details, “For example, standard PCR titer assays do not distinguish between full-length and partial genomes, which is a problem since the partial genome cannot express a functional therapeutic product and impedes AAV efficacy and safety. To solve the analytical challenge, Lonza recently developed an advanced three-dimensional mathematical linkage analysis with digital PCR to provide a comprehensive analysis on the AAV genome titer, identity, and integrity.”¹

Gu also points out that the company’s novel analytical method provides the quick detection of AAV titer and quality in the early stage of process development, enabling accelerated manufacturing and delivery of AAV drug products with high yield and quality to patients.

Triple transfection process

ProBio’s approach to AAV manufacturing initially centers around their triple transfection suspension system that provides the ability to achieve high viral vector yields with reproducibility and scalability. According to the company, this method is particularly beneficial for producing consistent and high-quality AAV vectors, which are critical for gene therapy applications.

Whitney Winters
SVP, U.S. Business Development
ProBio

“This system utilizes the PowerS-293 cell line alongside purpose-built Rep/Cap and helper plasmids (AAssistV) and client-provided gene of interest,” indicates Whitney Winters, PhD, SVP, U.S. Business Development. She continues, “Thus, plasmid 1 contains the gene of interest flanked by AAV inverted terminal repeats. Plasmid 2 carries the AAV rep and cap genes that are responsible for replication and capsid formation, respectively. Plasmid 3 provides needed adenovirus helper functions, such as E2A, E4 and VA RNA, which are critical for AAV replication and packaging yet lack the full adenovirus DNA in order to ensure safety. These three plasmids are co-transfected into the PowerS-293 cells, providing the critical components for AAV vector assembly with the cell culture.”

Wilson says that the company also emphasizes US-based sourcing to ensure that critical raw materials, including plasmids manufactured in-house, remain both cost-effective and readily available.

As with other vendors, scalability is also a key concern. Wilson explains, “We use single-use bioreactors ranging from 10 L to 200 L (scalable to 50-400 L). This minimizes contamination risk and accelerates turnaround, while advanced downstream purification workflows consistently produce high-quality viral vectors.”

Because clients’ needs can also include developmental stages, the company manufactures AAVs for use in the preclinical as well as IND through GMP phases Wilson also emphasized that “By integrating proprietary technologies, advanced process development, and single-use bioreactor systems, we can offer an efficient and scalable path from plasmid DNA design to final drug product fill-finish, all under one roof.”

 

Reference

1. Duong, T., Firmo, M., Li, CT. et al. Three-dimensional linkage analysis with digital PCR for genome integrity and identity of recombinant adeno-associated virus. Sci Rep 15, 2154 (2025).

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