Date of Award

12-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemical and Biomolecular Engineering

Committee Chair/Advisor

Dr. Mark A. Blenner

Committee Member

Dr. Jessica M. Larsen

Committee Member

Dr. Marc R. Birtwistle

Committee Member

Dr. Sarah W. Harcum

Abstract

Industrial Chinese hamster ovary (CHO) production of therapeutics requires cell lines with high secretory capacity to avoid an accumulation of improperly folded proteins, or endoplasmic reticulum (ER) stress. This presents a challenging engineering bottleneck. The unfolded protein response (UPR) is initiated to overcome ER stress and reestablish homeostasis. In this dissertation, the impacts of ER stress and the UPR on protein production in mammalian cells are detailed, and both selection- and rational-based strategies for enhancing the ER stress response in productive CHO cell lines are reviewed. This project aims to expand on recent research efforts for engineering ER stress-related responses in CHO cell lines to augment higher productivity in biomanufacturing. This work demonstrates recombinant protein overexpression and higher specific productivity results in unavoidable ER stress in fed-batch culture of two distinct CHO cell lines, one producing immunoglobulin G (IgG1) and one producing erythropoietin (EPO-Fc). High throughput RNA sequencing (RNASeq) and quantitative polymerase chain reaction (qPCR) were used to conduct differential gene expression analyses and study the effect of protein production over time versus a non-producing host cell line during fed-batch culture. These analyses showed enrichment and transient upregulation of mRNAs involved in ER stress, the UPR, and oxidative protein folding and processing. Analysis of temporal differential expression profiles indicated dependency on ATF6 pathway activation. The RNASeq analysis led to the identification of two engineering targets, ATF6β and WFS1, known regulators of the UPR. The IgG1-producing CHO cell line was used to analyze the effect of stable knockdown of either ATF6β or WFS1 on CHO cell growth and IgG1 production. This dissertation presents the first study to analyze the effect of WFS1 knockdown in producing CHO cells. Knockdown of ATF6β was found to improve specific productivity; however knockdown of WFS1 resulted in no improvement. Knockdown of ATF6β improved the UPR specifically during ER stress, but knockdown of WFS1 had negative impacts on UPR activation and product mRNA expressions. This dissertation closes with conclusions and future work in engineering the CHO cell ER stress response. It is anticipated this work will lead to advancements in CHO cell engineering for enhanced protein production of therapeutics.

Available for download on Sunday, December 31, 2023

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