2 Laboratoires Serono Sa, Serono Biotech Center, Zone Industrielle B, CH-1809 Fenil-Sur-corsier, Switzerland
3 Laboratoire de Spectrométrie de Masse Bio-Organique, ECPM, UMR CNRS 7509, Université Louis Pasteur, 25 rue Becquerel, 67087 Strasbourg Cedex, France
Received on June 28, 2002; revised on September 20, 2002; accepted on September 23, 2002
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Abstract |
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Key words: batch-to-batch consistency / glycoprotein / mass spectrometry / N-glycan structure / O-glycan structure
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Introduction |
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The oligosaccharide moieties of gonadotrophins have already been extensively studied (Pierce and Parsons, 1981; Sairam, 1983
; Ryan et al., 1987
; Baenziger and Green, 1988
; Hard et al., 1990
; Kobata, 1992
; Olijve et al., 1996
; Amoresano et al., 1996
; Liu and Bowers, 1997
; Jacoby et al., 2000
). The polymorphism of these glycoproteins is associated with the variation of sialic acid content and with other carbohydrate heterogeneities: N-glycans are of complex type with different numbers of antennae (from two to five) and various levels of sialylation. Fewer data are available on O-glycans (Amoresano et al., 1996
; Liu and Bowers, 1997
). Usually O-glycans are much more diverse than N-glycans and have no common core structures.
The presence of glycans is essential for the in vivo biological activity of the gonadotrophins because they are involved in their folding and secretion. Furthermore they are essential for the plasma half-life of the glycoprotein hormones (Thotakura and Blithe, 1995; Varki, 1993
; Calvo et al., 1986
; Cumming, 1991
; Mulders et al., 1997
). However, the relationship between charge and in vivo bioactivity is complex (D'Antonio et al., 1999
; Horsman et al., 2000
), the most sialic acid-rich glycoforms having a prolonged plasma half-life (Thotakura and Blithe, 1995
; Varki, 1993
; Calvo et al., 1986
; Cumming, 1991
; Mulders et al., 1997
) and the less acidic species having in vitro a greater receptor binding affinity, as shown in a recent study (Vitt et al., 1998
). Therefore, due to the key role of glycosylation in determining and modulating biological functions in vivo, one of the main challenges of the biotechnology industry is to develop a deep understanding of the molecular structure of the glycan side chains of therapeutic proteins (Cumming, 1991
; Hermentin et al., 1996
; Hermentin and Witzel, 1999
; Viseux et al., 2001
). The set-up of technologies that allow the careful monitoring of protein glycosylation during process development and improvement will indeed be a key success factor for achieving a tight control of the consistency of the product in terms of protein glycosylation and, in turn, of clinical efficacy.
This article reports the detailed characterization of the glycans of recombinant human (r-h) gonadotrophins (r-hFSH, r-hLH, and r-hCG) produced in Chinese hamster ovary (CHO) cell lines by combination of chromatography and mass spectrometry (MS). Different MS techniques, including matrix-assisted laser desorption ionization (MALDI) and electrospray (ES), have been used. In particular, the structure of an O-glycan of r-hCG, not yet described, has been unambiguously determined using nanoES ion trap (IT) MS (nanoES-ITMS).
Two glycan mapping methods have been developed that allow the separation of the glycans according to either their charge or sialylation level, or according to their antennarity or branching of the N-glycans. For r-hCG, owing to the presence of O-glycans, a specific glycan mapping method was developed that permits the complete resolution of the N-glycan from the O-glycan species.
Using these methods, two parameters have been defined for the quantitative assessment of the sialylation and antennarity of the N-glycans: the hypothetical charge number Z introduced in 1996 (Hermentin et al., 1996; Hermentin and Witzel, 1999
) and an antennarity index A, respectively. The high level of glycosylation consistency achieved in the production of r-hFSH (the active ingredient in Gonal-F) is reported.
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Results and discussion |
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Nevertheless, the interpretation of the MALDI-TOF mass spectra confirmed that the N-glycans are of complex type with di-, tri-, tetra- and even minor pentaantennary species. Fucosylated and nonfucosylated species were detected. The glycans are eluted as a function of the number of sialic acids they bear, which is linked to their charge. The groups of peaks corresponding to glycan species with different levels of sialylation are well separated without overlap. The separation of the glycans is performed essentially as a function of their charge but also as a function of their size. Indeed, within a group of charge, the glycan species elute in the order of decreasing molecular weight from tetra- to diantennary glycan forms, the corresponding fucosylated species eluting before the nonfucosylated species.
Minor glycan forms were detected in some of the individual fractions: these forms lacked one or two hexose residues (galactose residues, loss of 162 Da) and/or N-acetylhexosamine residues (N-acetylglucosamine, loss of 203 Da). These "degraded" forms are neither due to fragmentation processes in the mass spectrometer nor to a degradation process during hydrazinolysis but are forms really existing as minor species in gonadotrophins. These forms indeed have been detected on the entire r-hLH molecule by MS (data not shown).
Hypothetical charge number, Z
Thus MS experiments have confirmed that N-glycan species with the same sialylation level elute within the same group of peaks on the chromatogram. Moreover, because a single fluorescent label is fixed on each glycan species, the relative proportions of the areas on the chromatogram are representative of the relative proportions of glycan species. Therefore, the hypothetical charge number, Z, introduced in 1996 (Hermentin et al., 1996) has been calculated for r-hFSH and r-hLH. This Z number is defined as the sum of the products of the respective areas (A) in the neutral, mono-, di-, tri-, tetra-, and pentasialylated region of the N-glycan species, each multiplied by the corresponding charge:
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The validation experiments performed on both r-hFSH and r-hLH demonstrated that Z is an accurate parameter that characterizes the sialylation level of these two molecules. In addition, the precision in measuring Z has been determined to be better than 2%.
This method is also able to detect differences in the glycosylation pattern of batches with different bio-specific activities as shown in Figure 3. The r-hFSH batch with a high biospecific activity (Figure 3A) has a higher Z number than the r-hFSH batch with a lower biospecific activity (Figure 3B). This difference is reflected by the proportions of individual glycan species on the chromatograms, the r-hFSH batch with a lower biospecific activity having less sialylated glycans than the r-hFSH batch with a higher biospecific activity.
Separation of the N-glycans according to theirantennarity
The chromatograms of the N-glycans of r-hFSH and r-hLH separated according to their charge by anion exchange chromatography show that the N-glycans are separated also according to their antennarity, the tetraantennary species eluting before tri- and diantennary species within a group of isocharge species. However, due to the heterogeneity of the glycans, no clear separation between species having different antennarity levels is obtained. Therefore, a second dimensional separation has been developed.
After reducing the heterogeneity given by sialic acids with sialidase, the resulting neutral species were separated by reverse-phase chromatography with an acetonitrile gradient in 50 mM ammonium acetate (Figure 4 for r-hFSH). The different peaks on the chromatogram have been identified both with ESMS and MALDI-TOF MS. Both techniques gave complementary results allowing identification of the following species: di-, tri-, tetraantennary N-glycan species either fucosylated or not, as well as minor species including pentaantennary glycans and "degraded" forms lacking one or two hexose residue(s) (galactose residues, loss of 162 Da) and/or N-acetylhexosamine residues (N-acetylglucosamine, loss of 203 Da). These results are consistent with those obtained on the charged glycans. Because of possible different isomeric glycan structures, same isobaric structures have been detected in two different peaks on the chromatograms. In each peak, the most abundant species is represented on the chromatogram (Figure 4).
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Particular case of r-hCG: separation of N- and O-glycans
Recombinant hCG is both N- and O-glycosylated. The structures, and therefore the physicochemical properties of O- and N-glycans, are widely different. The structures of the O-glycans of hCG proposed by Liu and Bowers (1997) are presented in Table I. Both N- and O-glycans can be sialylated. However, the relevance of the sialylation of the O-glycans for the bioactivity of the molecule is minor (Nemansky et al., 1995
).
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The N-glycans were eluted as a function of their charge linked to the number of sialic acids. The N-glycans identified were N-complex type glycans with di-, tri-, and tetraantennarity forms and with a variable level of sialylation (from 0 to 2, 1 to 3, and 3 respectively). Both fucosylated and nonfucosylated species have been identified. As previously, within a group of glycans having the same level of sialylation (neutrals, monosialylated, disialylated, and trisialylated), the N-glycans are eluted from tetra- to diantennary forms, the corresponding fucosylated species being eluted before the nonfucosylated ones. As for r-hFSH and r-hLH, some artifactual peaks coming from the re-N-acetylation step in the hydrazinolysis process (Harvey, 1996, 2001
; Naven and Harvey, 1996
) complicated the mass spectra. Finally, some "degraded" minor forms lacking one or two hexose residue(s) and N-acetylhexosamine residue(s) were detected. These forms are present as minor forms on the r-hCG molecule and are not due to degradation during the glycan mapping process.
The O-glycans were submitted to both MALDI-TOF MS and ESMS techniques. Most of the O-glycan structures correspond to species with low molecular weights, which may interfere with the peaks of the matrix during the MALDI-TOF process. Using these two techniques, almost all the O-glycan structures proposed by Liu and Bowers (1997) have been identified from their mass measurement. In addition, five other possible structures for O-glycans of r-hCG have been proposed (Table II). The monosaccharide composition of these glycoforms, deduced from their mass measurement, includes the presence of a fucose residue, which has never been described for hCG. To confirm these possible structures, fragmentation experiments were performed by ITMS to determine the sequence of monosaccharides in the glycan (see later section).
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Separation of the N-glycans according to their antennarity. A second dimensional separation has been also developed for r-hCG. After reducing the heterogeneity given by sialic acids with sialidase, the resulting neutral species were separated by reverse-phase chromatography with an acetonitrile gradient in 50 mM ammonium acetate (Figure 6). By means of MS (both ESMS and MALDI-TOF MS), the different peaks on the chromatogram have been identified. N- and O-glycans were detected in different separate peaks and the following N-glycan structures were detected: di- and triantennary species either fucosylated or not. Minor "degraded" forms lacking one or two hexose residue(s) (galactose residues, loss of 162 Da) and /or N-acetylhexosamine residues (N-acetylglucosamine, loss of 203 Da) were also detected. These results are consistent with the one obtained on the charged glycans. Due to possible different isomeric glycan structures, same isobaric compositions have been detected in different peaks on the chromatograms. In each peak, the most abundant species is represented on the chromatogram (Figure 6).
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Characterization of O-glycans with ITMS
The strategy that was adopted to characterize the O-glycan structures of r-hCG relied first on the evaluation of the ability of an IT to perform multidimensional tandem MS (MSn) to elucidate glycan structures. Therefore fragmentations of glycans by nanoES-MSn were performed initially on well-known N-glycan structures. In a second step, this methodology was applied to the characterization of the high-performance anion exchange chromatography (HPAEC) fractions containing the different O-glycan structures of r-hCG. The characterization by tandem MS of glycans was performed both in positive and in negative ionization modes. Glycan fragmentation is possible in positive ion mode due to the basic reducing properties of the 2-AB moiety, which facilitates the protonation and the detection of the polysaccharide. The best results were obtained in positive mode for the N-glycans and in negative mode for O-glycans.
Evaluation of MSn analyses to characterize N-glycan structures. The ability of ITMS to perform MSn structural elucidation of glycans has been evaluated on different N-glycans for which the structures were known. The example of a neutral diantennary fucosylated complex type N-glycan (NA2F-2AB, average molecular mass 1907 Da) is presented here. The aim of this experiment was to localize the position of the labile fucose residue on the polysaccharide structure. MS2 was performed on the doubly charged ion at m/z 965.3 corresponding to NA2F-2AB cationized by a proton and a sodium (Figure 7A). All the MS2 fragments fit with the structure of the NA2F-2AB. Nevertheless, the localization of the fucose on the whole N-glycan structure needed further analysis. For that purpose, MS3 was attempted on the smaller fragments identified that still contained the fucose monosaccharide. The ion at m/z 1402.6, attributed to the fragments Y4/5ß, Y5
/4ß, Y3
, Y3ß according to Domon and Costello (1988)
nomenclature, was therefore fragmented. The interpretation of the MS3 spectrum did not allow a direct elucidation of the position of the fucose on the N-glycan structure (data not shown). However, because the observed ion at m/z 1037.4, attributed to the fragments Y3
/4ß, Y4
/3ß, contained the fucose, the fragmentation of Y3
/4ß, Y4
/3ß by MS4 analysis was performed (Figure 7B). In the corresponding MS4 spectrum, a fragment at m/z 510.2 was identified as the N-acetylhexosamine monosaccharide labeled with 2-AB and substituted by the fucose. Thus, the fucose was localized on the reductive part of the N-glycan NA2F-2AB.
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Characterization of O-glycans of r-hCG. Three high-performance liquid chromatography (HPLC) fractions containing the O-glycans of r-hCG were analyzed by nanoES-MSn. The first HPLC fraction contained two O-glycans at m/z 793.3 and m/z 1304.4. MS2 analyses were performed on both structures and the obtained fragment ions are summarized in Table III. The MS2 spectrum of the O-glycan labeled with 2-AB at m/z 793.3 (structure 1a in Table III) displayed fragment ions characteristic of the proposed structure previously described (Liu and Bowers, 1997), whereas MS2 fragment ions obtained for the O-glycan labeled with 2-AB at m/z 1304.4 (structure 1b in Table III) described a structure containing a fucose monosaccharide, which has not been previously described. The second HPLC fraction analyzed revealed an O-glycan at m/z 590.3. Its fragmentation by MS2 displayed fragment ions (Table III) corresponding to the disaccharide hexose/sialic acid labeled with 2-AB (structure 2 in Table III). The third HPLC fraction contained an O-glycan at m/z 1084.4, which was previously identified (Liu and Bowers, 1997
) (structure 3a in Table III). The MSn analyses allowed without any ambiguity its characterization. MS2 fragmentation of the O-glycan produced a single fragment ion Y1ß, Y2
at m/z 793.3 (Table III), which revealed the presence of a sialic acid.
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Batch-to-batch consistency of the glycoforms of thethree gonadotrophins
The glycan mapping methods discussed in this article are now applied routinely to monitor and control the consistency of sialylation of the three recombinant gonadotrophins in commercial manufacturing operation. As an illustration of the batch-to-batch consistency of the production of r-hFSH, Figure 9 reports the results of the analysis of more than 120 batches of drug substance over a period of 3 years: the sialylation level, expressed by the Z number, varies by less than 3.5%. For the same set of batches, the individual glycan species also demonstrate a remarkable consistency in their relative proportions (data not shown) with coefficients of variation in the range of 7% for the most abundant species (50% of disialylated glycans) and of 15% for the least abundant forms (46% of tetrasialylated or neutral glycans), which are measured with less precision. Comparable tight consistency has also been obtained for the sialylation of r-hLH and r-hCG in commercial production. The data available to date on the antennarity of the glycans of recombinant gonadotrophins indicate that this glycosylation parameter is also highly consistent: the antennarity level, expressed by the A index, varies by less than 1% over a period of 3 years of production as determined from a set of 10 batches of each of the three recombinant gonadotrophins r-hFSH, r-hLH, and r-hCG. Here also the individual glycan species demonstrate a remarkable consistency in their relative proportions (data not shown).
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Conclusions |
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Furthermore, the ability of nanoES-ITMS as a new powerful technique for the determination of the branching pattern of unknown glycan has also been demonstrated. An unexpected and not yet described structure for one of the O-glycans of r-hCG has been proposed and confirmed by nanoES-ITMS.
Quantitation of the level of sialylation and of antennarity of N-glycans can be obtained using the glycan mapping methods. The Z number, characteristic of the level of sialylation, and similarly the A index for the antennarity have been proposed as measurements of these parameters of glycosylation. The precision in the determination of Z number has been shown to be better than 2% and on A index around 1%. Using these methods, we have shown the high level of batch-to-batch consistency achieved for the glycosylation of three recombinant gonadotrophins in commercial production. These data demonstrate the tight control that can be achieved in the manufacturing of complex recombinant therapeutic glycoproteins. This consistency is a prerequisite to the delivering of a guaranteed dose of product from vial to vial because it opens the possibility to use very accurate physicochemical methods for the quantitation of the active substance, which is then no longer blurred by the known intrinsic variability of in vivo potency bioassays.
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Materials and methods |
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Glycan release and labeling
The glycans were released by hydrazinolysis on the fully automated GlycoPrep 1000TM instrument supplied by Oxford GlycoSciences. The N-glycans were released in the N*mode (at 100°C, 5 h). The N- and O-glycans of r-hCG were released in the N+O mode (at 95°C, 5 h). After lyophilization, the glycans were fluorescently labeled with 2-AB through a reductive amination reaction as described by Bigge et al. (1995).
Desialylation of sialylated N-glycans
Desialylation of the pool of sialylated glycans (from about 150 µg of glycoprotein) was performed with 0.05 U sialidase from V. cholerae at 37°C for 18 h at pH 5.5 in 250 mM ammonium acetate/20 mM calcium chloride. After desialylation, the glycans were dried using speed vacuum concentration.
Separation of charged glycans by AEC
AEC was carried out on two different columns, depending on the presence of O-glycans.
The charged N-glycans of r-hFSH and r-hLH were separated on a GlycoSep C weak anion exchanger column (4.6x100 mm, 5 µm, coated divinylbenzene resin, Glyko) using a gradient of ammonium acetate 500 mM, pH 4.5, in ultrapure water. A constant 20% of acetonitrile is maintained all along the chromatographic separation.
The charged N- and O-glycans of r-hCG were separated on a CarboPac PA-100 weak anion exchanger column (4x250 mm, 10 µm, quaternary ammonium functionalized latex, Dionex, Sunnyvale, CA) using a gradient of ammonium acetate 250 mM, pH 4.5, in ultrapure water. The 2-AB-labeled glycans were detected by fluorimetry (excitation: 330 nm;
emission: 420 nm).
Separation of desialylated glycans by Reverse-PhaseChromatography
After desialylation, the resulting neutral glycans were separated by reverse-phase chromatography on a GlycoSep R C18 column (4.6x150 mm, 3 µm, Glyko) using a gradient of ammonium acetate 50 mM, pH 6.0, containing 8% acetonitrile (eluent B), eluent A being ammonium acetate 50 mM, pH 6.0. The 2-AB-labeled glycans were detected by fluorimetry (excitation: 330 nm;
emission: 420 nm).
MALDI-TOF MS
MALDI-TOF mass spectra were acquired on a Biflex mass spectrometer (Bruker-Franzen Analytik GmBH, Brem, Germany) equipped with a 337-nm nitrogen laser, a reflectron, and a delayed extraction system. The system was operated either in the linear or in the reflector ion mode and either in the positive or the negative ion mode depending on whether the glycans were neutral or charged. Two different matrices have been used: 2,5-dihydroxybenzoic acid in water/acetonitrile (1/1, v/v) and 5-chloro 2-mercaptobenzothiazole (Xu et al., 1997) in tetrahydrofuran/ ethanol/water (1/1/1, v/v/v).
ESMS with triple quadrupole
ES mass spectra were acquired on a Quattro II triple quadrupole mass spectrometer (Micromass, Altrincham, UK). The cone voltage has been set up to prevent the fragmentation of the glycans in the interface. Fragmentation studies have been performed by running the ES mass spectrometer in the MS/MS mode. The parent ion was selected and collisioned with argon molecules at a pressure of about 5x103 mbars in the gas cell and with a collision energy optimized on a case-by-case basis (around 20 eV in general).
Nano ES-ITMS
Low energy collisions of multicharged ions were performed on an IT mass spectrometer ESQUIRE-LC system (Bruker-Franzen Analytik) equipped with a nanospray source. The gold/palladium-coated nanospray capillaries were from Protana (Odense, Denmark). Calibration of the analyzer was performed with the multicharged ions of the following five standard peptides: Leu-enkephalin, angiotensin, P-substance, bombesin, and ACTH, having monoisotopic molecular weights of 711.38 Da, 1045.54 Da, 1346.74 Da, 1619.81 Da, and 2464.20 Da, respectively.
To perform MSn experiments, isolation of the precursor ion was achieved by scanning frequencies of ions to eject all other ions from the trap. The precursor ion was fragmented by applying a resonance frequency on the end cap electrodes (peak to peak amplitude 0.82.5 V) matching the frequency of the selected ion. As a result, the kinetic energy of the precursor ion increases and dissociation due to collisions with the buffer gas helium (pressure 5x10-3 mbar) occurs. Sequences of isolation and fragmentation were repeated for MSn (n=1, 2, 3, 4) experiments to gain structural information on the selected fragment ions. Ions were scanned in standard resolution mode (m/z=0.6) with a scan speed of 13,000 Da/s. A total of 20 scans were averaged to obtain a mass spectrum.
Determination of in vivo bioactivity of r-hFSH
The in vivo bioactivity of r-hFSH was determined according to the Steelman and Pohley (1953) in vivo bioassay.
1 To whom correspondence should be addressed; e-mail: annick.gervais{at}serono.com
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Abbreviations |
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References |
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---|
Baenziger, J.U. and Green, E.D. (1988) Pituitary glycoprotein hormone oligosaccharides: structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin. Biochim. Biophys. Acta, 947, 287306.[ISI][Medline]
Bahr, U., Pfenninger, A., Karas, M., and Stahl, B. (1997) High-sensitivity analysis of neutral underivatized oligosaccharides by nanoelectrospray mass spectrometry. Anal. Chem., 69(22), 45304535.[CrossRef][ISI][Medline]
Bigge, J.C., Patel, T.P., Bruce, J.A., Goulding, P.N., Charles, S.M., and Parekh, R.B. (1995) Nonselective and efficient fluorescent labelling of glycans using 2-amino benzamide and anthranilic acid. Anal. Biochem., 230, 229238.[CrossRef][ISI][Medline]
Calvo, F.O., Keutmann, H.T., Bergert, E.R., and Ryan, R.J. (1986) Deglycosylated human follitropin: characterization and effects on Adenosine cyclic 3', 5'-phosphate production in porcine granulosa cells. Biochemistry, 25, 39383943.[ISI][Medline]
Creaser, C.S., Reynolds, J.C., and Harvey, D.J. (2002) Structural analysis of oligosaccharides by atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap mass spectrometry. Rapid Commun. Mass Spectrom., 16, 176184.[CrossRef][ISI][Medline]
Cumming, D.A. (1991) Glycosylation of recombinant protein therapeutics: control and functional implications. Glycobiology, 1(2), 115130.[Abstract]
D'Antonio, M., Borrelli, F., Datola, A., Bucci, R., Mascia, M., Polletta, P., Piscitelli, D., and Papoian, R. (1999) Biological characterisation of recombinant human follicle stimulating hormone isoforms. Hum. Reprod., 14(5) 11601167.
Domon, B. and Costello, C.E. (1988) A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconj., 5, 397409.[ISI]
Gennaro, L.A., Delaney, J., Vouros, P., Harvey, D.J., and Domon, B. (2002) Capillary electrophoresis/electrospray ion trap mass spectrometry for the analysis of negatively charged derivatized and underivatized glycans. Rapid Commun. Mass Spectrom., 16, 192200.[CrossRef][ISI][Medline]
Hard, K., Mekking, A., Damm, J.B.L., Kamerling, J.P., De Boer, W., Wijnands, R.A., and Vliegenthart, J.F.G. (1990) Isolation and structure determination of the intact sialylated N-linked carbohydrate chains of recombinant human follitropin expressed in Chinese hamster ovary cells. Eur. J. Biochem., 193, 263271.[Abstract]
Harvey, D.J. (1996) Matrix-assisted laser desorption/ionisation mass spectrometry of oligosaccharides and glycoconjugates. J. Chromatogr. A., 720, 429446.[CrossRef][ISI][Medline]
Harvey, D.J. (2001) Identification of protein-bound carbohydrates by mass spectrometry. Proteomics, 1, 311328.[CrossRef][ISI][Medline]
Hermentin, P. and Witzel, R. (1999) The hypothetical N-glycan charge. A number to characterise protein N-glycosylation. Pharm. Pharmacol. Commun., 5, 3343.
Hermentin, P., Witzel, R., Kanzy, E.J., Diderrich, G., Hoffmann, D., Metzner, H., Vorlop, J., and Haupt, H. (1996) The hypothetical N-glycan charge: a number that characterizes protein glycosylation. Glycobiology, 6(2), 217230.[Abstract]
Horsman, G., Talbot, J.A., McLoughlin, J.D., Lambert, A., and Robertson, W.R. (2000) A biological, immunological and physico-chemical comparison of the current clinical batches of the recombinant FSH preparations Gonal-F and Puregon. Hum. Reprod., 15(9), 18981902.
Jacoby, E.S., Kicman, A.T., Laidler, P., and Iles, R.K. (2000) Determination of the glycoforms of human chorionic gonadotropin ß-core fragment by matrix assisted laser desorption ionisation time-of-flight mass spectrometry. Clin. Chem., 46, 17961803.
Kobata, A. (1992) Structure and functions of the sugar chains of glycoproteins. Eur. J. Biochem., 209, 483501.[Abstract]
Liu, C.L. and Bowers, L.D. (1997) Mass spectrometry characterisation of the beta-subunit of human chorionic gonadotropin. J. Mass Spectrom., 32, 3342.[CrossRef][ISI][Medline]
Mulders, J.W.M., Derksen, M., Swolfs, A., and Maris, F. (1997) Prediction of the in vivo biological activity of human recombinant follicle stimulating hormone using quantitative isoelectric focusing. Biologicals, 25, 269281.[CrossRef][ISI][Medline]
Naven, T.J.P. and Harvey, D.J. (1996) Cationic derivatization of oligosaccharides with Girard's reagent for improved performance in matrix assisted laser desorption ionization and electrospray mass spectrometry. Rapid Commun. Mass Spectrom., 10, 829834.[CrossRef][ISI]
Nemansky, M., De Leeuw, R., Wijnands, R.A., and Van Den Eijnden, D.H. (1995) Enzymatic remodelling of the N- and O-linked carbohydrate of human chorionic gonadotropin. Effects on biological activity and receptor binding. Eur. J. Biochem., 227, 880888.[Abstract]
Olijve, W., De Boer, W., Mulders, J.W.M., and van Wezenbeek, P.M.G.F. (1996) Molecular biology and biochemistry of human recombinant follicle stimulating hormone (Puregon). Mol. Human Reprod., 2(5), 371382.[Abstract]
Patel, T., Bruce, J., Merry, A., Bigge, C., Wormald, M., Jaques, A., and Parekh, R. (1993) Use of hydrazine to release in intact and unreduced form both N- and O-linked oligosaccharides from glycoproteins. Biochemistry, 32, 679693.[ISI][Medline]
Pierce, J.G. and Parsons, T.F. (1981) Glycoprotein hormones: structure and functions. Annu. Rev. Biochem., 50, 465495.[CrossRef][ISI][Medline]
Piller, F. and Piller, V. (1993) Structural characterization of mucin-type O-linked oligosaccharides. In Fukuda, M. and Kobata, A. (eds), Glycobiology. The practical approach series. Oxford University Press, Oxford, UK, pp. 298299.
Rademaker, G.J., Pergantis, S.A., Blok-Tip, L., Langridge, T.I., Kleen, A., and Thomas-Oates, J.E. (1998) Mass spectrometry determination of the sites of O-glycan attachment with low picomolar sensitivity. Anal. Biochem., 257, 149160.[CrossRef][ISI][Medline]
Ryan, R.J., Keutmann, H.T., Charlesworth, M.C., McCormick, D.J., Milius, R.P., Calvo, F.O., and Vutyavanich, T. (1987) Structurefunction relationship of gonadotrophins. Rec. Prog. Horm. Res., 43, 383429.[ISI][Medline]
Sairam, M.R. (1983) Gonadotropic hormones: relationship between structure and function with emphasis on antagonists. In Li, C.H. (ed.), Hormonal Proteins and Peptides, vol. 11. Academic Press, New York, pp. 179.
Steelman, S.L. and Pohley, F.M. (1953) Assay of the follicle stimulating hormone based on the augmentation with human chorionic gonadotropin. Endocrinology, 53, 604616.[ISI]
Tarentino, A.L., Gomez, C.M., and Plummer, T.H. Jr. (1985) Deglycosylation of asparagine-linked glycans by peptide N-glycosidase F. Biochemistry, 24, 46654671.[ISI][Medline]
Thotakura, N.R. and Blithe, D.L. (1995) Glycoprotein hormones: glycobiology of gonadotrophins, thyrotrophin and free subunit. Glycobiology, 5, 310.[Abstract]
Varki, A. (1993) Biological roles of oligosaccharides: all theories are correct. Glycobiology, 3, 97130.[Abstract]
Viseux, N., Hronowski, X., Delaney, J., and Domon, B. (2001) Qualitative and quantitative analysis of the glycosylation pattern of recombinant proteins. Anal. Chem., 73, 47554762.[CrossRef][ISI][Medline]
Vitt, U.A., Kloosterboer, H.J., Rose, U.M., Mulders, J.W., Kiesel, P.S., Bete, S., and Nayudu, P.L. (1998) Isoforms of recombinant follicle-stimulating hormone: comparison of effects on murine follicular development in vitro. Biol. Reprod., 59, 854861.
Weiskopf, A.S., Vouros, P., and Harvey, D.J. (1997) Characterization of oligosaccharide composition and structure by quadrupole ion trap mass spectrometry. Rapid Commun. Mass Spectrom., 11, 14931504.[CrossRef][ISI][Medline]
Xu, N., Huang, Z.H., Watson, J.T., and Gage, D.A. (1997) Mercaptobenzothiazoles: a new class of matrices for laser desorption ionisation mass spectrometry. J. Am. Soc. Mass Spectrom., 8, 116.[CrossRef][ISI]
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