Production of CGMP-Grade Lentiviral Vectors.

Lentiviral vectors are important tools for gene transfer because of their ability to transduce a number of cell types without the need for host cells to be dividing (1, 2). As a result, investigators are using them as gene delivery vehicles in clinical applications (3–6). Although these vectors are used routinely in many research laboratories, large-scale production using current good manufacturing practice (CGMP) methods comes with a set of challenges that must be considered as more clinical trials using lentiviral vectors receive regulatory approval (7). One important consideration in designing a CGMP-compatible process is the need to have a manufacturing protocol that produces consistent lentivirus for multiple CGMP productions. Several methods are commonly used to transfect cells with lentiviruses: e.g., lipofection, electroporation, and calcium phosphate transfection. The selected method must consistently produce high-titer virus. Although several laboratories have produced stable packaging cell lines containing certain viral genes (8–13), development of such lines is time consuming. In addition, use of stable lines does not allow the flexibility to change envelope and accessory genes or to readily alter the ratios of helper genes expressed. With a mind toward generating a flexible process to produce lentivirus for a range of clinical protocols, we chose to use a four-plasmid system. Using self-inactivating (SIN) vesiculo-stomatitis virus G (VSV-G) pseudotyped lentiviral vectors to generate lentiviral particles (14), replication incompetent particles are produced by cotransfection of three helper plasmids encoding separate packaging functions as well as a separate transfer plasmid containing the vector backbone. Helper plasmids encode gag/pol and the rev-responsive element (pCgp), rev protein (pCMV-rev2), and the heterologous VSV-G envelope protein (pCMV-G) (15). To produce lentivirus suitable for transducing a range of patient-specific cells in diverse clinical trials, conditions must be optimized to produce the highest viral titers. We focused on maximizing titer and quantity of virus produced upstream while maintaining a consistent downstream process of concentration and purification. Although many parameters involved in lentiviral transfection have been assessed by others at the research scale using T-flasks, we developed a scalable method for efficient transfection at the multiliter scale using 10-layer Corning CellStack trays. This process allows for production of large quantities of lentivirus required for phase 1–2 clinical trials (16). Using antibiotics in production of materials for human clinical trials is undesirable. As a result, many clinical biologics are filtered as a final step in the manufacturing process. However, we and other investigators have observed that sterile filtration of lentivirus (0.2-µm pore size) decreases viral titer (17). Thus, we preferred not to invoke that sterilization method in our process. As a result, it was essential to take every precaution to ensure product sterility at all steps of the manufacturing process. To address those concerns in maintaining sterility of the lentiviral product — as well as for manufacturing staff safety — we devised a semiclosed manufacturing process that contains the virus during upstream production. Our semiclosed system can be used as part of a modular manufacturing process involving subbatches of lentiviral supernatant. This multiple-subbatch system is particularly helpful in addressing safety concerns that accompany production of large volumes of lentiviral material (100 L) by allowing for pooling of multiple 10-L subbatches made over several weeks. However, it is crucial to show consistent viral production across those subbatches. By optimizing upstream parameters, we developed a process that allows for extensive scale-up in a safe, sterile, and reproducible system to produce clinical-grade lentivirus. Our manufacturing process has produced several lots of lentivirus that have been or are currently being used in ex-vivo gene therapy trials.