Scalable AAV manufacturing is one of the most persistent bottlenecks in gene therapy. High viral titers through triple transfection are difficult to achieve consistently, and downstream purification using scalable chromatography has been a challenge for the field for years. The constraints get harder for emerging cell and gene therapy programs that also lack access to a robust, license free bioprocess. With support from a grant from the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL), Landmark Bio executed a pilot scale AAV9 batch using the NIIMBL Viral Vector Program's bioprocess framework, with the goal of producing an open source platform that any organization can use to make clinical scale material. In this session, two of the scientists who led the work share what the team learned, including an upstream optimization that delivered a greater than tenfold improvement in viral titer and a downstream effort that scaled production while optimizing chromatography for purity and yield. The result is a practical, transferable framework for AAV9 programs that need a viable path forward to the clinic.
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Senior Scientist, Process Development Landmark Bio |
Scientific Director, Process Development Landmark Bio |
Two technical realities make AAV manufacturing harder than most modalities. Achieving consistent high titers through triple transfection is difficult, with productivity sensitive to plasmid ratios, transfection conditions, host cell state, and scale dependent factors that do not always translate cleanly from bench to bioreactor. On the downstream side, purifying AAV drug substance to acceptable quality using scalable chromatography techniques has challenged the field for years, particularly the separation of full from empty capsids. For emerging cell and gene therapy programs, those technical challenges are compounded by limited access to robust, license free bioprocesses, which means many teams are starting from scratch on a process that demands deep expertise to get right.
With support from a grant from the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL), Landmark Bio executed a pilot scale AAV9 batch using the NIIMBL Viral Vector Program's bioprocess framework. The goal was to confirm and optimize the framework, then make it available as an open source platform that any organization can use to produce clinical scale material. The intent was practical rather than academic: give programs that lack a robust internal AAV9 process a viable starting point, supported by data from a real pilot scale execution.
The upstream study used the AAViator and RevIT transfection system and achieved a greater than tenfold improvement in viral titer compared to the baseline process. That kind of step change in productivity has direct implications for downstream economics, batch sizing, and the number of clinical scale runs required to support a program. It also reduces some of the variability that traditionally makes triple transfection processes difficult to scale, which improves both reproducibility and the predictability of campaign planning.
The downstream effort focused on scaling production and optimizing chromatography for purity while maximizing yield. Those two goals often pull in opposite directions, especially with AAV, where aggressive purification steps can drive losses that compound across the train. The optimization work focused on finding chromatography conditions that hold up at scale, deliver the purity required for a clinical program, and preserve enough yield to make the overall economics workable for a CGT developer.
AAV9 has specific manufacturing and analytical considerations that do not always translate cleanly from work done on other serotypes. Capsid structure, surface chemistry, stability profile, and the behavior of the virus on chromatography media can all differ from AAV2 or AAV5 reference processes. That means upstream conditions optimized for one serotype may not deliver the same productivity for AAV9, and downstream methods that work for full versus empty separation on one serotype may need to be reworked for another. The AAV9 specific work in this project was designed to surface those differences and build a process that holds up specifically for the serotype, rather than relying on platform assumptions from other serotypes.
Many emerging cell and gene therapy programs face a structural problem: the AAV processes available to them are either internal to large developers or licensed in ways that add cost, complexity, or limitations on use. An open source, license free process that has been demonstrated at pilot scale changes that calculus. It gives programs a starting point that is technically credible, reduces the time and capital required to build a viable AAV9 process from scratch, and creates a shared framework the field can iterate on. For programs that need to demonstrate a path to the clinic without a long process development runway, that kind of foundation matters.
The blueprint covers the full path from process development through clinical-grade GMP manufacturing, with attention to the decision points that matter most at each stage. In process development, the focus is on confirming the upstream and downstream framework, optimizing the unit operations that drive titer and purity, and qualifying analytical methods that will travel into GMP. In tech transfer, the emphasis shifts to building the documentation, training, and operational structure that allow a process to execute consistently in a regulated environment. At GMP, the blueprint is supported by the same team that developed the process, which compresses the translation gap that traditionally introduces risk between PD and manufacturing.
In a traditional model, process development, analytics, and GMP manufacturing live in separate teams, often at separate sites, with information moving through documentation rather than people. That structure creates handoff points where critical context can be lost. Landmark Bio's integrated model keeps process development, advanced analytics, and GMP manufacturing under one team. The same scientists who optimize the upstream and downstream unit operations remain involved as the process moves toward GMP execution. Analytical methods are developed in parallel with process steps rather than after the fact. When something unexpected happens during a GMP run, the people with the deepest understanding of the process are on hand to troubleshoot. That structural integration is what reduces translation gaps and protects program timelines.
AAV9 analytical packages have to address identity, capsid content (full versus empty ratio), genomic and infectious titer, purity, residual host cell protein and DNA, and potency. Each of those measurements has its own technical challenges, and several depend on assays that need to be qualified in step with the process they characterize. Building those analytical methods alongside the process, rather than after it, ensures that decisions made during development are based on data the team can trust, and that the analytical package supporting GMP release is ready when the process is.
The findings from this project offer a practical, transferable framework for further optimization, not a fixed protocol. Programs can apply the upstream and downstream learnings as a starting point, adapt them to their specific construct and manufacturing context, and use the data generated as a reference for what is achievable at pilot scale. For programs that want hands on support, Landmark Bio can engage at any stage, from process design through GMP execution, with the integrated team that ran the original work. If the session raised questions specific to your AAV9 program, or if you would like to discuss where your program is right now, the team is glad to find time to connect.