Isolation and characterization of unknown oligosaccharides from recombinant therapeutic glycoproteins expressed from chinese hamster ovary cell line

Graça, Telmo Gonçalo Henriques

http://hdl.handle.net/10400.1/684

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Authors

Graça, Telmo Gonçalo Henriques

Advisor(s)

Melo, Eduardo

Conradt, Harald Sigurd

Abstract(s)

The glycosylation pattern (sialylation, antennarity, presence of truncated/incomplete structures, and the type of peripheral substitutions of glycans) of a recombinant glycoprotein therapeutic protein can affect its immunogenicity, bioactivity pharmacokinetic profile, stability and solubility. The main focus of the present work was to identify and to structurally characterize unknown structural components of N-linked oligosaccharides attached to recombinantly expressed therapeutic glycoproteins that can serve as reference standards for monitoring and quantification of these structures in human biopharmaceuticals from CHO cells or other expression systems. The N-linked oligosaccharide structures were isolated from a CHO cell culture supernatant obtained from a GMP-compatible production process for recombinant human erythropoietin. The total protein from the cell culture supernatant was digested with trypsin and the glycopeptides were isolated by reversed phase chromatography (RP-HPLC). The N-glycans were enzymatically released from glycopeptides using polypeptide N-glycosidase F and were separated from the peptides by RP-HPLC. In a first step N-linked oligosaccharides were fractionated according to their charge using a MonoQ anion exchange chromatography column. In a second step fractions from the Mono Q chromatography were further subfractionated by antennarity applying an aminopropyl bonded phase matrix. Throughout all fractionation steps N-glycans pools were analyzed by high pH anion exchange chromatography with pulsed amperometric detection (HPAECPAD). Mass spectrometric analysis was applied for analysis of total N-glycans in the cell culture supernatant and was also used for the structural elucidation of the newly detected Nglycans. Three unusual N-linked oligosaccharides representing about 10 % of the total carbohydrate material were identified and were characterized. Two of the glycan structures were identified as phosphorylated oligomannosidic structures (Man5GlcNAc2 and Man6GlcNAc2). A third structure was identified as being a monosialylated and phosphorylated hybrid-type glycan with a proximal 1-6 linked Fuc. None of the unusual oligosaccharides herein described had previously been reported in the literature. The three N-glycan structures were recovered with purities above 95% and in sufficient quantities to be used in the future as oligosaccharide reference standards in release tests for human glycoprotein biopharmaceuticals produced in biotechnological production processes involving CHO cell lines.