Principles of a Scaleable Transient Transfection and Expression Technology for Mammalian Cells

Abstract

We report the first use of DNA-calcium phosphate co-precipitates for DNA transfer to suspended mammalian cells in bioreactors or spinners. Our goal was a scaleable transient transfection method with efficient DNA transfer and rapid product formation. In a batch process CaPO₄-DNA complexes were directly injected into the culture medium of human immortalized kidney cells¹ (293 HEK). Low transfection rates were observed at times, especially at pH values of the culture medium lower than 7. However, at high calcium concentrations, sensitivity to low pH was decreased. By adjusting the calcium concentration in the medium, precipitate quality as a prerequisite for efficient transfection could be addressed. Indeed, we found product titers from suspended cells to be similar to those achieved in plates: In a 2L suspension, containing 293 HEK cells transfected with 1mg of purified plasmid DNA, we obtained 2 mg of recombinant Plasminogen after 5 days.