A proline metabolism selection system and its application to the engineering of lipid biosynthesis in Chinese hamster ovary cells

James D Budge,Joanne Roobol,Gurdeep Singh,Théo Mozzanino,Tanya J Knight,Jane Povey,Andrew Dean,Sarah J Turner,Colin M Jaques,Robert J Young,Andrew J Racher,C Mark Smales

doi:10.1016/j.mec.2021.e00179

Abstract

Chinese hamster ovary (CHO) cells are the leading mammalian cell host employed to produce complex secreted recombinant biotherapeutics such as monoclonal antibodies (mAbs). Metabolic selection marker technologies (e.g. glutamine synthetase (GS) or dihydrofolate reductase (DHFR)) are routinely employed to generate such recombinant mammalian cell lines. Here we describe the development of a selection marker system based on the metabolic requirement of CHO cells to produce proline, and that uses pyrroline-5-carboxylase synthetase (P5CS) to complement this auxotrophy. Firstly, we showed the system can be used to generate cells that have growth kinetics in proline-free medium similar to those of the parent CHO cell line, CHOK1SV GS-KO™ grown in proline-containing medium. As we have previously described how engineering lipid metabolism can be harnessed to enhance recombinant protein productivity in CHO cells, we then used the P5CS selection system to re-engineer lipid metabolism by over-expression of either sterol regulatory element binding protein 1 (SREBF1) or stearoyl CoA desaturase 1 (SCD1). The cells with re-engineered proline and lipid metabolism showed consistent growth and P5CS, SCD1 and SREBF1 expression across 100 cell generations. Finally, we show that the P5CS and GS selection systems can be used together. A GS vector containing the light and heavy chains for a mAb was super-transfected into a CHOK1SV GS-KO™ host over-expressing SCD1 from a P5CS vector. The resulting stable transfectant pools achieved a higher concentration at harvest for a model difficult to express mAb than the CHOK1SV GS-KO™ host. This demonstrates that the P5CS and GS selection systems can be used concomitantly to enable CHO cell line genetic engineering and recombinant protein expression.

Highlights

Recombinant protein drugs are manufactured using a range of expression systems
Both these observations are in agreement with that expected based on the report of Kao and Puck (1967) who showed that in the absence of proline Chinese hamster ovary (CHO) cells had virtually no growth whilst viability was initially maintained for 2–3 days but decreased with a half-life of 1.3 days
After a lack of developments over the last several decades there has been an upsurge of interest with a number of groups reporting the recent development of alternatives to glutamine synthetase (GS) and Dihydrofolate reductase (DHFR) metabolic selection sys tems for use in CHO cells (Kaufman and Sharp, 1982a; Josse et al, 2018; Cockett et al, 1990; Birch et al, 2008)

Summary

Introduction

Recombinant protein drugs are manufactured using a range of expression systems. A kaleidoscope of processes and technologies required for the generation and purification of recombinant material from CHO cells has been explored to improve productivity, ensure consistent product quality and reduce process timelines (Vito et al, 2020; Povey et al, 2014; Pekle et al, 2019; Shukla et al, 2018; Budge et al, 2021). There are problems with antibiotic-based selection systems: antibiotics can alter host cell gene expression that may impact produc tivity yields and/or product quality (Ryu et al, 2017), mask low-level contamination with adventitious agents, the antibiotic itself is a contaminant in the manufacturing process (Mignon et al, 2015; Davies, BY license (http://creativecommons.org/licenses/by/4.0/)

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