Viral vectors are a critical component of cell and gene therapies. Unfortunately, process development and manufacturing of viral vectors are a major bottleneck in the industry. One of the current challenges in the process development and manufacturing of viral vectors is viral titer quantification. Many assays for viral titer quantification are low-throughput and high-cost assays with poor robustness and reproducibility.
HIV-1-derived lentiviral vectors (LVVs) and other retroviral vectors were first used in the 1990s for cell and gene therapy, and they are the most commonly used ex vivo gene delivery vehicle for chimeric antigen receptors (CARs) in CAR-T cell cancer immunotherapies. Since the first use of LVVs in cell and gene therapies, assays have been developed to measure both physical and functional titers of LVVs.
Physical titers estimate the number of viral particles in a sample by quantifying viral proteins or nucleic acid. Measuring lentiviral p24 by ELISA or other immunoassays, and lentiviral RNA by qPCR are well-established and commonly used methods to determine the physical titer, since these assays can deliver same-day results. However, the numbers resulting from physical titers are typically overestimates because they also detect non-infectious viral particles and free, unassembled p24 protein.
In contrast to physical titers, functional titers measure the number of viral particles capable of infecting the target cells. Thus, functional titers may offer more practical information on potency and quality of the viral vector. For this purpose, a target cell line must be transduced with serial dilutions of viral samples. The integrated provirus is quantified by qPCR (quantitative DNA-polymerase chain reaction) of the cells’ genomic DNA. The drawbacks to this method are the labor-intensive steps that include the need to culture target cells, perform genomic DNA isolation, design and validate primers and probes, and generate suitable standards and controls.
Flow cytometry analysis of transduction efficiency is a popular alternative to traditional functional titer characterization by qPCR. In this method, flow cytometry is utilized to detect cells expressing the transduced transgene and quantify the LVV transduction efficiency to provide a functional titer. Despite the simplicity of this assay, there are various pitfalls that can cause inconsistencies and misinterpretation of the results. Here, we discuss these considerations and propose solutions to improve the robustness and efficiency of functional viral vector titration.