Improving monoclonal antibody quality and efficacy: Studies on a camelid heavy-chain monoclonal antibody

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Mishra, Neha
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Glycosylation is a critical quality attribute of biotherapeutics as it contributes to their quality and efficacy. The work described in this thesis aims to investigate these aspects of an EG2-hFc chimeric (camelid heavy chain Fab-human Fc) single domain monoclonal antibody by increasing the antibody-dependent cell cytotoxicity response. Two methods of generating non-fucosylated antibodies are compared. The first method involves the fucosyltransferase inhibitor, 2 fluoro peracetylated fucose (2FF) with Chinese Hamster Ovary (CHO) cells producing EG2-hFc and the second uses the prokaryotic RMD gene, which was transfected into CHO EG2-hFc cells. Both methods successfully generated reduced fucosylation, but neither produced completely non-fucosylated antibodies. The fucosylation levels of the RMD transfected CHO EG2-hFc cells could be reversed with increasing concentrations of fucose added to the cells. To analyse the fucosylation pattern of EG2-hFc samples, electrospray ionization mass spectrometry (ESI-MS) was applied. It was observed that the addition of fucose was inhibited by 2FF in a concentration-dependent manner. An attempt was made to develop a non-radioactive ADCC assay using engineered NK-92 effector cells. The optimization assays performed as expected, with the cytotoxicity% increasing with the increasing concentrations of antibody, but experimental assays using EG2 samples did not result in the same cytotoxicity%. Therefore, this LDH-based ADCC assay requires further optimization before it can be a standard for measuring the ADCC of an antibody. To investigate the effect of bioprocess parameters such as redox potential on glycosylation of CHO EG2-hFc, a study was designed to detect the internal redox potential, which may be one factor influencing the intracellular machinery in the ER and Golgi where glycan processing occurs. Redox sensitive green fluorescent proteins were targeted to the organelles of CHO EG2-hFc cells. Flow cytometry data provided a proof-of-concept that the roGFP probes can be utilized to determine the redox status. Together, these results advance the understanding of fucosylation for ADCC and provided the basis for the development of tools to analyse ADCC and redox state of cells producing a potential therapeutic.
Glycosylation, Monoclonal Antibody, Fucosylation, ADCC, Product Quality
Comparison of two glycoengineering strategies to control the fucosylation of a monoclonal antibody N Mishra, M Spearman, L Donald, H Perreault M Butler, Journal of Biotechnology: X, Volume 5, 2020, 100015
Mass spectrometric analysis of core fucosylation and sequence variation in a human– camelid monoclonal antibody, L. J. Donald, M. Spearman, N. Mishra, E. Komatsu, M. Butler and H. Perreault, Mol. Omics, 2020.