Stibbs, Dale John; (2023) Continuous Manufacturing of Lentiviral Vectors for Cell and Gene Therapy Applications. Doctoral thesis (Eng.D), UCL (University College London).

Text Dale Stibbs EngD Thesis.pdf - Accepted Version Access restricted to UCL open access staff until 1 October 2024. Download (17MB)

Abstract

Lentiviral vectors (LVs) are efficient tools for mediating long-term, stable gene expression in mammalian cells. These properties have resulted in extensive interest in their application for the manufacture of gene-modified cell therapies. LVs are usually produced using transient transfection. However, the high costs of the plasmid DNA (pDNA) and transfection reagent make this approach undesirable at scale. This can be addressed using stable producer cell lines expressing all the required LV genes. This work aimed to establish a continuous LV production process using the WinPac-RDpro-GFP cell line that constitutively expressed third-generations LVs. Process development in flasks identified seeding at 3 x 104 cells.cm-2 maximised infectious LV titres and was amenable for scaling. Modulating the medium exchange rate found that 1, 2 and 3 vessel volumes per day (VVD) resulted in similar LV titres of 0.69 ± 0.08 TU.cell-1. Scaling the process from a T-25 flask to a multilayer flask with a surface area of 6,320 cm2 led to a 2-fold reduction in the infectious titre. It was postulated that this was due to poor gas exchange within the multilayer flask that impacted cell growth. The process in flasks was transferred to the iCELLis Nano® bioreactor. Process development through implementing perfusion culture, increasing the medium exchange rate to 1.5 VVD and reducing the culture pH to 6.85 from 7.20 resulted in a 2.6-fold increase in infectious titres compared to the 1 VVD pseudo-perfusion process. The ratio of physical to infectious particles was 132 ± 8, which indicates a high quality of vector supernatant. Across three independent processes, the batch-to-batch variability was low, with the coefficient of variation (CV) of total infectious and physical LVs being 6.4% and 10.0%, respectively. These were comparable to the CVs obtained in T-25 and multilayer flasks, indicating that LV production using stable cell lines resulted in high process reproducibility. A second issue is that the material produced requires concentration to facilitate downstream processing. Single-pass tangential flow filtration (SPTFF) provides an in-line concentration method. To realise the potential of this technology, a microscale SPTFF system was designed to facilitate process development with reduced feed volumes. The system had a membrane area of 59.5 cm2, 11.2-fold lower than the laboratory-scale system. Modulating the feed flux to concentrate 300 mL of LV supernatant found that recoveries of infectious LVs increased with increasing feed fluxes. The lowest recoveries were recorded at 4 L.m-2.h-1 at (4.3 ± 0.9)%, with the highest recoveries found at 100 L.m-2.h-1 of (73.6 ± 2.8)%. The low recoveries were attributed to LVs becoming deposited within the system due to the low shear rates. Implementing a wash step increased recoveries of infectious LVs, with total recoveries from both steps ranging from (74.2 ± 2.7)% to (87.0 ± 2.2)% at feed fluxes between 15 and 100 L.m-2.h-1. The lower recoveries at feed fluxes of 4 and 8 L.m-2.h-1 were attributed to the long processing times and short half-life of the LVs. The SPTFF processes achieved total protein removals between (90.0 ± 1.6)% to (45.0 ± 2.6)%. Similarly, double-stranded DNA (dsDNA) removal ranged between (93.3 ± 0.8)% to (46.1 ± 0.2)%. For protein and dsDNA, the removal decreased with increasing feed fluxes. Analysis of the permeate from the system identified that dsDNA appears to be in or on the membrane. Conversely, the protein transversed the membrane and was recovered in the permeate. Overall, the results demonstrate the advantages of implementing continuous production and concentration for LV production, and the work represents a step towards establishing an integrated continuous bioprocess.

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