Stable Constitutive BiP Expression Prolongs Late-Stage GLP-1–Fc Accumulation in CHO Cells
TL;DR
Stable constitutive expression of host-derived BiP was associated with prolonged late-stage GLP-1–Fc accumulation in the producer population examined, and these kinetics are compatible with a model in which the timing of ER chaperone supply influences the maintenance of productive output.
Abstract
Chinese hamster ovary (CHO) cells are the principal platform for manufacturing recombinant biopharmaceuticals, but sustained production can exceed the protein-folding capacity of the endoplasmic reticulum (ER). Binding immunoglobulin protein (BiP/GRP78), encoded by Hspa5, is a major ER chaperone that supports folding and modulates unfolded protein response (UPR) signaling, yet increasing BiP abundance has produced beneficial or inhibitory outcomes depending on the recombinant product. Here, we introduced an additional Chinese hamster Hspa5 coding sequence under the constitutive EEF1A1 promoter into a CHO line producing a dulaglutide analogue comprising a modified GLP-1 peptide fused to human IgG4 Fc and compared the resulting stable DUL-BiP population with an empty-vector DUL-Neo control. At constant 37 °C, the two populations showed similar viable cell density and viability trajectories, whereas DUL-BiP cells continued extracellular GLP-1–Fc accumulation for approximately two days after accumulation in DUL-Neo cells had slowed and reached a 40% higher final titer in this experiment. Two independent temperature-shift experiments showed the same direction of the final-titer difference, although the increases were smaller and were accompanied by reduced peak viable cell density. Semi-quantitative immunoblot analysis of samples from the constant-temperature experiment showed a comparatively stable total BiP signal in DUL-BiP cells and a pronounced late increase in DUL-Neo cells, together with temporal changes in cleaved ATF6, eIF2α phosphorylation, and CHOP in both populations. Intracellular GLP-1–Fc did not progressively accumulate. Because the immunoblot time courses were derived from one biological fed-batch experiment, they are interpreted descriptively rather than as independent quantitative biological replicates. Stable constitutive expression of host-derived BiP was therefore associated with prolonged late-stage GLP-1–Fc accumulation in the producer population examined. These kinetics are compatible with a model in which the timing of ER chaperone supply influences the maintenance of productive output, while the underlying mechanism and generality to other producer populations or recombinant products remain to be established.
