There is a large bottleneck in gene therapy manufacturing, therefore specialised technical knowledge in process and analytical development combined with knowledge of GMP facilities is critical for bringing these therapies to the clinic. Ales Strancar, Managing Director at Sartorius BIA Separations demonstrated how technology can be used to improve production of adeno-associated virus (AAV) vectors which are key for gene therapy.
Recovery in AAV manufacturing is critical due to the high cost of gene therapy treatments, yet existing analytical methods are often insufficient. Therefore, Sartorius has developed a PATfix system. This unique tool relies on chromatography-based technology for real-time monitoring of AAV capsids, allowing for precise detection of impurities like histone-DNA complexes and residual proteins.
Strancar explained that most AAV production is intracellular meaning it requires cell lysis. For cell lysis, detergents are most commonly used but they can clog filters during scale-up. Stamcar suggested salts as an effective alternative to detergent.
Stancar said: “So we need to find something which sticks to any DNA-related impurity,” he continued, “With the fluorescent detector, we can trace the DNA-related impurities.”
Cation exchange chromatography can balance purity and stability and does not require the more specific conditions required by affinity-based methods. Stancar noted: “With the cation exchange, you need to reduce the pH down to 4 or even 3.5.
The lower the pH, the better the purity but of course we are out of the comfort zone of the stability, so we are destroying a bit of the product.”
This shows that finding the right compromise between stability and purity remains challenging. Even though scientific literature posits that AAV capsids are stable, real-world evidence shows that under low pH conditions required for purification, they are less stable than expected.
Furthermore, Stancar stressed that differentiating between empty, partial, and full capsids is necessary for product quality. Advanced detectors like fluorescent and multi-light scattering tools help differentiate these properties. Cation exchange chromatography offers substantial advantages over affinity methods, delivering higher recovery rates (up to 30% more) and comparable or better purity, making it a cost-effective and reliable choice.
Despite advancements, a universal AAV manufacturing platform remains elusive and could take years to develop. However, tools like the PATfix system optimise chromatography conditions and focus on recovery and purity. This can significantly reduce costs for manufacturers and improve the quality of AAV vectors. Stancar concluded: “In many cases replacing affinity with Cation exchange would result in better recovery, meaning much cheaper manufacturing.”
