GlycoFlow Glycoengineering of therapeutic antibodies by flow biocatalysis.

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The advent of biopharmaceuticals is revolutionizing health care, opening up entirely new avenues against diseases that are difficult to treat using traditional drugs. Marketed therapeutic antibodies are proteins that recognize and bind specific molecules on diseased cells, thereby eliciting a beneficial immunological response. A lesser-known fact is that antibodies are usually not single homogenous proteins. They also require glycosylation, which means the antibody is linked to a complex structure of various sugars (i.e. glycans). This glycosylation is essential for generating efficacious therapeutic antibodies. Therefore, these biopharmaceuticals are manufactured using elaborate and costly protein expression systems that carry the biological machinery needed to install these sugars. Because living cells are used, the resulting product comprises a heterogenous population with multiple glycan structures that can show significant differences, with up to a 50-fold decrease in efficacy when the glycans are fucosylated (decorated with a fucose sugar). Such a heterogeneity imposes a significant burden on the development and use of therapeutic antibodies, insofar as it impacts efficacy and safety profiles, manufacturing costs, batch variability as well as regulatory requirements.Consequently, better approaches are needed to control the structures of glycans linked to antibodies, as well as the consistency of the glycan population. Current strategies exploit genetically modified expression cell lines (e.g. to reduce the level of fucosylation) that still produce highly variable glycosylation profiles. Alternatively, in vitro batch chemo-enzymatic modifications have proven successful at generating homogenously glycosylated antibodies, but they suffer from complex purification processes and scalability issues.We propose a GlycoFlow platform in which therapeutic antibodies could be glycoengineered using flow biocatalysis. By passing a solution of antibodies through modular cartridges of glycan-processing enzymes, we aim to manipulate the structures of sugars directly in continuous flow, generating optimized and homogenous glycosylated antibodies. Continuous flow technologies are game-changers for the modular synthesis of pharmaceutical ingredients with high efficiency and consistency, but have yet to be leveraged for glycoengineering of biopharmaceuticals.To achieve this goal, we have assembled a diverse team of young and experienced experts in glycosciences, glycan-processing enzymes, antibody biochemistry, and protein engineering. Our team will take advantage of the historical expertise in flow chemistry at UdeM, coupled with world-leading researchers from Concordia and McMaster to deliver innovative solutions for the next-generation of biopharmaceuticals. Specifically, we will develop flow technologies that yield homogenously glycosylated antibodies as well as simpler solutions towards afucosylated and sialylated glycans, both of which have showed increased efficacy and improved anti-inflammatory properties.