Dynamics of unfolded protein response in recombinant CHO cells

Abstract

Genes in the protein secretion pathway have been targeted to increase productivity of monoclonal antibodies in Chinese hamster ovary cells. The results have been highly variable depending on the cell type and the relative amount of recombinant and target proteins. This paper presents a comprehensive study encompassing major components of the protein processing pathway in the endoplasmic reticulum (ER) to elucidate its role in recombinant cells. mRNA profiles of all major ER chaperones and unfolded protein response (UPR) pathway genes are measured at a series of time points in a high-producing cell line under the dynamic environment of a batch culture. An initial increase in IgG heavy chain mRNA levels correlates with an increase in productivity. We observe a parallel increase in the expression levels of majority of chaperones. The chaperone levels continue to increase until the end of the batch culture. In contrast, calreticulin and ERO1-L alpha, two of the lowest expressed genes exhibit transient time profiles, with peak induction on day 3. In response to increased ER stress, both the GCN2/PKR-like ER kinase and inositol-requiring enzyme-1alpha (Ire1α) signalling branch of the UPR are upregulated. Interestingly, spliced X-Box binding protein 1 (XBP1s) transcription factor from Ire1α pathway is detected from the beginning of the batch culture. Comparison with the expression levels in a low producer, show much lower induction at the end of the exponential growth phase. Thus, the unfolded protein response strongly correlates with the magnitude and timing of stress in the course of the batch culture.

Fig. 1

Engineering view of protein secretion pathway in endoplasmic reticulum. Under stress, accumulation of unfolded proteins inside ER leads to activation of the unfolded protein response (UPR) pathway. ER homeostasis is restored back by a co-ordinated activation of three different pathways; endoplasmic reticulum associated folding (ERAF), endoplasmic reticulum associated degradation (ERAD) and UPR. C denotes chaperones and D denotes degradation enzymes

Fig. 2

Growth characteristics of the two CHO cell lines. a Viable cell densities (×10⁶cells/ml), b Viability (%), c Total cell densities (×10⁶ cells/ml) and d Specific death rate (day⁻¹) of the high and low producer in batch culture. Solid lines denote high producers (HP) while dash dotted lines denote low producers (LP)

Fig. 3

Comparison of a Antibody titers (μg/ml) and b Specific productivities—q_P (pg/cell/day) between HP (solid lines) and LP (dash dotted lines) in a batch culture

Fig. 4

Detailed view of protein secretion pathway. On translocation of the unfolded protein into the ER, multiple chaperones help in folding, glycosylation and formation of disulfide bridges. In the event of an ER overload, accumulation of unfolded proteins leads to ER stress. Three membrane transducers, PERK, Ire1α and ATF6, are bound to GRP78 under normal conditions. ER stress leads to dissociation of GRP78 and subsequent activation of PERK and Ire1α by dimerization and phosphorylation. Activation of ATF6 is by translocation to golgi complex after dissociation from GRP78 where it is cleaved by serine proteases to an active transcription factor ATF6p50. Downstream targets of each of these pathways are shown. AARE amino acid response element; ARE anti-oxidant response element; ERSE endoplasmic reticulum stress element; ATF/CRE activating transcription factor/cAMP responsive element. Abbreviations for the genes are explained in the text

Fig. 5

mRNA profile of a IgG heavy chain (HC) and b light chain (LC) in HP (solid lines) and LP (dash dotted lines).*Denotes p value < 0.05 with respect to day 1 expression levels in high producer, whereas **refers to low producer data. The ratio of IgG HC mRNA to LC mRNA is shown in c

Fig. 6

Transcriptional profile of chaperones a GRP78 b GRP94 c CNX d CRT e UGGT1-V1 f ERDJ4 and g ERO1α in the high and low producer cell lines during the course of batch culture. Symbols used are same in Fig. 5

Fig. 7

Transcriptional profile of key PERK/GCN2 pathway genes, a ATF4, b CHOP, c GADD34 and d NRF2. Refer to Fig. 5 for symbols

Fig. 8

Transcriptional profile of a XBP1s. b XBP1u and c XBP1_T in the two cell lines. d Shows the ratio of XBP1s to XBP1_T during the course of the batch culture. Refer to Fig. 5 for symbols