Optimisation of a simple method to transiently transfect a CHO cell line in high-throughput and at large scale

• A process that can be used as a guide to optimise transient transfection conditions.
• A simple, robust transfection protocol using “in situ” addition of DNA and PEI Max.
• Protocol is essentially the same from screening scale to Wave Bioreactors.
• Mild hypothermic conditions give a significant increase in volumetric yield.
• The procedure is applicable to a host of different secreted proteins.

Transient expression of heterologous proteins in mammalian systems is a powerful way to generate protein reagents quickly. However, it has historically suffered from poor yields in comparison to methods where the recombinant gene is stably integrated into the genome and high expressing clones isolated. Transient methods have been well described for HEK-based systems. In this paper we show the use of a design of experiments (DoE) approach to quickly analyse the effect of a range of different parameters on protein expression from a CHO-based transient system. We show that this system is amenable to a very simple transfection procedure by independent direct addition of DNA and transfection reagent to the culture vessel. In addition we show that expression can be improved by reducing the temperature of the culture conditions post-transfection. The process is demonstrated to be transferrable from 3 ml cultures in deep 24-well plates through cultures in CultiFlask Bioreactors, shake flasks and up to 25 L culture in Wave Bioreactors. Data are shown to illustrate the utility of the system with a number of different classes of protein.