While gene therapies are offering unique and unprecedented treatment options for patients, these versatile toolboxes are coming with high complexity on a molecular level, which finally poses significant challenges to provide access to such therapies on a global scale. Gene therapies are currently where monoclonal antibodies were 25 years ago and we need a shift of our development paradigms and transformative innovation for reliable supply, more sustainable cost of manufacturing as well as enhanced molecular understanding of these next-generation therapeutics.

In the presentation we will explore the journey of up-scaling an Adeno-Associated Virus (AAV) production process. We will share an overview of the experiences and insights gained during the scale-up work, highlighting the complexities faced during the most challenging steps of the upstream and downstream processes. Furthermore, we will discuss the valuable lessons learned and the innovative solutions we are implementing to address the remaining challenges effectively.

rAAVs are at the forefront of gene therapy, but the manufacturing processes for these remain a significant challenge. Specifically, the focus of product developers is on scalability and achieving high productivity consistently to ensure economic viability. While this field is still emerging, innovative approaches in upstream processing have shown potential. Here, we explore the evolving landscape of upstream rAAV production, highlighting the promising strategies to enhance efficiency and scalability.

Gene therapy is an emerging treatment modality for a wide range of monogenetic disease indications, including those in the kidney. Meeting the demand for these clinical indications requires innovative manufacturing strategies to boost rAAV productivity. Our success in triple plasmid transfection has elevated AAV productivity in small-scale platforms, and we outline our scaling and implementation approach at our CDMO to address these emerging therapeutic needs.

• ​Industrializing Gene Therapies - what have we learned from commercial products
• Cell line development to process development
• Technology gaps in process development - upstream to downstream
• Formulation and Stability

• ​Moving from Upstream to Downstream Processing
• Formulation Strategies
• Fill Finish

Recombinant adeno-associated virus (rAAV) vector is the most widely used viral vector for in vivo gene therapy. This work is aimed to develop a powerful rAAV production platform based on transient transfection of HEK293 cells in suspension, delivering large amounts of rAAV. After only a few months of development through a holistic approach, an end-to-end process was defined. This so-called “TaRGeT’s AAV platform” showed high and consistent productivity up to 5x10¹¹ vg(vector genome)/mL in bulk harvest. Finally, this process has been successfully scaled-up from shake flask to 2L then to 50L stirred-tank bioreactors.

We developed a novel three-plasmid packaging system, Higher-Expression Recombinant AAV (HERA) system, which increased the rAAV yield by 5 to 10-fold (to 1E15vg/L across different serotypes) and reduced rcAAV to undetectable level. The HERA system has been evaluated in Neurophth’s suspension HEK293 platform for multiple preclinical and clinical batches. Herein, we present process and quality attributes summaries of historical batches to demonstrate the HERA system not only significantly reduced manufacturing scale, but also achieved improved quality profile

Adeno-associated virus (AAV) vectors are widely used as a gene therapy vector. One of the current limitations on the use of AAV vectors is their small packaging capacity impairing delivery of therapeutic genes over 3.5kb in size. Herein, dual AAV co-delivery strategies are discussed and presented, namely the use of protein trans-splicing systems, to deliver larger genes. AAV vector doses are also analysed.

New modalities are a challenging task for downstream processing. Alternative purification strategies that can improve the purification yield, such as affinity chromatography or the use of new adsorbent materials are regarded nowadays as enabling technologies to overcome the capacity bottleneck in biomanufacturing. The current talk will focus on the development of new engineering ligands and new matrices aimed at the purification of viral vectors; some case studies will be discussed.

Process intensification and integration are often viewed as tools to improve bioprocess efficiency. This talk will explore the use of perfusion bioreaction and continuous chromatography to seamlessly integrate and connect upstream and downstream stages. AAV expression, harvesting, and clarification processes are integrated using tangential flow depth filtration. The cases of continuous AAV affinity capture and polishing will be presented, with an emphasis on the challenges and opportunities for future developments.

Bionanoparticles, like virus-like particles, are promising candidates for future vaccines and gene therapies. For the production of safe pharmaceuticals, a major focus lies on the downstream processing. Within our research, we are investigating the fundamental principles of bionanoparticle purification with the goal of moving from resin-based chromatography to more environmentally friendly, biodegradable, membrane-based methods. This shift enhances process efficiency and aligns with our commitment to sustainable production.

In this presentation, we show how advanced machine learning and hybrid modeling approaches can be exploited to significantly improve process understanding, performance, and automated operation as digital twins. All presentations will be centered on industrial implementation examples for mAb, cell & gene therapy, and mRNA processes with numerous big pharma and CDMO partners allowing to quantify efficiency gains in and improved understanding in process development.

The lytic nature of IC-BEVS limits its use for continuous bioprocessing. Here, a continuous multi-stage bioreactor process was established to produce influenza hemagglutinin-displaying virus-like particles (HA-VLPs) using IC-BEVS. Different process designs (varying cell line and rBAC construct) and residence times (RT=18, 36, and 54 hours) were tested. The best-performing design allowed consistent rBAC (108-109 pfu/mL) and HA-VLPs (34±14 HA titer/mL) titers; RT=54 hours outperformed other RTs. HA-VLPs were efficiently identified by electron microscopy after 20 days of continuous operation. This work showcases successful continuous HA-VLPs production using IC-BEVS, paving the way for establishing continuous, integrated setups using this expression system.

We present a novel purification method for recombinant multimeric virus-like particles (VLPs) using metal-ion affinity precipitation. The study focused on VLPs made of turnip yellow mosaic virus coat protein, produced in Escherichia coli. Remarkably, a mere 15 µM (or lower) of transition-metal salt, such as nickel chloride, proved sufficient to induce precipitation, leading to an impressive recovery rate of up to 70% with a purity exceeding 0.90.