Polyelectrolyte complex
particles assembled from plasmid DNA (pDNA)
and poly(ethylenimine) (PEI) have been widely used to produce lentiviral
vectors (LVVs) for gene therapy. The current batch-mode preparation
for pDNA/PEI particles presents limited reproducibility in large-scale
LVV manufacturing processes, leading to challenges in tightly controlling
particle stability, transfection outcomes, and LVV production yield.
Here we identified the size of pDNA/PEI particles as a key determinant
for a high transfection efficiency with an optimal size of 400–500
nm, due to a cellular-uptake-related mechanism. We developed a kinetics-based
approach to assemble size-controlled and shelf-stable particles using
preassembled nanoparticles as building blocks and demonstrated production
scalability on a scale of at least 100 mL. The preservation of colloidal
stability and transfection efficiency was benchmarked against particles
generated using an industry standard protocol. This particle manufacturing
method effectively streamlines the viral manufacturing process and
improves the production quality and consistency.
With an increasing number of lentiviral vector (LVV)-based cell and gene therapy candidates reaching clinical trials, scalable suspension cell culture processes using stirred tank reactors (STRs) are needed to meet future demands. However, to cost-effectively scale LVV production in STRs requires process development which can be expensive and time consuming to perform in bench-top bioreactors. To address these issues, a multi-parametric approach for process development using a micro scale bioreactor system (Ambr ® 15 cell culture system, Sartorius) was assessed. Since the medium exchange process step cannot be linearly or methodically scaled-down from a bench-scale STR to a microbioreactor due to system differences, this study focused on adjusting to those differences by developing and testing three different medium exchange protocols. The implementation of one approach (Process 2.0) using an automated cell settling medium exchange protocol produced results which closely aligned with an established LVV bench-scale process in transfection efficiency and productivity, as well as lowered variability between vessels in the cell culture workstation. In summary, this study demonstrates the suitability of the Ambr 15 ® system as a process screening tool which has the potential to reduce costs and timelines of the development of scalable LVV production systems in suspension culture.
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