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.

Transient CHO expression platform for robust antibody production and its enhanced N-glycan sialylation on therapeutic glycoproteins.

Large-scale transient expression in mammalian cells is a rapid protein production technology often used to shorten overall timelines for biotherapeutics drug discovery. In this study we demonstrate transient expression in a Chinese hamster ovary (CHO) host (ExpiCHO-S™) cell line capable of achieving high recombinant antibody expression titers, comparable to levels obtained using human embryonic kidney (HEK) 293 cells. For some antibodies, ExpiCHO-S™ cells generated protein materials with better titers and improved protein quality characteristics (i.e., less aggregation) than those from HEK293. Green fluorescent protein imaging data indicated that ExpiCHO-S™ displayed a delayed but prolonged transient protein expression process compared to HEK293. When therapeutic glycoproteins containing non-Fc N-linked glycans were expressed in transient ExpiCHO-S™, the glycan pattern was unexpectedly found to have few sialylated N-glycans, in contrast to glycans produced within a stable CHO expression system. To improve N-glycan sialylation in transient ExpiCHO-S™, we co-transfected galactosyltransferase and sialyltransferase genes along with the target genes, as well as supplemented the culture medium with glycan precursors. The authors have demonstrated that co-transfection of glycosyltransferases combined with medium addition of galactose and uridine led to increased sialylation content of N-glycans during transient ExpiCHO-S™ expression. These results have provided a scientific basis for developing a future transient CHO system with N-glycan compositions that are similar to those profiles obtained from stable CHO protein production systems. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2724, 2019.