Small-molecule control of antibody N-glycosylation in engineered mammalian cells.

N-linked glycosylation in monoclonal antibodies (mAbs) is crucial for structural and functional properties of mAb therapeutics, including stability, pharmacokinetics, safety and clinical efficacy. The biopharmaceutical industry currently lacks tools to precisely control N-glycosylation levels during mAb production. In this study, we engineered Chinese hamster ovary cells with synthetic genetic circuits to tune N-glycosylation of a stably expressed IgG. We knocked out two key glycosyltransferase genes, α-1,6-fucosyltransferase (FUT8) and ß-1,4-galactosyltransferase (ß4GALT1), genomically integrated circuits expressing synthetic glycosyltransferase genes under constitutive or inducible promoters and generated antibodies with concurrently desired fucosylation (0-97%) and galactosylation (0-87%) levels. Simultaneous and independent control of FUT8 and ß4GALT1 expression was achieved using orthogonal small molecule inducers. Effector function studies confirmed that glycosylation profile changes affected antibody binding to a cell surface receptor. Precise and rational modification of N-glycosylation will allow new recombinant protein therapeutics with tailored in vitro and in vivo effects for various biotechnological and biomedical applications.

Dissecting N-Glycosylation Dynamics in Chinese Hamster Ovary Cells Fed-batch Cultures using Time Course Omics Analyses.

N-linked glycosylation affects the potency, safety, immunogenicity, and pharmacokinetic clearance of several therapeutic proteins including monoclonal antibodies. A robust control strategy is needed to dial in appropriate glycosylation profile during the course of cell culture processes accurately. However, N-glycosylation dynamics remains insufficiently understood owing to the lack of integrative analyses of factors that influence the dynamics, including sugar nucleotide donors, glycosyltransferases, and glycosidases. Here, an integrative approach involving multi-dimensional omics analyses was employed to dissect the temporal dynamics of glycoforms produced during fed-batch cultures of CHO cells. Several pathways including glycolysis, tricarboxylic citric acid cycle, and nucleotide biosynthesis exhibited temporal dynamics over the cell culture period. The steps involving galactose and sialic acid addition were determined as temporal bottlenecks. Our results show that galactose, and not manganese, is able to mitigate the temporal bottleneck, despite both being known effectors of galactosylation. Furthermore, sialylation is limited by the galactosylated precursors and autoregulation of cytidine monophosphate-sialic acid biosynthesis.

Reactivity-driven cleanup of 2-Aminobenzamide derivatized oligosaccharides.

N-glycan profiling is commonly accomplished by the derivatization of the enzymatically released oligosaccharides with a fluorophore, thereby facilitating their analysis by hydrophilic-interaction liquid chromatography (HILIC). These fluorescent dyes are often present in large excess during derivatization reactions, and their removal is typically required to minimize chromatographic interference. Herein, we report a reactivity-driven 2-phase extraction protocol with the aldehyde reagent octanal, which demonstrated efficient 2-aminobenzamide cleanup as well as high derivatized N-glycan recovery. This cleanup method can be performed within minutes, and therefore provides an alternative sample preparation route for N-glycan profiling with improved time efficiency and operational simplicity.

Challenges in the Conversion of Manual Processes to Machine-Assisted Syntheses: Activation of Thioglycoside Donors with Aryl(trifluoroethyl)iodonium Triflimide.

The steps needed to adapt a stable iodonium promoter for use in automated fluorous-assisted solution-phase oligosaccharide synthesis are described. Direct adaptation of the originally reported batch procedure resulted in the formation of an orthoester or protecting group transfer to the glycosyl acceptor. Fortunately, the addition of inexpensive ß-pinene as an acid scavenger avoided both of these side reactions. The utility of this newly developed protocol was applied to the automated solution-phase synthesis of a ß-glucan fragment.

Aryl(trifluoroethyl)iodonium Triflimide and Nitrile Solvent Systems: A Combination for the Stereoselective Synthesis of Armed 1,2-trans-ß-Glycosides at Noncryogenic Temperatures.

Armed thioglycosides can be activated with aryl(trifluoroethyl)iodonium triflimide in 2:1 CH2Cl2/pivalonitrile or a solvent combination of CH2Cl2, acetonitrile, isobutyronitrile, and pivalonitrile (6:1:1:1) at 0 °C for glycosylation reactions that proceed in good yield and moderate to excellent selectivity (up to 25:1 ß/α). Comparison to other common glycosylation promoters reveals that both the mixed solvent and the iodonium salt promoter are required for stereoselectivity.

An air- and water-stable iodonium salt promoter for facile thioglycoside activation.

The air- and water-stable iodonium salt phenyl(trifluoroethyl)iodonium triflimide is shown to activate thioglycosides for glycosylation at room temperature. Both armed and disarmed thioglycosides rapidly undergo glycosylation in 68-97% yield. The reaction conditions are mild and do not require strict exclusion of air and moisture. The operational simplicity of the method should allow experimentalists with a limited synthetic background to construct glycosidic linkages.

Selective synthesis of 1,2-cis-α-glycosides without directing groups. Application to iterative oligosaccharide synthesis.

A method for the highly selective synthesis of 1,2-cis-α-linked glycosides that does not require the use of the specialized protecting group patterns normally employed to control diastereoselectivity is described. Thioglycoside acceptors can be used, permitting iterative oligosaccharide synthesis. The approach eliminates the need for lengthy syntheses of monosaccharides possessing highly specialized and unconventional protecting group patterns.