Manipulation of Endoplasmic Reticulum Folding and Assembly Machinery for Improved Production of Complex Biotherapeutics

Chinese Hamster Ovary (CHO) cells ability to produce complex glycoproteins which do not illicit an immune response in humans makes them ideal hosts cell factories for the production of recombinant therapeutic proteins. Their responses are well characterised through extensive use in biopharmaceutical platforms, simplifying regulatory approval. Modern therapeutics are shifting towards unnatural multi-specific and fragment-based antibody modalities, presenting new challenges to host CHO cell factories. Cellular production bottlenecks can prevent promising therapeutics from progressing to market, due to diminished product titre or quality. Despite diverse cell engineering approaches to facilitate production, solutions are often highly context specific and unique to individual molecule and CHO hosts evaluated. This thesis is divided into two research branches aiming to facilitate the rapid identification of context specific solutions to aid the production of modern therapeutics. Through the design of an industry applicable transient Simultaneous Overexpression and Silencing Co-transfection (SOSC) gene screening platform, a High Throughput (HTP) workflow is developed. This facilitates rapid identification of product specific genetic engineering targets, providing insights into context specific sensitivity of effector gene manipulation. This platform was applied, evaluating stable Monoclonal Antibody (mAb) producers of opposing expression difficulty, identifying several molecule specific and one host cell engineering target. Despite this, relation of the experimental results back to the host specific transcriptomics dataset was unsuccessful. In parallel, compounds which alleviate pathologic protein aggregates in disease models were evaluated as novel tools to reduce aggregation rate in Difficult to Express (DTE) mAbs. This aimed to demonstrate whether aggregate formation in proteopathic disease is mechanistically similar to that of modern DTE therapeutics. An initial Proof of Concept (POC) study evaluating a small library of proteostasis regulators found most compounds resulted in non-toxic growth arrest. Upon delayed supplementation, this resulted in several product specific titre and Specific Productivity (qP) increases. Next, a diverse library of compounds associated with disaggregation in models of proteopathic disease was identified. Preliminary assessments identified 53 hits, and the subsequent evaluation found 20 % of the total library improved and/or titre of a model DTE mAb.