Optimisation of a simple method to transiently transfect a CHO cell line in high-throughput and at large scale.
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.
Discovery Sciences, AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK.
W Mark
WM
Abbott
Discovery Sciences, AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK.
Brian
B
Middleton
Discovery Sciences, AstraZeneca, Pepparedsleden 1, 431 83 Mölndal, Sweden.
Fredrik
F
Kartberg
Discovery Sciences, AstraZeneca, Pepparedsleden 1, 431 83 Mölndal, Sweden.
Josefine
J
Claesson
Discovery Sciences, AstraZeneca, Pepparedsleden 1, 431 83 Mölndal, Sweden.
Robert
R
Roth
Discovery Sciences, AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK. Electronic address: David.Fisher2@astrazeneca.com.
David
D
Fisher
D000818:Animals; D019149:Bioreactors / Q000191:economics; D016466:CHO Cells / Q000166:cytology / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000191:economics / Q000379:methods; D003412:Cricetulus; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics; D015870:Gene Expression; D011094:Polyethyleneimine / Q000737:chemistry; D014162:Transfection / Q000191:economics / Q000379:methods*
D011094:Polyethyleneimine; D004247:DNA
Design of experiments; Polyethyleneimine; Transient gene expression