A new Chinese hamster ovary cell line expressing α2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins

Bragonzi, Alessandra; Distefano, Gianfranco; Buckberry, Lorraine D.; Acerbis, Giulia; Foglieni, Chiara; Lamotte, Damien; Campi, Gabriele; Marc, Annie; Soria, Marco R.; Jenkins, Nigel; Monaco, Lucía

doi:10.1016/s0304-4165(00)00023-4

Cited by 94 publications

(63 citation statements)

References 32 publications

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“…However, the proportion of terminal galac- (m/z 2245). Moreover, 6-linked galactose is absent, which is in accordance with the absence of (␣2-6) sialyltransferase in the CHO cell line (28).…”

Section: Maldi-ms Analysis Of N-glycans Released By Pngase-f-supporting

confidence: 79%

Glycosylation Pattern of Mature Dimeric Leukocyte and Recombinant Monomeric Myeloperoxidase

Antwerpen

Slomianny

Boudjeltia

et al. 2010

Journal of Biological Chemistry

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The involvement of myeloperoxidase (MPO) in various inflammatory conditions has been the scope of many recent studies. Besides its well studied catalytic activity, the role of its overall structure and glycosylation pattern in biological function is barely known. Here, the N-glycan composition of native dimeric human MPO purified from neutrophils and of monomeric MPO recombinantly expressed in Chinese hamster ovary cells has been investigated. Analyses showed the presence of five N-glycans at positions 323, 355, 391, 483, 729 in both proteins. Site by site analysis demonstrated a well conserved micro-and macro-heterogeneity and more complex-type N-glycans for the recombinant form. Comparison of biological functionality of glycosylated and deglycosylated recombinant MPO suggests that glycosylation is required for optimal enzymatic activity. Data are discussed with regard to biosynthesis and the threedimensional structure of MPO.Myeloperoxidase (MPO, 5 EC 1.11.1.7) is a heme-containing glycosylated oxidoreductase that catalyzes the production of hypochlorous acid (HOCl) in the presence of hydrogen peroxide (H 2 O 2 ) and chloride anions (1). The macromolecule is a homodimer with each monomer consisting of a light and a heavy chain. The dimer is stabilized by a disulfide bridge and by several interactions between the carbohydrate moieties located at the dimer interface (2). MPO is a lysosomal enzyme present in the azurophilic granules of human neutrophils and monocytes and is synthesized in the promyelocytic state during maturation of the polymorphonuclear (PMN) leukocytes (3).During phagocytosis of exogenous structures, neutrophils release their granule content (e.g. MPO) into the phagosome. Respiratory burst is initiated by activation of NADPH-oxidase that reduces molecular oxygen and releases superoxide radical anions. Upon superoxide dismutation, hydrogen peroxide is produced that mediates MPO-driven oxidation of chloride to antimicrobial HOCl.More recent data have indicated that these reactions are not restricted to the phago-lysosome. Indeed, under oxidative stress (i.e. excessive and uncontrolled production of various reactive oxygen-derived species, ROS), MPO can escape from neutrophils and circulate in extracellular fluids. Thereby it might cause oxidative modification to host tissues and biomolecules. The potential deleterious effect of this "circulating" MPO has been demonstrated in various pathological conditions involving chronic inflammation, such as atherosclerosis, demyelinating disorders of the central nervous system or endstage renal disease (4 -9).In many of these experiments, recombinant human MPO (r-MPO) expressed in a Chinese hamster ovary (CHO) cell line has been used (10) because recombinant production delivers higher amounts of protein as compared with the classical purification from neutrophils (11). However, although having the same catalytic activity as the dimeric leukocyte enzyme (h-MPO) (12-14), biosynthesis of r-MPO in CHO cell lines lacks some proteolytic steps. As a consequence, r-...

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Section: Maldi-ms Analysis Of N-glycans Released By Pngase-f-supporting

confidence: 79%

Glycosylation Pattern of Mature Dimeric Leukocyte and Recombinant Monomeric Myeloperoxidase

Antwerpen

Slomianny

Boudjeltia

et al. 2010

Journal of Biological Chemistry

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“…In vivo experiments have shown that the extent and type of sialylation influences serum half-lives of recombinant proteins (40). Hence, the pharmacokinetic properties of our N-glycosylated scDb molecules might be further improved using expression systems leading to increased production of fully sialylated N-glycans with ␣-2,6-linked terminal sialic acid residues (41).…”

Section: Discussionmentioning

confidence: 99%

N-Glycosylation as Novel Strategy to Improve Pharmacokinetic Properties of Bispecific Single-chain Diabodies

Stork

Zettlitz

Müller

et al. 2008

Journal of Biological Chemistry

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The therapeutic efficacy of recombinant antibodies such as single-chain Fv fragments and small bispecific or bifunctional molecules is often limited by rapid elimination from the circulation because of their small size. Here, we have investigated the effects of N-glycosylation on the activity and pharmacokinetics of a small bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. We could show that the introduction of N-glycosylation sequons into the flanking linker and a C-terminal extension results in the production of N-glycosylated molecules after expression in transfected HEK293 cells. N-Glycosylated scDb variants possessing 3, 6, or 9 N-glycosylation sites, respectively, retained antigen binding activity and bispecificity for target and effector cells as shown in a target cell-dependent IL-2 release assay, although activity was reduced ϳ3-5-fold compared with the unmodified scDb. All N-glycosylated scDb variants exhibited a prolonged circulation time compared with scDb, leading to a 2-3-fold increase of the area under curve (AUC). In comparison, conjugation of a branched 40-kDa PEG chain increased AUC by a factor of 10.6, while a chimeric anti-CEA IgG1 molecule had the longest circulation time with a 17-fold increase in AUC. Thus, N-glycosylation complements the repertoire of strategies to modulate pharmacokinetics of small recombinant antibody molecules by an approach that moderately prolongs circulation time.Whole antibodies, especially chimeric, humanized or fully human IgG molecules, exhibit a long circulation time in the human body that can reach a half-life of 27 days (1, 2). In contrast, antibody fragments, e.g. Fab fragments, or recombinant formats, such as single-chain Fv fragments (scFv) 3 or bispecific derivatives thereof (tandem scFv, diabodies, single-chain diabodies), are rapidly cleared from circulation (2-4). This is mainly due to the small size leading to rapid renal clearance and the lack of recycling processes mediated by the neonatal Fc receptor (FcRn). Thus, repeated injections or infusions are required to maintain a therapeutically effective dose in the body (5). Several strategies to improve pharmacokinetic properties and thus dosing and therapeutic efficacy of recombinant antibodies have been developed in recent years. These strategies can be divided into: 1) those based on reducing renal clearance by increasing the apparent size of the therapeutic molecule, and 2) those that in addition implement FcRn-mediated recycling processes (6 -8).PEGylation of proteins is a well-established strategy to improve pharmacokinetic properties by increasing the molecular mass and the hydrodynamic radius (9 -11). Several PEGylated proteins are in clinical use, e.g. PEGylated interferon ␣-2a (PegIntron, PEGASYS) and PEGylated granulocyte-colony stimulating factor (Neulasta), or are under clinical development, e.g. a PEGylated anti-TNF Fab fragment (certolizumab pegol) (9, 12) Furthermore, direct fusion to albumin or an albumin bin...

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“…Since CHO cells lack 2,6 sialyltransferase activity [40] but express the polysialyltransferase ST8SiaIV [41], these sialic acid moieties are most likely 2,8-linked. In our previous mass spectrometrical analysis, about 4% of the sialylated N-glycans of sICAM-1-CT were found to be non-fucosylated N-glycans carrying either three sialic acid residues on maximally two antennae or four sialic acid residues on maximally three antennae [35].…”

Section: Discussionmentioning

confidence: 99%

Consequences of Soluble ICAM-1 N-Glycan Alterations on Receptor Binding and Signaling Kinetics in Mouse Astrocytes

Schürpf¹,

Callewaert²,

Meyer³

et al. 2008

TOGLYJ

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Soluble intercellular adhesion molecule-1 (sICAM-1) is elevated in the cerebrospinal fluid of patients with severe brain trauma and mouse sICAM-1 induces the production of macrophage inflammatory protein-2 (MIP-2) in mouse astrocytes. The production of MIP-2 is greatly enhanced when sICAM-1 contains sialylated complex-type N-glycans (sICAM-1-CT) as produced by Chinese hamster ovary (CHO) cells. By contrast, sICAM-1 from the Lec1 mutant of CHO cells (sICAM-1-HM), containing only high mannose-type N-glycans, is relatively inactive. Here we show that the Nglycans of sICAM-1-CT are mostly 2,3-sialylated bi-, tri-, and tetraantennary complex-type structures with varying amounts of core fucosylation. Unexpectedly, sICAM-1-CT and sICAM-1-HM bound equivalently to mouse astrocytes. Enhanced MIP-2 induction by sICAM-1-CT was associated with a more rapid, higher level, and prolonged MIP-2 response as well as sICAM-1-CT accumulation at the plasma membranes of mouse astrocytes. These results show that glycosylation of sICAM-1 contributes to its signaling properties at the astrocyte cell surface, and suggest that altered glycosylation which might arise as a result of inflammation could regulate the bioactivity of sICAM-1.

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A new Chinese hamster ovary cell line expressing α2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins

Cited by 94 publications

References 32 publications

Glycosylation Pattern of Mature Dimeric Leukocyte and Recombinant Monomeric Myeloperoxidase

Glycosylation Pattern of Mature Dimeric Leukocyte and Recombinant Monomeric Myeloperoxidase

N-Glycosylation as Novel Strategy to Improve Pharmacokinetic Properties of Bispecific Single-chain Diabodies

Consequences of Soluble ICAM-1 N-Glycan Alterations on Receptor Binding and Signaling Kinetics in Mouse Astrocytes

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