(634d) Therapeutic Protein Production Is Enhanced By Debottlenecking Secretion in Cells Stressed By Overproduction

Therapeutic proteins from insulin to COVID-19 antibodies have reshaped modern health care. The ability to replace deficient proteins or to bind problematic proteins allows for the treatment of countless diseases. Despite recent successes in clinical use, therapeutic proteins are still limited by costs associated with manufacturing, purification, storage, and administration. Many therapeutic proteins must be made in mammalian cells to ensure biocompatible post-translational modifications, further increasing costs. Protein overexpression can induce endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR). The UPR decreases global protein production, increases chaperone production to refold misfolded proteins, increases protein degradation pathways, and, if necessary, initiates apoptosis. Activation of the UPR is unavoidable in therapeutic protein production, but a comprehensive understanding of the effects of the UPR on cell function would allow for mitigation of the negative effects of the UPR on protein production.

Minimizing ER stress can be achieved through proper management/selection of media compositions, agitation techniques, non-product gene expression, host cell line, and product gene sequence. An area that is understudied is the impact of UPR on the secretion of protein products. The UPR activates proteasomal and lysosomal degradation. In proteasomal degradation, ubiquitinated proteins must cross the ER lumen to enter the cytoplasm where interaction with the proteasome degrades the misfolded proteins. In lysosomal degradation parts of the ER form vesicles that rapidly mature into and merge with acidic lysosomes, resulting in increased rigidity of the lysosomal membranes. Rigid membranes protect the cell and neighboring cells from harm from the misfolded proteins by providing a barrier to cross membrane transport and secretion. This leads to a build up of material in the lysosome rather than the cytoplasm for proteasomal degradation or culture media, the destination for recombinant protein recovery, for removal.

In this work, we demonstrate that ER stress decreases therapeutic protein production through lysosomal sequestration of the protein product. In vitro transcription was used to produce mRNA for secreted and intracellular EGFP and luciferase proteins. mRNA was then transfected into HeLa cells using Lipofectamine 2000 and 4 hours after transfection, media containing low but increasing levels of tunicamycin, a common ER stress inducer, was added to the cells. Media and cell lysate were collected at time intervals up to 48 hours post ER stress induction. The EGFP-mRNA transfected cells were stained with LysoTracker Red and Fillipin III to track the lysosomes and cholesterol, respectively. HeLa cells under ER stress retain a higher fraction of functional EGFP and luciferase in cholesterol dense lysosomes than cells not experiencing ER stress. Importantly, the effects of ER stress on productivity were mitigated by increasing the level of negatively charged phospholipids in lysosomal membranes. These results provide evidence that therapeutic protein production can be improved by alleviating UPR suppression of secretion.