Lentiviral vectors (LVVs) are broadly used in research and therapeutic development, yet substantial interlaboratory variability continues to hinder the comparison of vector quantity, potency, and impurity measurements, underscoring the need for standardized reference materials. To address this need, the Lentiviral Vector Reference Material (LVVRM) initiative was launched as a structured, multi-phase effort, with earlier phases focused on large-scale upstream production and downstream preparation of a GFP-encoding LVV. In the final phase of the LVVRM initiative described herein, bulk material produced during prior large-scale manufacturing runs was thawed, pooled, subjected to sterile filtration, and dispensed into single-use vials under controlled conditions designed to maintain stability and consistency. Each lot then underwent comprehensive quality control testing. Sterility, identity, purity, physical and functional titers, and safety attributes were assessed using established characterization frameworks appropriate for lentiviral vector reference materials. The resulting LVVRM (ATCC® VR-3382™) met quality standards and release criteria, providing a well-characterized reagent suitable for assay calibration, method qualification, and longitudinal performance monitoring across laboratories.
Tag: <span>lentiviral vector</span>
Lentiviral vectors (LVV) are widely used in an increasing number of approved cell and gene therapies, and benchmarking is important in evaluating production processes and products. We have produced a lentiviral vector reference material (LVV-RM) that expresses the common marker gene (green fluorescent protein [GFP]) to enable data comparisons and support LVV research programs. To generate that reference material, we have applied our previously developed fed-batch process to a 100 L production using an inducible LVV-producer cell line. The LVV material was harvested three days post-induction from a 200 L single-use bioreactor. A downstream purification process was also developed and scaled-up to meet production requirements: It consisted of nuclease digestion, clarification by depth filtration, chromatography capture using CIM-QA monolith anion exchangers, and ultrafiltration/diafiltration using a hollow fiber membrane prior to bulk-filling the final product. A total of 4.4 L of diafiltered and concentrated LVV product was obtained (9.2 E10 particles/mL, 3.6 E7 TU/mL) and stored at -80°C. Nearly 8000 vials are now available to the LVV community via the American Type Culture Collection (ATCC).
The rapidly growing interest for cell and gene therapies demands the development of robust, scalable, and cost-effective bioprocesses for viral vector production. For the production of lentiviral vector (LVV) at high titers, we have developed an inducible packaging system in suspension HEK293 cells from which we can also generate stable producer cell lines, in serum-free conditions. To evaluate the potential of this platform, we have generated a stable cell line that produces an LVV encoding a green fluorescent protein (GFP) and obtains 10E+07 to 10E+08 transduction units (TU)/mL at the 4 L, 10 L and 50 L scales. Functional LVV titers were maintained across all scales in bioreactors with different configurations and geometries indicating process robustness. Further, the addition of 10% feed increased the volumetric productivity by 3.5-fold in comparison to batch production, making our platform suitable for large-scale LVV production and showing a real potential for commercial manufacturing.