What Does It Take To Make Higher Quality AAV?
By Life Science Connect Editorial Staff
The development of highly effective AAV vectors is a journey wrought with excitement and challenges, due in no small part to the rapid pace of scientific innovation within the in vivo gene therapy field. In particular, many are struggling to improve the overall quality of AAV therapeutics. In a discussion with Anna Rose Welch, director of Cell & Gene Collaborative, Justin Horvath, senior director of external manufacturing and manufacturing sciences for REGENXBIO, and Andrew Tustian, senior director of viral production core at Regeneron, examined the current state of AAV quality control and improvement, as well as which quality-related advancements the industry could be on the brink of realizing.
Questioning Well Characterized: How Well Do We Really Know Our AAV Viral Vectors?
AAV manufacturing and innovation have come a long way, but characterizing vectors continues to be a challenge due to a range of factors, such as heterogeneous populations, molecule size and structure, and the dynamic nature of vector production. Both speakers emphasized that there are a range of advanced characterization technologies in use today, including next-generation sequencing and DNA electrophoresis. “There's been a lot of methods traditionally looking for residual DNA through PCR-based methods, but the technology has advanced to give you other options to really understand your DNA population,” Horvath said. Tustian also described mass spectrometry as a game changer for AAV analysis.
During the discussion, an audience poll revealed that more than half of attendees weren’t sure what it means to be “well-characterized.” Horvath attributed this uncertainty to the level of innovation that has taken place in the past eight years or so. “Now we're getting into deeper characterization and understanding what we can do and what the value of those analytical methods is,” he said.
Tustian reflected on the progress made in understanding critical quality attributes (CQAs), particularly as they pertain to full, partial, and empty capsids. He noted that while there was previous debate about the potential benefits of having empty vectors acting as decoys for the host immune system, recent studies have highlighted the potential risks associated with high dosing, including hepatoxicity, thrombotic microangiopathy, and neurotoxicity. In turn, Tustian believes the industry “wants to minimize the percentage of empty particles as much as it can, and the level to which each company minimizes partial/empty capsids is open for discussion and probably dependent on dose level and route of administration,” he said.
However, Horvath also insisted that the industry needs to expand its scope beyond full and empty capsids to other equally important quality-related discussions, including the relationship between potency and other characterization methods. “The better we can understand the influence of some of these deeper characterization technologies and measure them directly and get to the potency of our gene therapies will help,” he said.
Tustian added that immunogenicity remains a big concern for the AAV space. As such, having an assay designed to measure the immunogenicity of a specific capsid can help determine the likelihood of an immune response. “Then you can use that assay to link back to more basic quality attributes, including capsid DNA degradation,” he said.
In response to a question from the audience, both men confirmed that, in addition to empty capsids, regulators are focused on factors such as comparability, the validated state of analytical methods (particularly content and potency methods), and the qualification of the potency assay. In particular, Tustian noted the importance of consistent assays from early to late stages of development and the need for early understanding of characterization methods to address challenges in comparability throughout the product life cycle.
Space Invaders: The Quest For Stable Producer Cell Lines
Another big challenge facing the industry is achieving the necessary yields of viral vectors without having to sacrifice quality. Tustian noted that the demand for higher doses requires a logarithmic increase in process productivity. As the dosage increases, the production requirements grow exponentially. Horvath agreed, adding: “If you’re after a neuromuscular condition and the doses are very high, you probably have a lot more concern around the yield and potentially around the quality piece of that; if you’re going for an ocular indication, there may be less concern around the yield.”
When it comes to navigating the transition from developing therapies for rare diseases to addressing larger patient populations, both speakers acknowledged the need for increased scalability and higher purity in the final drug product, particularly for gene therapies targeting more prevalent conditions. “Generally, if you're looking at gene therapies reaching beyond the rare diseases, or especially to neuromuscular conditions, you need access to larger scales,” Horvath added.
To expand AAV gene therapies’ relevance from rare diseases to more prevalent diseases, Horvath shed light on the continuous efforts to enhance productivity and control in upstream manufacturing, emphasizing the shift from adherent to suspension-based technology. He also underscored the significance of innovating in various facets, including cell line selection, transfection efficiency, and downstream process optimization. In particular, Horvath noted the ongoing debate about using SF9 or HEK 293 platforms.
“The industry is not fully harmonized on a platform at this point,” he said. “And some of that depends on the serotype you're producing; some of it depends on the disease indication you're going after, as well as a company’s technical capabilities and internal expertise.”
They also touched on the vision of a standardized producer cell line, similar to the model seen in monoclonal antibodies. Horvath highlighted the necessity of solving complex biological questions to achieve stable producer cell lines, stating, “It’s really solving the biology question of how you can program these cells and keep them healthy to produce what you want.”
Tustian likened it to playing Space Invaders: “I don't think one innovation is going to unlock this technology... you need to blast each one, one-by-one, until you get closer and closer.”