Here, we present data on the performance of a novel Sf9-based Baculovirus expression system based upon a yeastolate-free, animal origin-free, chemically-defined, high-density culture medium that allows for Sf9 cells to reach densities nearly twice as high as those attained in traditional yeastolatecontaining media. Sf9 cells adapted to grow to high densities in the yeastolate-free media were generated and a new, high-efficiency Bacmid transfection reagent was developed to allow for the generation of high titer baculovirus stocks. Together, with the addition of protein expression enhancer these improvements allow for the optimization of a new expression protocol that takes advantage of the high cell densities achievable with the new chemically-defined medium and adapted Sf9 cells, as well as high multiplicity of infection (MOI), to significantly improve protein titers and enable lot -to- lot consistency of both cell growth and protein expression in a defined media formulation.

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