Scalable Transient Protein Expression

Abstract

Transient transfection is a well-established method to rapidly express recombinant proteins from mammalian cells. Accelerating activity in biotherapeutic drug development, demand for protein-based reagents, vaccine research, and large initiatives in structural and functional studies of proteins have propelled the need to generate moderate to high amounts of recombinant proteins and other macromolecules in a flexible and rapid manner. Progress over the last 10-15 years has demonstrated that transient transfections can be reliably and readily scaled up to handle milliliters to tens of liters of cells in suspension culture and obtain milligrams to grams of recombinant protein in a process that requires only days to weeks. This review will summarize developments in this field, properties of the components of a transient expression system that enable maximal protein production, and detailed protocols for this application.

Transient CHO expression platform for robust antibody production and its enhanced N‐glycan sialylation on therapeutic glycoproteins

“…It is known that the transfection efficiency in CHO cells is significantly lower than that in HEK293 cells . To better understand the transfection efficiency in ExpiCHO‐S™ and Expi293F™, green fluorescent protein (GFP) vector was transfected into these cells as described in Materials and Methods.…”

“…The delayed expression in ExpiCHO‐S™ cells is not due to low DNA transfection efficiency as both cell lines exhibited high GFP expression at 76 h. Similar transfection efficiency in both ExpiCHO‐S™ and Expi293F™ has also been reported recently, consistent with the data shown in this study. ExpiCHO‐S™ cells grow significantly faster than Expi293F™ cells, with an average doubling time of 17 h vs. 25 h of Expi293F™ . Rapid cell division might negatively affect protein expression, but the delayed protein prediction observed in the transfected ExpiCHO‐S™ cells doesn't seem to be attributed to cell growth.…”

“…Stable cell line expression generates consistent product quality with high yields and has been used as a preferred approach for generating large quantities of protein in the biopharmaceutical industry. To provide milligram to gram quantities of protein products in early therapeutic development, transient protein production systems have been employed because protein can be supplied in weeks . This rapid protein production technology facilitates biotherapeutics drug discovery by significantly shortening research timelines as compared to stable cell line generation.…”

“…Human embryonic kidney (HEK) 293 cells, established as a result of sheared adenovirus DNA transformation, is the most widely used cell line for transient expression . HEK293 transient expression achieves significantly higher protein titers than transient CHO, likely due to its better transfection efficiency and cell robustness . In recent years, a number of strategies have been developed to improve transient CHO expression levels.…”

“…In this study, we have expressed a number of therapeutic candidate proteins in the ExpiCHO‐S™ transient system. The authors show that the ExpiCHO‐S™ transient system achieved expression titers as high as those of transient Expi293F™, a HEK293 clone capable of achieving high cell densities and titers . Furthermore, some antibodies produced by ExpiCHO‐S™ had higher titers and better protein quality with less aggregation than those from Expi293F™.…”