This presentation outlines the development of a commercial manufacturing process for RGX-202, a systemically delivered AAV8 vector targeting Duchenne Muscular Dystrophy. It will cover the scale-up from laboratory to commercial production, focusing on optimizing both upstream and downstream processes to ensure high yield, purity, and efficiency. Advances in downstream processing, including improvements in purification and recovery methods, will be discussed in the context of enhancing scalability and maintaining product consistency.

Traditional AAV production in HEK293 cells relies on a 3-plasmid system requiring extensive transfection optimization to outperform traditional bioreactor titers of 5-8E14 vg/L. By implementing a 2-plasmid approach, the Alexion upstream platform consistently achieves titers exceeding 2E15 vg/L across diverse capsids and genes of interest, demonstrating robust productivity. This streamlined method can accelerate development, simplify scale-up, and enhance speed to clinic for multiple gene therapy programs while lowering manufacturing costs.

Assessing manufacturability is an important step toward enabling efficient process development and timely clinical manufacturing for AAV gene therapy. Additionally, to accelerate timelines post-candidate nomination, process development may commence prior to this milestone, sometimes on more than one candidate. In this presentation we share one such case study regarding process development for an AAV gene therapy using a novel capsid, that led to a successful manufacturing campaign. We will discuss team strategy while managing uncertainty along with lessons learned to accelerate manufacturing timelines.

Increasing yields is one of gene therapy's greatest challenges—and addressing this helps to make gene therapy more accessible to broader patient populations. This talk will explore strategies used to improve yield and the empty to full particle (E/F) ratio by leveraging design of experiments (DoE) to systematically evaluate transfection parameters including total DNA, plasmid ratios, complexation time, transfection reagent, and enhancers.

Critical Quality Attributes (CQAs) are key characteristics of a drug product that must be maintained within specified limits to ensure the product's safety, efficacy, and quality. Appropriate CQA sets the tone of manufacturing development. We discuss the role and significance of CQAs in robust manufacturing development for gene therapy. By testing appropriate CQAs early in the development process, the risk of unexpected quality issues and potential safety incidences are reduced.

Tech transfers are an important aspect of CMC development. In this presentation, I will present the essential elements for the success of a tech transfer from sending site to the receiving site and the evaluation of the CDMOs for their capability and capacity for successful production of the first cGMP batch.

Adenovirus is one attractive viral vector for gene therapy and therapeutic vaccines. We have engineered A-549 adenoviral vector complementing cells for the production of E1-deleted adenoviral vectors and developed bioprocesses in cell suspension yielding free of replication-competent adenovirus. We have tested several production modes and increased initial titers in batch mode from mid 109 vp/mL up to 7,0 x 1010vp/mL in a bioreactor perfusion culture at the 3 L scale.

We performed a genome-wide CRISPR-based knockout screen to identify genes that can be targeted in human embryonic kidney (HEK) 293 producer cells to modulate rAAV production. We discovered that the knockout of a group of heparan sulfate biosynthesis genes previously implicated in rAAV infectivity decreased rAAV production. Additionally, we identified several vesicular trafficking proteins for which knockout in HEK 293 cells increased rAAV yields.

Cryopreservation is an essential part of cell-based biomanufacturing. At Ultragenyx, we utilize mammalian producer cell lines to produce recombinant AAV gene therapy products. We observed several MCB and WCB with poor thaw recovery and initiated an investigation of cell-banking, transfer & handling, and thaw procedures. Cell banking density, freeze/thaw cycles, media, transfer durations, and other factors were shown to have significant impact on thaw recovery. We applied these learnings to develop a more robust manufacturing process and improved control on shipping and handling procedures to enable us to improve our GMP cell bank recovery performance.

Efficient purification processes are needed for the increasing number of recombinant adeno-associated virus (rAAV) therapies. Impurities such as host cell proteins and empty capsids copurify with rAAV and should be reduced during manufacturing. Optimization strategies in downstream purification and supporting data for improved vector purity and recovery will be presented in this talk. The process ensures production of high-quality rAAV for clinical use and is scalable from 1L to 1000L.

Adeno-associated viruses (AAV) are among the leading vectors for gene therapy. The purification of AAV remains a bottleneck as it typically requires multiple individual process steps, often resulting in product loss and high costs. Current downstream processes are usually serotype-specific and rely primarily on expensive affinity resins. To address these limitations, we developed a serotype-independent capture method using steric exclusion chromatography that can be combined with a subsequent full/empty separation step. This alternative approach eliminates the need for intermediate concentration and buffer exchange, thereby reducing the number of process steps required while achieving high-purity full AAV particles. 

This presentation explores the downstream platform process across HEK transfection and Pinnacle PCL cell-line platforms, focusing on key differences in process for AAV vector manufacturing. We will discuss purification process differences, impurity removal, and viral clearance strategy for reproducible vector manufacturing intended for clinical and commercial use. Key considerations to address challenges will be discussed, offering insights for advancing AAV GT vector manufacturing.

This talk will address the unique challenges in developing effective virus filtration strategies for rAAV gene therapy products. We’ll examine the role of virus filtration in enhancing viral clearance robustness and its increasing regulatory emphasis in AAV manufacturing. Key topics include evaluating commercially available virus filters for AAV manufacturing, assessing their throughput and process yield, and demonstrating robust clearance of model viruses like Adenovirus type 5 and Simian virus 40.

Adeno-associated virus (AAV) is highly inefficient at packaging its genome, with up to 90% of the formed AAV capsids being empty. The upstream cell lysis generates significant impurity burdens for downstream. A purification process, including harvest clarification, ultrafiltration/diafiltration, affinity chromatography for AAV capture, and anion exchange chromatography (AEX) for AAV polishing was developed for different serotypes. This talk will highlight:

• New chromatography and membrane filtration modalities that are used to overcome the manufacturing process challenges
• An increase in resin loading was found to enhance virus recovery from the affinity capture. 
• Over 40% increase in full capsids was achieved through AEX polishing.
  

This presentation will focus on the latest advancements in AAV polishing technology, crucial for enhancing the purity and potency of adeno-associated virus (AAV) vectors used in gene therapy. It will cover innovative methods for downstream processing, including chromatography and filtration techniques that effectively remove impurities and improve vector yield. The session will also discuss the impact of these technologies on the overall quality and scalability of AAV production.