Updated transient gene expression protocol and the development of a small-scale transient gene expression protocol -evaluated by testing of longevity molecules as potential protein expression enhancers

Abstract

Abstract Transient gene expression (TGE) is commonly used to quickly produce protein-based drugs, such as antibodies, that require post-translational modifications. We have previously published a protocol for efficient and inexpensive TGE of multispecific and multivalent antibodies, which we have improved upon and described in the first part of this paper; by replacing the expensive Expi293 expression with BalanCD HEK293 medium, the medium cost was decreased by approximately 90%, and in addition, the harvesting procedure was shortened from 2.5 hours to 15 minutes by mixing the harvested cell media with the mineral compund diatomaceous earth that effectively absorbs and sequester cells and cell debris. The cell media can then be quickly filtered without the need to exchange obstructed filters and without the previously required 1-hour centrifugation step. In the second part of this paper, a small-scale TGE protocol was developed to surmount the cost limitation of testing many culture conditions. The small-scale TGE protocol uses 6-well plates which is the cheapest alternative for scaling down the protein expression, and consumes 83% less material compared to transfections done with the smallest available shaking flasks for cell culture. To test the small-scale TGE protocol we evaluated substances, belonging to a category called longevity molecules, as potential protein expression enhancers. Though the longevity molecules failed to increase protein expression in the conditions tested, our results corroborates the functionality of the small-scale TGE protocol and provides a simple methodology to expediently evaluate factors that can lead to improved protein production protocols in the future.