Citation

  • Authors: Heldt, CL., et al.
  • Year: 2019
  • Journal: J Chromatogr B Analyt Technol Biomed Life Sci 1124 173-179
  • Applications: in vitro / DNA / PEIpro, PEIpro residual test
  • Cell types:
    1. Name: Expi293F
      Description: Human embryonic kidney Fibroblast
      Known as: Expi 293-F, Expi, HEK-293 Expi
    2. Name: HEK-293S

Method

Expi293 cells were grown in 500 ml shake flasks culture in Expi293 medium at 200 rpm. Plasmid co-transfection for AAV2 production was achieved at a DNA:PEIpro ratio of 1:2. For AAV9 production, suspension HEK-293 cells were grown and transfected in 10L bioreactors, AAV2 and AAV9 particles were harvested at 72 h post-transfection.

Abstract

Adeno-associated virus (AAV) is a strong candidate for single-gene mutation gene therapy. AAV comes in several serotypes that target different organs in the body. The current purification methods for AAV vectors often rely on serotype dependent affinity chromatography. However, it is desired to create a platform for AAV purification that mirrors the evolution of antibody platform processes. To do this, any serotype dependent steps need to be removed from the process. The harvest and initial capture steps that can satisfy all of the needs of a platform AAV process is the use of low pH and Triton in the harvest, followed by filtration and cation exchange chromatography (CEX) for initial capture. The low pH hydrolyses and removes the host cell DNA, a difficult contaminate to remove. CEX then provides a concentration and capture step. The only step that remains is to determine the polishing and final formulation. This harvest strategy provides a serotype independent purification that removes both host cell DNA and host cell proteins and is friendly to scale-up for future AAV processes.

Pubmed