Over the last two decades, glycoengineering has proven itself as an effective method for the production of biopharmaceuticals with improved efficacy, circulatory half-life, and safety profiles. Chinese hamster ovary (CHO) cells are the workhorse for biopharmaceutical production in a multibillion USD industry, and a deep understanding of CHO cell glycosylation is necessary for the design of effective glycoengineering strategies and the development of increasingly accurate predictive in silico models. In this thesis, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry was used to analyse the N-glycomic profiles of CHO-K1 and CHO-S cells as well as CHO-K1 and CHO-S cells engineered to recombinantly produce IgG1 monoclonal antibodies. To my knowledge, this was the first analysis to show how both genetic divergence in these industrially important cell lines, and antibody production, result in significant differences in N-glycomic profiles, particularly in the level of antennal branching and terminal elaboration in complex N-glycans. This study also served as the basis for a novel in vivo glycoengineering strategy designed to increase galactosylation in antibody-producing CHO-S cells and improve downstream antibody effector functions. The working hypothesis behind the strategy was that overexpressing glucose-6-phosphatase or glucose-6-phosphate translocase could increase antibody galactosylation. The outcome of this novel approach is as of yet inconclusive and optimisation efforts could not be completed due to the time constraints imposed by the COVID-19 pandemic. However, the comparison of CHO-K1 and CHO-S cells was extended to the mass spectrometric N-glycomic analysis of protein backbone glycoengineered IgG1-Fc constructs with novel N- glycosylation sites characterised by hypersialylated N-glycans that provide new functionalities, such as sialic acid receptor binding and inhibition of influenza hemagglutination. Finally, mass spectrometric N-glycomic analyses were performed in a proof-of-concept study that demonstrated how a novel artificial Golgi reactor with immobilised enzymes generated biopharmaceuticals with highly homogeneous and bespoke N-glycan profiles in vitro.