Citation

  • Authors: Valkama AJ. et al.
  • Year: 2020
  • Journal: Mol Ther Methods Clin Dev 17 717-730
  • Applications: in vitro / DNA / PEIpro
  • Cell type: HEK-293T
    Description: Human embryonic kidney Fibroblast
    Known as: HEK293T, 293T

Method

Dish/vessel: iCELLis Nano. iCELLis 500. DNA : 200 ng/cm². 300 ng/cm². Reagent Amount: 200 ng/cm². 300 ng/cm². Ratio: 1:1. four-plasmid system (pVSVg. pGag-Pol. pRev. and LV plasmid expressing (GFP or HSV-TK). Virus titration in HeLa cells.

Abstract

The interest in lentiviral vectors (LVs) has increased prominently for gene therapy applications, but few have reached the later stages of clinical trials. The main challenge has remained in scaling up the manufacturing process for the fragile vector to obtain high titers for in vivo usage. We have previously scaled up the LV production to iCELLis 500, being able to produce up to 180 L of harvest material in one run with perfusion. The following challenge considers the purification and concentration of the product to meet titer and purity requirements for clinical use. We have developed a downstream process, beginning with clarification, buffer exchange, and concentration, by tangential flow filtration. This is followed by a purification step using single membrane-based anion exchange chromatography and final formulation with tangential flow filtration. Different materials and conditions were compared to optimize the process, especially for the chromatography step that has been the bottleneck in lentiviral vector purification scale-up. The final infectious titer of the lentiviral vector product manufactured using the optimized scale-up process was determined to be 1.97 × 109 transducing units (TU)/mL, which can be considered as a high titer for lentiviral vectors.

Pubmed