High Intracellular Seed Train BiP Levels Correlate With Poor Production Culture Performance in CHO Cells

Abstract

Consistent cell culture performance is a prerequisite to ensure product quality consistency and achieve productivity goals for the manufacture of recombinant protein therapeutics, including monoclonal antibodies. Here a peculiar observation is reported where high levels of intracellular BiP in seed train cultures are consistently predictive of poor cell culture performance in the subsequent inoculum and production cultures for a monoclonal antibody produced in CHO cells. This investigation suggests that in this cell line the high intracellular BiP levels in the seed train are triggered by a slightly lower culture pH, which interferes with proper antibody folding and secretion. While the seed train culture does not display any obvious signs of the problem at slightly lower culture pH, inoculum trains, and production cultures sourced from these low pH seed trains display significantly lower cell growth and cell size. High intracellular BiP levels may interfere with UPR signaling, thereby hampering a proper and timely UPR response in the production media. Studies of other problematic cell lines have shown a similar correlation between intracellular BiP accumulation and poor production performance. The authors believe intracellular BiP levels in seed train should hence be low in order to increase the success rate in production.

Activation of the PERK branch of the unfolded protein response during production reduces specific productivity in CHO cells via downregulation of PDGFRa and IRE1a signaling

“…Our investigation into one of our antibody‐expressing cell lines, mAb1 CHO DG44, revealed that transcriptional downregulation, and hence lower expression of PDGFRa protein, was the likely cause of poor growth outcomes during production when the cells were sourced from seed train cultures exposed to low pH (Figure 1a,b). Previously we have shown that this poor growth outcome correlated with increased intracellular BiP levels, which is indicative of UPR activation 16 . When the UPR was chemically induced, PDGFRa protein levels also decreased due to transcriptional downregulation, a phenomenon that could be reversed by chemical inhibition of the PERK branch of the UPR pathway, suggesting that PERK activation mediates PDGFRa downregulation (Figures 1c–f and S1a–c).…”

“…Previously we have shown that this poor growth outcome correlated with increased intracellular BiP levels, which is indicative of UPR activation. 16 When the UPR was chemically induced, PDGFRa protein levels also decreased due to transcriptional downregulation, a phenomenon that could be reversed by chemical inhibition of the PERK branch of the UPR pathway, suggesting that PERK activation mediates PDGFRa downregulation (Figures 1c-f and S1a-c). This was further confirmed when chemical induction of UPR in a PERK KO cell line did not result in downregulation of PDGFRa expression (Figure 1g).…”

“…16 When exposed to low pH conditions, high intracellular BiP levels were detected in this cell line that correlated to low growth profiles during production and poor bioprocess outcomes. 16 To better understand the underlying mechanism of reduced production culture growth in low versus high pH conditions, seed train cultures were maintained under high and low pH conditions and subjected to proteomic analysis by mass spectrometry. Significant reductions in expression of PDGFRa protein levels were observed under low pH conditions (Figure 1a), which was linked to transcriptional attenuation of the PDGFRa gene (Figure 1b).…”

“…Previously, we have shown that UPR activation in the seed train culture of a particular CHO cell line, triggered by reduction in the pH media, was indicative of poor bioprocess outcomes during production. 16 In this manuscript, we report further mechanistic studies that linked low pH exposure and UPR to downregulated expression of platelet-derived growth factor alpha (PDGFRa) in these cells. Our findings revealed that PDGFRa signaling is important for CHO cell growth and productivity.…”

Enhancing CHO by Systems Biotechnology

“…The data also reveals cells to be oxidatively stressed at the high density, thus giving indications for possible optimization strategies for such processes. Tung et al then present data on slight variations in pH during the seed train of an industrial scale process, which results in increased changes in the BiP concentration, thus revealing problems associated with proper protein folding und ER related stress under these conditions. While other standard process parameters such as growth and productivity were not obviously affected, such stress‐situations can still impact on the final process performance.…”