Our previous post on the best way to manufacture lentiviral vectors (LVVs) for cell and gene therapies (CGTs) discussed the challenges of optimizing a scaled-up LVV manufacturing process. LVVs are essential in the manufacture of cellular immunotherapies (e.g., CAR-Ts). This post focuses on improving three broad areas of manufacturing LVVs.
Cell culture and LVV production using stable producer lines
LVVs can be produced using transient transfection of cells with all needed plasmids to construct the virus. This typically includes four plasmids for envelop and packaging genes plus the gene of interest. Alternatively, LVVs can be produced using “packaging” cell lines that already contain viral packaging components, and only need to be transfected by a single plasmid-carrying gene of interest to generate LVV particles.
When large quantities of LVVs are required for applications in CGTs (e.g., producing a cellular immunotherapy for cancer), stable producer lines are typically used to reduce costs (see https://doi.org/10.1016/j.bej.2020.107868) and increase process robustness. Stable producer lines have integrated all viral genes into the host cell genome. Stable producer lines reduce the cost of goods sold (COGS) for production, by doing the following:
- Simplifying upstream production: Transient transfection of plasmids involves optimization of plasmid ratios to cells and results can be variable. Upstream production steps for stable producer lines are less complex and only involve the addition of an induction reagent to initiate viral vector production.
- Reducing the costs for reagents and labour: Stable producer lines eliminate the need for large amounts of expensive raw materials (e.g., GMP-grade plasmids, which may be 20-25 per cent of COGS). The process can be simplified and automated, reducing the need for skilled labour, which also helps to bring costs down.
- Increasing flexibility for scaling up: Stable producer lines can be grown as suspension cultures using a serum-free medium in traditional bioreactor systems. Knowledge and expertise from similar bioreactor processes for vaccine and other recombinant protein expression can be adapted, and processes made robust, minimizing lot-to-lot variability.
However, continuous production of LVVs results in cytotoxicity for the cells and, as a result, virus-producing cells will lose viability over time.
Two ways to reduce cytotoxicity in stable producer lines include using inducible systems and less toxic alternative envelope proteins (e.g., RD114). Inducible systems act as on/off switches for the expression of viral transgenes. These improvements in controlling expression of packaging genes will reduce cytotoxicity but may require the use of antibiotics in the process.
Purification using multi-modal chromatography
Ion exchange chromatography is a commonly used polishing chromatography method due to its ability to remove impurities from a heterogeneous mixture. It is used to remove host cell protein and DNA impurities and also to prevent viral particles from aggregating. The conditions need to be carefully optimized as high salt concentrations could impact LVV infectivity.
Alternatively, multi-modal chromatography, which combines two or more purification methods, can be used. CCRM has tested Capto™ Core 700, a gentler form of multi-modal chromatography resin that optimizes polishing for improved recovery of LVV. It consists of beads with ligand-activated cores and inactive outer shells that employ size exclusion, ion exchange, and hydrophobic interactions together. The beads bind impurities (e.g., protein and DNA) within positively charged and hydrophobic cores while excluding viruses due to 700 kDa pores on the resin. LVV is collected in the flow-through under conditions that maintain infectivity and higher recovery.
Quality control assays
As a quality check during upstream manufacturing, several assays are used together to accurately assess viral titre and purity.
LVV production depends on viral titre, which measures the amount of virus in a given volume. Two primary testing methods include:
- Infectious titre: This assay determines infectivity of viral particles by transducing HEK293T cells with serially diluted virus.
- Total Virus particle titre: This assay quantifies the number of viral particles in a given volume performed by measuring the amount of p24 protein present in a test sample. This does not reflect infectivity of virus particles.
The ratio of these two titre measures indicates the purity of the viral preparation.
Other impurities are also measured, including:
- Host cell protein
- Host cell DNA
CCRM has performed several optimization studies to adapt these assays to LVVs produced from suspension cultures. These improvements will ensure the assays can be integrated into large-scale, suspension culture-based manufacturing protocols.
Using existing assays for viral titre during upstream and downstream processing is relatively straightforward and established. However, the technology transfer can lead to bottlenecks when working with a contract development and manufacturing organization (CDMO). Liquid-handling robotic systems can be used to automate assays and thereby speed the processing of multiple samples.
CCRM’s experienced process development team can help to incorporate quality control assays into LVV manufacturing processes. Contact us to learn how our team can help you improve your LVV manufacturing process.