In an introductory post on lentiviral vector (LVV) manufacturing for cell and gene therapies (CGTs) we touched upon the challenges with transfection-based protocols for producing LVVs at large scale. Here we will take a closer look at the use of stable producer cell lines as an alternative to transient transfection for the manufacture of LVVs.

What Is a Stable Producer Cell Line?

For applications in CGTs where large quantities of LVVs are required (e.g. the production of cellular immunotherapy for cancer), using stable producer lines is one of the primary ways to reduce costs and increase safety and reproducibility. The first stable producer lines or “packaging lines” were created in the early 1990s. To understand what they are and how they work, we need a brief lesson in virology. The LVV system is based on HIV-1 – a well known virus that causes disease in humans. Functional virus is produced when the following four genes are expressed: 1) gag-Pol – encoding structural proteins and enzymes; 2) Rev – encoding accessory protein for nuclear export; 3) VSV-G – encoding for a common envelope protein that allows for the virus to enter target cells; and, 4) Promoter elements and transgenes. The goal of creating stable producer lines is to achieve controlled production of functional virus by having all four genes integrated into the host cell DNA to increase reproducibility while driving down per-batch cost.

Addressing the Challenge of Cytotoxicity with Stable Producer Lines

Continuous production of LVVs is cytotoxic due to the expression of VSV-G envelope proteins and the viral proteases encoded by the gag-pol genes. Even with traditional transient transfection, virus producing cells will lose viability over time. The issue of cytotoxicity becomes an even greater concern in stable producer lines, where sustained production of viruses is the goal. The primary strategies to mitigate cytotoxicity in stable producer lines have been the use of inducible systems and alternative envelope proteins that are less toxic (e.g. RD114). Inducible systems, like the Tet-On and Tet-Off systems, have been genetically engineered into stable producer lines to act as on/off switches for the expression of viral transgenes that can be controlled by the introduction of antibiotics or small molecules into the culture medium. The introduction of these systems was an important advancement in stable producer line technology because they allow for fine-tuned control of gene expression. However, they add an additional level of complexity to these systems that requires considerable expertise to engineer.

What Are the Process Development Considerations for the Use of Stable Producer Lines?

While there are considerable challenges for large-scale use of transient transfection systems, the process changes required when using a stable producer line should also be taken into account. For instance, prior to beginning upstream processing, a selection step is required to identify a clonal population of cells that will be efficient at producing virus. In a scenario where a new stable producer line is being created, there is considerable development work required to characterize the cell line for safety and performance. Further, for applications in CGTs, it is imperative that the developer of a new stable producer line starts with a qualified cell line to avoid delays when moving from early development to manufacturing for clinical trials.

How Do Stable Producer Lines Create Efficiencies and Reduce Costs in the Production of CGTs?

1. Simplified Upstream Production: transient transfection of plasmids requires more steps that can introduce variability. In contrast, the upstream steps for stable producer lines are less complex, consisting of cell expansion and induction of viral gene expression. Following the expansion step, a qualified master cell bank can be created, cryopreserved, and used in subsequent production runs, thereby increasing the reproducibility of the process.

2. Lower Costs for Reagents and Labour: when producing LVVs for use in CGTs, GMP-grade transfection reagents and plasmid DNA are a significant cost driver that need to be removed from the final product. Stable producer lines eliminate the requirement for these expensive raw materials. Further, there is inherent variability in the raw materials used for transient transfection, which translates to variability in the efficiency of virus production from batch to batch. Finally, transient transfection is technically difficult and requires skilled labour with molecular biology expertise. In contrast, the single induction step required for LVV production in stable producer lines is simpler, reduces the requirements for skilled labour, and is more amenable to automation.

3. More Flexibility for Scale-Up: commercial scale production of LVVs is greatly facilitated by moving away from the use of adherent cell culture and adapting protocols for suspension culture. Stable producer lines can be modified for use in suspension culture, thus greatly facilitating scale-up in a bioreactor system. Further, many stable producer lines have been optimized for use with chemically defined media – reducing variability and reliance on the use of animal serum.

When Is the Time to Switch from Transient Transfection to Stable Producer Lines for LVV Manufacturing?

Ultimately the answer is “it depends.” While transient transfection systems have drawbacks, the end use of the product and the therapy developer’s resources will dictate if and when a stable producer line is used for the production of LVVs. As with any process change, the most cost-effective time to make this decision is early in development. A change in production systems would require the developer to demonstrate equivalency between LVVs produced using transient transfection versus stable producer lines – a process that would introduce costs, delays, and require additional interactions with regulators.

Another question for developers to consider is whether to develop a novel stable producer line in-house. The industry is not yet at the point where this is common practice. Considerable investment is required to generate a novel stable producer line, as this process is not trivial and requires specialized expertise in genetic engineering. As the indications for CGTs expand and technology advances, therapy developers may want to gain an edge on the competition with a propriety stable producer line. However, given the current state of the industry, whether to make an investment in creating a novel stable producer line and, at what point to do so, remains an important early decision.

Working with CDMOs like CCRM, which have significant process development experience in LVV manufacturing, can help therapy developers navigate these complex questions and make the best decision for their product.