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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.

The presence of the blood brain barrier (BBB) creates a nigh on impenetrable obstacle for large macromolecular therapeutics that need to be delivered to the brain milieu to treat neurological disorders. To overcome this, one of the strategies used is to bypass the barrier with what is referred to as a “Trojan Horse” strategy, where therapeutics are designed to use endogenous receptor-mediated pathways to piggyback their way through the BBB. Even though in vivo methodologies are commonly used to test the efficacy of BBB penetrating biologics, comparable in vitro BBB models are in high demand, as they benefit from being an isolated cellular system devoid of physiological factors that can on occasion mask the processes behind BBB transport via transcytosis. We have developed an in vitro BBB model (In-Cell BBB-Trans assay) based on the murine cEND cells that helps delineate the ability of modified large bivalent IgG antibodies, conjugated to the transferrin-receptor binder scFv8D3, to cross an endothelial monolayer grown on porous cell culture inserts (PCIs). Following the administrating of bivalent antibodies to the endothelial monolayer, a highly sensitive ELISA method is used to determine the concentration in the apical (blood) and basolateral (brain) chambers of the PCI system, allowing for the evaluation of apical recycling and basolateral transcytosis respectively. Our results show that antibodies conjugated to scFv8D3 transcytose at considerably higher levels compared to unconjugated antibodies in the In-Cell BBB-Trans assay. Interestingly, we are able to show that these results mimic in vivo brain uptake studies using identical antibodies. In addition, we are able to transversely section PCI cultured cells, allowing for the identification of biomarkers pertinent to transcytosis of the antibodies. Furthermore, studies using the In-Cell BBB-Trans assay revealed transcytosis of the transferrin-receptor targeting antibodies is dependent on endocytosis. In conclusion, we have designed a simple, reproducible In-Cell BBB-Trans assay based on murine cells that can be used to rapidly determine the BBB penetrating capabilities of transferrin-receptor targeting antibodies. We believe the In-Cell BBB-Trans assay can be used as a powerful, pre-clinical screening platform for therapeutic neurological pathologies.