Upscaling of lentiviral vector production by tangential flow filtration.
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 serum-free 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 low-speed 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.
Geraerts
Martine
M
Laboratory for Molecular Virology and Gene Therapy, K.U. Leuven, Belgium.
Michiels
Martine
M
Baekelandt
Veerle
V
Debyser
Zeger
Z
Gijsbers
Rik
R
D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D002498:Centrifugation; D003342:Corpus Striatum / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000302:isolation & purification*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids; D019076:Transgenes; D014773:Virology / Q000379:methods
D016895:Culture Media, Serum-Free; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins