Structure-function relationships of the soluble form of the antiaging protein Klotho have therapeutic implications for managing kidney disease.

The fortuitously discovered antiaging membrane protein αKlotho (Klotho) is highly expressed in the kidney, and deletion of the Klotho gene in mice causes a phenotype strikingly similar to that of chronic kidney disease (CKD). Klotho functions as a co-receptor for fibroblast growth factor 23 (FGF23) signaling, whereas its shed extracellular domain, soluble Klotho (sKlotho), carrying glycosidase activity, is a humoral factor that regulates renal health. Low sKlotho in CKD is associated with disease progression, and sKlotho supplementation has emerged as a potential therapeutic strategy for managing CKD. Here, we explored the structure-function relationship and post-translational modifications of sKlotho variants to guide the future design of sKlotho-based therapeutics. Chinese hamster ovary (CHO)- and human embryonic kidney (HEK)-derived WT sKlotho proteins had varied activities in FGF23 co-receptor and ß-glucuronidase assays in vitro and distinct properties in vivo Sialidase treatment of heavily sialylated CHO-sKlotho increased its co-receptor activity 3-fold, yet it remained less active than hyposialylated HEK-sKlotho. MS and glycopeptide-mapping analyses revealed that HEK-sKlotho is uniquely modified with an unusual N-glycan structure consisting of N,N'-di-N-acetyllactose diamine at multiple N-linked sites, one of which at Asn-126 was adjacent to a putative GalNAc transfer motif. Site-directed mutagenesis and structural modeling analyses directly implicated N-glycans in Klotho's protein folding and function. Moreover, the introduction of two catalytic glutamate residues conserved across glycosidases into sKlotho enhanced its glucuronidase activity but decreased its FGF23 co-receptor activity, suggesting that these two functions might be structurally divergent. These findings open up opportunities for rational engineering of pharmacologically enhanced sKlotho therapeutics for managing kidney disease.

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.