Engineering Glycoprotein Biotherapeutics

Glycosylation can modulate the efficacy, safety and half-life in serum of biotherapeutics. The industry is progressively implementing the "Quality by Design" paradigm, which requires critical quality attributes in the end-product such as glycoform distribution to be rationally integrated into the design of manufacturing processes. For instance, antibody-dependent cell-mediated cytotoxicity can be enhanced by the presence of terminal galactose residues on monoclonal antibody glycans, which highlights the significance of developing manufacturing design in order to exert control over a biotherapeutic's glycomic profile.
This PhD project involves a collaboration between the Biopolymer Mass Spectrometry laboratory lead by Dr. Stuart Haslam and Dr Cleo Kontoravdi's laboratory (Chemical Engineering, ICL), in which various approaches aimed at improving biotherapeutic manufacturing will be investigated. The Kontoravdi laboratory has an in silico model which assist in the design of feeding strategies of CHO cell cultures and predict the resulting antibody yields and the glycomic profiles of both antibodies and host-cell proteins. The effectiveness of such feeding strategies in terms of IgG and CHO host cell glycosylation will be investigated. MALDI mass spectrometry (MS) is the method of choice for this project for investigating the glycomic profiles of host-cell proteins and recombinant biotherapeutics. Genetic engineering in CHO cells will also be investigated, aiming to produce a stable mutant with improved antibody galactosylation, in which genes producing polylactosamine extensions are silenced or knocked out. This should reduce the proportion of galactose directed towards glycosylating host-cell proteins and redirect galactose towards biotherapeutic galactosylation. MS will be used to determine whether genetic experiments were successful.
An additional approach aims to produce a glycoengineered KU70 Pichia pastoris cell line, where a hypermannosylation gene (OCH1) is knocked out and a type I mannosidase is recombinantly expressed. In this way the cells should produce a Man5 glycoform in abundance. The Man5 glycan will then be further processed in an artificial Golgi reactor (AGR), where incubation in subsequent chambers with immobilised N-acetylglucosaminyltransferases and mannosidases can produce the desired biotherapeutic glycoforms. The Kontoravdi laboratory is working on the design and initial prototypes of an AGR, which have been shown to modify free oligosaccharides, and now needs to be tested with glycoproteins.