A scalable suspension insect cell transfection method for production of baculoviruses with low amplification passages

摘要

Baculovirus expression vector systems (BEVS) have been widely used for production of recombinant proteins in insect cells. However, baculoviruses superinfection and repeated passages originate defective interfering particle (DIP) mutants, which is a limitation to a continuous large-scale production. Accordingly, a classical chemical transfection method performed on monolayer ofSpodoptera frugiperdainsect cells (Sf9) was modified to produce recombinant baculoviruses with high efficiency. Modifications consist to transfect exponentially growing cells in suspension after concentration by tenfold through centrifugation. Ten different constructions of recombinant baculoviruses with insert varying in size from 180?bp to 2,395?bp, were obtained through employment of theBac-to-Bacexpression system (ThermoFisher/Invitrogen). The transfection efficiency of the modified protocol varied from 45 to 57%, independent of the insert size, while classical method present transfection efficiency of 2 to 20%. After transfection of 6?×?106cells, the recombinant baculoviruses titer obtained with modified method was about 2?×?107pfu/ mL in a total volume of 12?mL, which is scalable to 24 liters of 1?×?108pfu/ mL, after only two amplification rounds, contributing to improve large scale heterologous protein production in insect cells, with low amplification passages.