Optimising producer cell line culture and transfection for improved viral vector manufacture
Creators: name: Evans, Angharad
Publisher: name: Loughborough University
Year: 2022
Subjects: subject: 90499 Chemical Engineering not elsewhere classified
subject: FOS: Chemical engineering

description: The field of gene therapy has changed drastically since the first human gene transfer experiment conducted by Rosenberg et al., over 30 years ago. The field’s increasing popularity has brought to light the ever-increasing demand for more efficient and cost-effective solutions to the manufacturing of cell and gene therapies. The need to address these issues has highlighted the numerous challenges that the industry currently faces when striving to develop novel gene therapies. The objective of this research was to investigate improving achievable outputs of cell manufacture for two of the most used adherent-based producer cell lines, HEK293 and HEK293T, with further studies examining the impact on lentiviral vector outputs. Investigations into improving cell culture outputs resulted in the generation of a standardised culture process with predictable, scalable, high yield outputs with a lower cost production. Standardisation of the cell culture process comprised of the implementation of set seeding densities, set culture times, set reagent volumes and defined mixing procedures, all of which led to the creation of a standard operating procedure for adherent HEK culture for use within the Centre for Biological Engineering at Loughborough University. Consistent cell densities of 3.34x105 ± 8.40% cells/cm2 for HEK293T and 2.06x105 ± 13.23% cells/cm2 for HEK293 were achieved, with the low cost of output cells (HEK293T) ranging between £4.15x10-5 and £4.98x10-5 per individual passage. Furthermore, it was found that the more cells that were produced within a standardised process, the lower the cost to create those cells, with a 42% reduction in cost of creation being observed. Qualities such as these would prove extremely beneficial in the development of scalable clinical grade manufacturing processes. Further studies aimed to identify and address key areas of variation within the transfection processes and examine factors that could influence the point of infection for transfection and the impact on lentiviral vector outputs. Factors such as type of production cell used, the seeding density used, length of incubation prior to transfection, phase of cell cycle and presence of serum at point of transfection were investigated for their impact on transfection success. Additional factors were investigated with the aim of assessing their impact on lentiviral transduction success. The factors being assessed were the type of filter membrane used for clarification of viral supernatant, presence of lentiviral infection enhancing polymers, and volume of viral supernatant added to cells. Baseline investigations generated titres of 7.02x103 TU/ml in HEK293 cells. Post experimentation, improved titres of 4.67x104 TU/ml and 4.00x105 TU/ml were achieved for HEK293 and HEK293T cells, respectively. Batch to batch variability in lentiviral titres are a known phenomena within the industry, especially with the use of anchorage dependant production systems. This variation was also observed within this body of work and emphasised the current difficulties being faced when developing accessible, scalable, and standardised manufacturing procedures for the generation of viral vectors using producer cell lines.