The relationship between mTOR signalling pathway and recombinant antibody productivity in CHO cell lines

Abstract

BackgroundHigh recombinant protein productivity in mammalian cell lines is often associated with phenotypic changes in protein content, energy metabolism, and cell growth, but the key determinants that regulate productivity are still not clearly understood. The mammalian target of rapamycin (mTOR) signalling pathway has emerged as a central regulator for many cellular processes including cell growth, apoptosis, metabolism, and protein synthesis. This role of this pathway changes in response to diverse environmental cues and allows the upstream proteins that respond directly to extracellular signals (such as nutrient availability, energy status, and physical stresses) to communicate with downstream effectors which, in turn, regulate various essential cellular processes.ResultsIn this study, we have performed a transcriptomic analysis using a pathway-focused polymerase chain reaction (PCR) array to compare the expression of 84 target genes related to the mTOR signalling in two recombinant CHO cell lines with a 17.4-fold difference in specific monoclonal antibody productivity (q p ). Eight differentially expressed genes that exhibited more than a 1.5-fold change were identified. Pik3cd (encoding the Class 1A catalytic subunit of phosphatidylinositol 3-kinase [PI3K]) was the most differentially expressed gene having a 71.3-fold higher level of expression in the high producer cell line than in the low producer. The difference in the gene’s transcription levels was confirmed at the protein level by examining expression of p110δ.ConclusionExpression of p110δ correlated with specific productivity (q p ) across six different CHO cell lines, with a range of expression levels from 3 to 51 pg/cell/day, suggesting that p110δ may be a key factor in regulating productivity in recombinant cell lines.

mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells

“…This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status and the amount of 4E-BP1 present are important in the co-regulation of eIF4E with 4E-BP1. With respect to 4E-BP1 phosphorylation, mammalian cells contain multiple 4E-BP1 isoforms, but the isoform pattern observed in rodent cells is simpler [43,44]. Consistent with the rat model, our work and that of others [45] show that in CHO cells, a total of three 4E-BP1 species are usually detected: a hyperphosphorylated form γ, a middle form β and a hypophosphorylated α form (see Figures 2B and 6A).…”

“…The proteins ULK1, Atg13 and FIP200 complex link mTORC1 signalling to autophagy [43]. Our study indicates that a low-producing cell line (CHO52), where cell viability decreases during batch culture earlier than other cell lines, exhibits markers of autophagy (conversion of LC3-I into LC3-II) at an earlier stage compared with those cell lines where viability does not decrease at similar times (including both higher producing cell lines and a Null-producing cell line).…”

CHO cells knocked out for TSC2 display an improved productivity of antibodies under fed batch conditions

“…Several studies suggested that mTOR inhibition occurs in low producing CHO cells under batch or fed batch growing conditions, and that high producing clones regulate transcription of the PI3K/AKT/mTOR pathway to promote its activity (Edros et al, 2014). It promotes proliferation and the anabolic pathway by enhancing synthesis and inhibiting catabolism, primarily autophagy.…”

Scale-down of CHO cell fed-batch cultures

“…This is in agreement with Lloyd et al 56 , who argued that the cell size is the major cellular determinant of productivity rather than the cell cycle phase. In addition to this, Edros et al 89 and Khoo and Al-Rubeai 90 showed that the increase in specific productivity is directly related to the increase in cell volume due to increase in transcription, translation, and secretion machinery. Kim et al 58 reported that the enhancement of the specific thrombopoietin productivity is linearly correlated with the increase in cell size for nine CHO cell clones generated using the same transfection process.…”

“…Furthermore, Kim et al 58 showed that the enhancement of the qp was linearly correlated with an increase in cell size for nine CHO cell clones generated using the same transfection process. Edros et al 89 and Khoo and Al-Rubeai 90 showed that the increase in qp was directly related to the increase in cell volume due to enhancement of the transcription, translation, and secretion machinery.…”