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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, 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 27, 2016

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Journal of Proteome Research

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: [email protected] 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 uhFSH 1

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compared 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, 2


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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 3



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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 4


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insight into the critical glycosylation structures of hFSH. Experimental section Chemicals and reagents One lot of Puregon®-HP of 50 IU/0.5mL and two lots of Puregon®-HP of 100 IU/0.5mL (rhFSH) (Organon, Oss, Netherlands) were purchased in China. All strengths were presented as liquor. One lot of high purity uhFSH (>98%) was obtained directly from the manufacturer, Shanghai Techwell Biopharmaceutical Company (Shanghai, China), as active pharmaceutical ingredient. Clean gel IEF was obtained from Amersham Biosciences (Piscataway, NJ). Dithiothreitol (DTT), iodoacetamide (IAM), NH4HCO3, trypsin, 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA) were purchased from Sigma-Aldrich (St. Louis, MO). Chymotrypsin was obtained from Roche (Penzberg, Germany). PNGase F, from New England Biolabs (Ipswich, MA); 10kDa MWCO centrifugal filters, from Millipore (Bedford, MA); and porous graphitized carbon (PGC) columns, from Grace (Columbia, MD). Glycoclean column and 2-AB labeling kit were obtained from Prozyme (San Leandro, CA). All other reagents were purchased from Sigma (St. Louis, MO) or Fluka (Bucho, Switzerland). Purification of rhFSH The rhFSH isolation by SEC column was performed as previously described.16 For this study, three lots of Puregon® injection were dispensed and combined, then loaded on to a Sephadex G-25 column (GE Healthcare life science) at a flow rate of 2 mL/min under isocratic condition of buffer A (2 mM phosphate, pH 7.4). The baseline of the flow through was monitored at 215 and 280 nm until a return to baseline was observed. The eluted 5



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rhFSH was collected and applied to a 10 kDa MWCO Centrifuge device (50 mL). The centrifuge was spun for 1 h at 3,000 rpm at 4°C and washed with buffer of 5 mM phosphate containing 150 mM NaCl pH 7.4. After concentration, fractions were combined and stored at -80°C. The isolated rhFSH concentration was determined by SEC-HPLC (superdex 75, GE Healthcare life science), and the protein purity, quality and recovery were compared to Puregon®. Intact protein and α/β-subunit mass analysis For intact molecular weight assay, the hFSH samples were subjected to 10% SDS-PAGE under non-reducing condition. Gels were stained by Coomassie R-250. The subunits of hFSH were analyzed in linear mode by the 4800 Plus MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Framingham, MA).14 The reduced hFSH subunits were further analyzed by the Acquity UPLC system connected on-line to a quadrupole time-of-flight tandem mass spectrometer (Xevo G-2S, Waters Corporation, Milford, MA). The column was a Waters Acquity UPLC BEH C4 column (2.1 mm × 100 mm, 1.7 µm particle). The flow rate was 0.2 mL/min using a gradient from 15% to 25% solvent B (100% acetonitrile with 0.1% formic acid) in 25 min at a column temperature of 80°C. Solvent A was 0.1% formic acid in water. For mass spectrometric analysis, a MS data acquisition method was employed with a scan range m/z 500-2500. Isoelectric focusing (IEF) for hFSH isoform pattern distribution A commercial dried polyacrylamide gel, Clean gel IEF, was rehydrated in a solution containing an appropriate composition of carrier ampholytes necessary to create the desired pH gradient (3–6). The separated proteins and pI marker were stained by 6


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Coomassie blue. Quantitative determination of sialic acid The sialic acid contents of both hFSHs were quantitatively estimated using a colorimetric resorcinol-hydrochloric acid method. The method was followed ChP 2010 Vol III: Appendix VI C, 35. The mean relative content and SD were determined for two replicates per sample. Protease and PNGase F digestion For N-glycosylation site identification and O-glycopeptide analysis, both hFSHs were reduced by incubating with 10 mM DTT for 30 min at 57°C and alkylated with 20 mM IAA at room temperature for 1h in the dark. One molar NH4HCO3 was diluted in the sample to make its final concentration to 50 mM. Deglycosylation was performed by adding 300IU PNGase F at 37°C overnight. Trypsin was added at 1:50 w/w and incubated at 37°C for 14 h. Digestion was quenched by adding 10% TFA. For N-glycopeptide analysis, samples of each hFSH were reduced and alkylated. Chymotrypsin was added at 1:25 w/w in 100 mM Tris-HCl, 10mM CaCl2 (pH 7.8). Enzymatic digestion was performed overnight at 25°C. Releasing and labeling of N-glycans The oligosaccharides of hFSH were released by PNGase F and desalted on a non-porous graphitized carbon. Then 2-AB labeling and purification of N-glycans were performed as described in previous study.17 Profiling and relative quantification of N-glycans Analysis of 2-AB labeled N-glycans was performed on a Waters Acquity UPLC BEH 7



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glycan column (2.1 mm × 150 mm, 1.7 µm particle) using an Acquity UPLC with a fluorescence detector (Waters Corporation, Milford, MA).18 The glycans were analyzed using mobile phase A, 50 mM ammonium formate (pH 4.4), and mobile phase B, acetonitrile. The gradient was 70-53% B in 35 min with a flow rate of 0.4 mL/min and column temperature of 40°C. Samples were injected in 80% acetonitrile. The fluorescence detection was carried out using an excitation wavelength of 330 nm and an emission wavelength of 420 nm. The eluted positions of the N-glycans were determined in glucose units (GU) by comparison with a standard dextran hydrolyzate 2-AB labeled (dextran ladder).19 The LC-MS data were acquired on Xevo G-2S (Waters Corporation, Milford, MA). The interested parent ions were selected for MS2 data acquisition.20 Identification of N-glycan sites A multiplexed data acquisition method (MSE) was employed for mass spectrometric analysis of tryptic digest of hFSHs. The instrument and analyzed method were described previously.20 Intact N-glycopeptide analysis N-Glycopeptides were analyzed by the Acquity UPLC system connected on-line to Xevo G-2S. The column was also a Waters Acquity UPLC BEH glycan column (2.1 mm × 150 mm, 1.7 µm particle). The experiment was performed as described elsewhere.21 The flow rate was 0.2 mL/min using a gradient of 1% to 40% solvent B (100% acetonitrile with 0.1% formic acid) in 63 min, followed by an increase to 90% B in 4 min, and then to 90% B in 6 min. Solvent A was 0.1% formic acid in water. Intact O-glycopeptide analysis 8


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O-Glycopeptides were analyzed by nano LC-MS/MS on an Orbitrap Fusion Tribrid MS (Thermo Fisher Scientific, San Jose, CA) coupled to an EASY-nLC System (Thermo Fisher Scientific, San Jose, CA). Peptide mixtures were loaded onto a Magic C18 spray tip 15 cm × 75 µm i.d. column (Michrom Bioresources) and separated at a flow rate of 350 nL/min using a gradient of 8% to 22% solvent B (100% acetonitrile with 0.1% formic acid) in 54 min, followed by an increase to 35% B in 15 min, and then to 90% B in 10 min and held for another 6 min. Solvent A was 0.1% formic acid in water. Data acquisition was performed under data dependent acquisition (DDA) with HCD-pd-ETD. The parameters settings were: top speed mode with 3 s cycle time; FT MS: scan range (m/z) = 350−2000; MS resolution = 120K; MS2 resolution=30K; other parameters followed a previous report.22 To restrict the ETD MS2 data acquisition to true O-glycopeptide precursors, the preceding HCD can be applied in producing diagnostic glyco-oxonium ions (138.0545, 204.0867 and 366.1396) using as a filtering criterion to trigger ETD in the HCD-pd-ETD mode. LC-MS data processing N-glycan data were processed using UNIFI 1.7 with Glycobase 3+ (Waters Corporation, Milford, MA) for N-glycan structure. The peak area of chromatography was calculated for relative glycan quantification. The mean relative content and SD were determined for three replicates per sample. The MS2 data analysis was performed with MassLynx 4.1 data system. The data of intact hFSH subunits were deconvoluted and analyzed by UNIFI 1.7. The observed N-glycans structures were set as amino acid modification for deconvolution (mass error

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