Upscaling of lentiviral vector production by tangential flow filtration

✍ Scribed by Martine Geraerts; Martine Michiels; Veerle Baekelandt; Zeger Debyser; Rik Gijsbers

• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 251 KB
• Volume: 7
• Category: Article
• ISSN: 1099-498X
• DOI: 10.1002/jgm.778

✦ Synopsis

Background HIV-1-derived vectors are promising tools for gene transfer into the brain. Application of these vectors for gene therapy or for the creation of animal models for neurodegenerative diseases requires standardization and upscaling of lentiviral vector production methods.

Methods

In this study, serum-free HIV-1 vector production was efficiently upscaled by use of cell factories and the introduction of tangential flow filtration (TFF) prior to centrifugation.

Results

Vector titers (TU/ml) and p24 values (pg p24/ml) for a serumfree HIV-1 vector produced in cell factories and using TFF prior to centrifugation were comparable to those of small-scale productions. TFF allowed a 66-fold concentration of the vectors with complete vector recovery. Further concentration of the vector (30-fold) was achieved either by lowspeed centrifugation or by ultracentrifugation. Combination of TFF and ultracentrifugation resulted in a vector recovery of 90-100% and titers that increased 1800-fold and 900-fold for transducing units and p24 concentration, respectively.

Conclusions With this new standardized method for lentiviral vector production and concentration, 1 ml of concentrated vector is routinely produced with titers of 10 9 -10 10 TU/ml starting from 2 l of cell-culture medium. Moreover, stereotactic injection of this vector in mouse striatum resulted in a large transduced brain volume in the absence of any immune response.