Development of a semi-automated high throughput transient transfection system

• Using a Biomek FXP liquid handler, we developed a semi-automated high throughput PEI-mediated 293T transient transfection process which transfects 96 tubespins in a batch.
• A spinner bag agitated on a magnetic stir plate prevented cell settling during the cell dispensing step and produced comparable cell seeding densities among different tubes.
• We programmed a complex addition method so that each tube received the DNA:PEI complex within a 9–10 min incubation range.
• The semi-automated transfection process was applied to express 520 variants of a human IgG1 (hu IgG1) antibody.

Transient transfection of mammalian cells provides a rapid method of producing protein for research purposes. Combining the transient transfection protein expression system with new automation technologies developed for the biotechnology industry would enable a high throughput protein production platform that could be utilized to generate a variety of different proteins in a short amount of time. These proteins could be used for an assortment of studies including proof of concept, antibody development, and biological structure and function. Here we describe such a platform: a semi-automated process for PEI-mediated transient protein production in tubespins at a throughput of 96 transfections at a time using a Biomek FXP liquid handling system. In one batch, 96 different proteins can be produced in milligram amounts by PEI transfection of HEK293 cells cultured in 50 mL tubespins. Methods were developed for the liquid handling system to automate the different processes associated with transient transfections such as initial cell seeding, DNA:PEI complex activation and DNA:PEI complex addition to the cells. Increasing DNA:PEI complex incubation time resulted in lower protein expression. To minimize protein production variability, the methods were further optimized to achieve consistent cell seeding, control the DNA:PEI incubation time and prevent cross-contamination among different tubespins. This semi-automated transfection process was applied to express 520 variants of a human IgG1 (hu IgG1) antibody.