In the field of cell and gene therapy, there are two main types of viral vectors: adeno associated virus (AAV) and Lentivirus (LV). The manufacture of these vectors is dependent on the regulatory requirements dictated by its end use. AAVs are most commonly used to deliver gene therapies – meaning they will be administered directly to a patient. In contrast, LV is typically used as an input material to genetically modify cells used for cell therapies (e.g. CAR-Ts) and is not included in the final product delivered to the patient.
In this post we will focus on LV because of its importance in the manufacture of cellular immunotherapies (e.g. CAR-Ts). However, many of the same considerations will apply to AAV manufacturing as well.
Today, clinical and commercial-scale processes for the manufacture of LV remain based on methods adapted from the basic research laboratory. Upstream processing begins with transient transfection of adherent HEK293 cells, cultured in serum-containing medium, with 3-4 plasmids encoding the main components of the viral vector. The virus is produced by the HEK293 cells and released into the medium, where LV is harvested in two collection steps at 48- and 72-hours post-transfection.
Next, the harvest of LV from the collected medium is achieved by a number of downstream processing steps. The first purification enzymatically breaks down residual plasmid DNA. This is a critical safety step to ensure that the size of any remaining non-viral DNA fragments are reduced below a minimal functional size to prevent unwanted gene expression. Next, ultrafiltration is used to concentrate the virus, followed by chromatography to remove impurities. Since chromatography requires dilution in a buffer solution, a second concentration step is then performed. Finally, the virus undergoes sterile filtration to yield concentrated viral vectors with minimal impurities.
Sounds simple, right!? In fact, each of these steps requires optimization to allow for process scale-up and maximal recovery of functional virus, free of contaminants. The main challenge for optimization is how to effectively translate protocols that are reliable and reproducible at small scale to a scale compatible with commercial product manufacture.
Ultimately, the aim of process optimization is to achieve the industry standard of 20%-30% recovery of the final product.
Challenges for Optimization of Scaled-up LV Manufacturing Processes
- Scaling out to scale up: The use of adherent cultures makes it difficult to scale up linearly, instead, scale-up of LV production is accomplished by scaling-out. Multi-layer cell factories are often used to increase the available surface area for culture of HEK293 cells. Using this method, batches of 60-100L can be produced during the upstream steps.
- The use of serum: HEK293 cells are typically grown in medium containing animal-derived serum. This is an obvious drawback due to the inherent risk of introducing pathogens and lot to lot variability, as well as potential supply chain issues. Developers must ensure a reliable and safe supply of serum and identify a back-up should a shortage arise with their primary supplier and show equivalency of the secondary supplier.
- Transient transfection is inherently variable: Even at small scale, transfection efficiency will vary due to uneven uptake of plasmids by the cells. This problem is compounded at larger scales. Transfection efficiency is influenced by the choice of transfection reagent and quality of plasmid DNA. To ensure optimal and reliable transfection efficiency, establishing a supply chain for high-quality plasmid DNA and your chosen transfection reagent is of critical importance.
- Loss of product during downstream processing: Since virus is lost or degraded in every downstream processing step, optimization is required for unit operation. In this post, we will not cover every factor that can contribute to sub-optimal recoveries; however, we will highlight the sensitivity of viruses to pH, salt concentration and temperature as key considerations. Determining the optimal buffer to be used during downstream processing of your virus will improve recovery.
- Requirements for quality control (QC) testing: The amount of product needed to carry out QC testing is often underestimated. Determining the QC tests that are required for your product (e.g. residual testing, viral titer, potency) early in the manufacturing plan will help you to design a process that yields enough virus to carry out this critical step.
To provide more comprehensive solutions to these scale-up challenges, the industry is moving towards the use of three new technologies: 1) suspension cultures grown in bioreactors—improving manufacturing logistics, labour costs and process footprint; 2) stable producer cell lines—able to reliably produce viral vectors at more consistently high levels without the need for transient transfection; and 3) animal component free, chemically-defined medium—eliminating reliance on animal serum. The adoption of these new technologies for viral vector manufacture will require process development innovations that CCRM is already hard at work on. To date, we have developed a bioreactor-based viral manufacturing platform using stable producer cell lines grown in suspension culture with chemically-defined medium.
We believe this approach provides a robust, scalable and commercially-viable solution for viral vector manufacture that will enable new cell and gene therapy products.