Comparative assessment of glycosylation of recombinant human FSH and highly purified FSH

Hong Wang, Xi Chen, Xiaoxi Zhang, Wei Zhang, Yan Li, Hongrui Yin, Hong Shao, and Gang Chen

J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00921 • Publication Date (Web): 26 Jan 2016

Downloaded from http://pubs.acs.org on January 28, 2016

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Comparative assessment of glycosylation of a recombinant human FSH and a

highly purified FSH extracted from human urine

Hong Wang†, Xi Chen‡, Xiaoxi Zhang¶, Wei Zhang¶, Yan Li§, Hongrui Yin†, Hong Shao†,

Gang Chen*†

† Shanghai Institute for Food and Drug Control, Shanghai, 201203, China

‡ Waters Corporation, Shanghai, 201206, China

¶ Thermo Fisher Scientific, Shanghai, 201206, China

§ Shanghai Techwell Biopharmaceutical Corporation, Shanghai, 201108, China

* Corresponding author: Tel.:+86-21-50798175; fax: +86-21-50798176.

E-mail address: chengang@smda.gov.cn

Abstract

Glycosylation is an important PTM and is critical for manufacture and efficacy of therapeutic glycoproteins. Glycan significantly influences the biological properties of human follicle-stimulating hormone (hFSH). Using a glycoproteomic strategy, this study compared the glycosylation of a putative highly purified FSH (uhFSH) obtained from human urine with that of a recombinant human FSH (rhFSH) obtained from Chinese hamster ovary (CHO) cells. Intact and subunit masses, N-glycans, N-glycosylation sites, and intact N- and O-glycopeptides were analyzed and compared by mass spectrometry. Classic and complementary analytical methods, including SDS-PAGE, isoelectric focusing, and the Steelman-Pohley bioassay were also employed to compare their intact molecular weights, charge variants, and specific activities. Results showed that highly sialylated, branched, and macro-heterogeneity glycans are predominant in the uhFSHcompared with rhFSH. The O-glycopeptides of both hFSHs, which have not been described previously, were characterized herein. A high degree of heterogeneity was observed in the N-glycopeptides of both hFSHs. The differences in glycosylation provide useful information in elucidating and in further investigation of the critical glycan structures of hFSH.

Key words: follicle-stimulating hormone, uhFSH, rhFSH, glycosylation, mass spectrometry

Introduction

Human follicle-stimulating hormone (hFSH) plays a key role in the development and function of the reproductive system and is used clinically to stimulate follicular maturation for in vitro fertilization and treatment of an ovulatory women.1 hFSH is a heterodimeric structure consisting of non-covalently linked α- and β-subunits. Each subunit contains two N-glycosylation sites carrying sialylated complex type N-glycans.2 The oligosaccharide composition, branching pattern, and number of sialic acid residues of hFSH are markedly variable giving rise to multiple glycoforms of hFSH. Two classes of hFSH-containing pharmaceutical preparations currently exist; those derived from the urine of post-menopausal women (uhFSH) and those manufactured using recombinant DNA technology (rhFSH). These preparations possess identical amino acid sequence,although their terminal sialylation, pI, half-life, and effect in clinical application vary.3 Comparative clinical studies have revealed the differences in oocyte quality and clinical outcome between rhFSH and uhFSH.4

The oligosaccharide moiety is critical in determining the pharmacological properties, including stability, solubility/bioavailability, in vivo activity, pharmacokinetics, and immunogenicity of therapeutic glycoproteins.5 The two major types of oligosaccharide attached to therapeutic glycoproteins are the N-linked and O-linked glycans. Glycans significantly influence the biological properties of hFSH. Glycans attached to the α-subunit are critical for dimer assembly, integrity, secretion, and signal transduction, whereas β-subunit glycans are important for dimer assembly and secretion.6 The sialylation and complexity of hFSH oligosaccharides affect endocrine activity and expression of genes regulating granulosa cell function.7,8 The classic Steelman–Pohley bioassay in rats showed that FSH exhibiting high level of sialylation possesses long half-life in vivo biopotency.9 Moreover, the threshold of ovarian follicles and the dynamics of follicular growth are influenced by FSH glycosylation.10 Some studies have revealed that uhFSH has longer half-life than rhFSH because it contains more sialic acids.11 Considering the importance of hFSH glycosylation to the biological activity of hFSH, evaluating the difference in glycosylation in rhFSH and uhFSH intended for clinical use is essential.

However, analysis of protein glycosylation is a challenging task because of the variable glycoside linkages, branching, and numerous isomers in hFSH. Liquid chromatography-mass spectrometry (LC-MS) has become an invaluable combination of technologies for detection, quantification, comparison, and further elucidation of glycan structure. Alternative fragmentation technologies, including higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), can extensively characterize not only N-glycopeptides but also O-glycopeptides.12 Moreover, several studies have already mapped hFSH glycosylation. The oligosaccharide composition of commercial FSH preparations was evaluated using RP-HPLC/IT-TOF MS.13 The results showed the highly sialylated and branched glycans in uhFSH compared with rhFSH expressed in rodent cell lines. Site-specific analysis of all four glycosylation sites in rhFSH was also accomplished using Q-TOF MS.1 In addition, the glycan moiety of rhFSH produced in CHO cells was analyzed using a combination of LC and MS techniques, including both matrix-assisted laser desorption ionization (MALDI) and electro-spray ionization (ESI) MS.14 Quantitation of the level of sialylation and of antennarity of N-glycans was obtained using glycan mapping methods. FSH glycosylation micro-heterogeneity in pituitary and urinary hFSH was evaluated using nano-ESI-MS.15

Using a glycoproteomic strategy, we compared the glycosylation pattern of uhFSH with that of rhFSH produced in CHO cells. The N-glycan chains labeled with 2-aminobenzamide (2-AB) were analyzed and quantified using hydrophilic interaction chromatography (HILIC)-based LC-MS. The N-glycosylation sites and the site-specific glycan structures were also revealed. The novel HCD product-dependent ETD (HCD-pd-ETD) workflow was used in revealing the O-glycosites and site-specific O-glycans. In addition, the charge variants and sialic acid contents were determined, and in vivo bioassay was performed. Higher level of sialylation, antennary, and macro-heterogeneity were detected in uhFSH than in rhFSH. Moreover, NeuGc residue was found in rhFSH and is possibly immunogenic. Highly heterogeneous N-glycosylation patterns were also observed in both hFSHs. Two O-glycosylation sites were discovered in rhFSH, whereas five O-glycosylation sites were discovered in uhFSH. O-glycosylation in both hFSHs has not yet been described. The differences in glycosylation provide deeper insight into the critical glycosylation structures of hFSH.