Purification of recombinant baculoviruses for gene therapy using membrane processes.
Recombinant baculoviruses (rBVs) are widely used as vectors for the production of recombinant proteins in insect cells. More recently, these viral vectors have been gaining increasing attention due to their emerging potential as gene therapy vehicles to mammalian cells. Their production in stirred bioreactors using insect cells is an established technology; however, the downstream processing (DSP) of baculoviruses envisaged for clinical applications is still poorly developed. In the present work, the recovery and purification of rBVs aiming at injectable-grade virus batches for gene therapy trials was studied. A complete downstream process comprising three steps--depth filtration, ultra/diafiltration and membrane sorption--was successfully developed. Optimal operational conditions for each individual step were achieved yielding a scalable DSP for rBVs as vectors for gene therapy. The processing route designed hereby presents global recovery yields reaching 40% (at purities over 98%) and, most importantly, relies on technologies easy to transfer to process scales under cGMP guidelines.
Vicente
T
T
IBET/ITQB-UNL, Oeiras, Portugal.
Peixoto
C
C
Carrondo
M J T
MJ
Alves
P M
PM