Date of Award

2023

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Roberta King

Abstract

Monoclonal antibodies (mAbs) make up a large part of biopharmaceutical sales due to their great success in treating cancer, autoimmune, and cardiovascular diseases. Due to an increasing number of mAbs using innovative approaches in drug development, combined with changes in the drug market, there is an increased need for optimizing process intensification and quality control. Thus, a profound understanding of cellular metabolism and how this impacts the protein product’s critical quality attributes (CQA) would be of great value. Among the quality attributes of mAbs, post-translational modifications such as N-linked glycosylation are of particular importance as they affect the activity, efficacy, and immunogenicity of mAbs. This thesis aims to drive understanding of the N-linked glycosylation process under mild hypothermia conditions in CHO cells by applying 13C isotopic tracing and flux balance analysis (FBA), which has become tremendously successful in metabolic engineering. Mammalian cells, especially Chinese hamster ovary (CHO) cells, have become the primary host in the biopharmaceutical production of mAbs. The first chapter of this thesis is a review paper that offers a comprehensive summary of the steps of upstream processes, future directions, and adaption challenges of emerging new technologies in the pharmaceutical industry. Next, a prospective paper describes a constraint-based systems biology model FBA that could be used to estimate metabolic reaction rates by using the rates as inputs to multivariate batch evolution models (BEM). As a result, we show that metabolic activity is reproducible across batches and can be monitored to detect an induced macroscopic process deviation such as a temperature shift. This new paradigm has the potential to offer increased process flexibility without compromising safety and efficacy, such as for scale-up changes. These findings suggest monitoring metabolic activity is a promising avenue for biomanufacturing process monitoring and control. Lastly, we utilize 1,2-13C2 glucose and 1,6-13C2 glucose tracers as feeds in the Ambr15 bioreactor system to cultivate CHO cells to experimentally investigate the effect of mild hypothermia (33°C) on mAb N-linked glycosylation, using flux balance analysis to identify differences in cell metabolism. Many publications have applied tracers to elucidate the metabolic state during cell exponential, stationary, and late lag phases of the culture, but no studies have been conducted to understand the mild hypothermia culture impact by utilizing a 13C tracer. We conclude that as the titer decreases, the simpler glycoforms G1, G1F, G2, and G2F decrease, while the complex sialylated glycoforms G1FS1, G2FS1, and G2FBS1 increase. The structures of complex N-glycans were modified under lower temperatures, affecting levels of branching and terminal residues like sialic acid and galactose. The increase in complex glycoforms could be beneficial in fucosylation, which is a crucial determinant of antibody-dependent cell-mediated cytotoxicity (ADCC), and the reduced core fucosylation generally exhibits increased binding affinity to FcγRIIIa receptors on immune cells. For maintaining product quality and dependable therapeutic performance, consistent glycosylation profiles are critical. When process changes such as altered temperature or pH result in increased complex glycoforms, this changes the attributes of the product protein. As such, it is crucial to ensure that this change will not alter the therapeutic properties of the drug, such as efficacy or half-life. The outcome of this study is a metabolic understanding of previously uncharacterized variation in a CHO bioprocess resulting in inconsistent glycoforms. This biological understanding can used to identify key biomarkers for improved process monitoring and to develop strategies to reduce glycoform variation by controlling and focusing on the metabolic state of the cells over the bioreactor process parameters.

Download

Available for download on Sunday, January 19, 2025

Share

COinS