Glycosylation of human recombinant gonadotrophins: characterization and batch-to-batch consistency

Annick Gervais1,2, Yves-alexis Hammel2, Sophie Pelloux2, Pierre Lepage2, Gianni Baer2, Nathalie Carte3, Odile Sorokine3, Jean-marc Strub3, Roman Koerner3, Emmanuelle Leize3 and Alain Van Dorsselaer3

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


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Conclusions
 Materials and methods
 References
 
The glycan moiety of human recombinant gonadotrophins (r-hFSH, r-hLH, and r-hCG) produced in CHO cell lines has been characterized by a combination of chromatographic and mass spectrometric techniques, including both matrix-assisted laser desorption ionization and electrospray. Two glycan mapping methods have been developed for the three gonadotrophins that allow separation of the glycans according to either their charge or sialylation level or their antennarity. A method was also developed for r-hCG that permits the complete resolution of the N-glycan from the O-glycan species. Whereas the structure found for the N-glycans of the gonadotrophins was in agreement with the complex type model, the structure for an O-glycan of r-hCG, not yet described, has been unambiguously determined using nanoelectrospray ion trap mass spectrometry. Using these two glycan mapping methods, the high level of batch-to-batch consistency achieved for the glycosylation of the three recombinant gonadotrophins in commercial production has been shown. These data demonstrate the tight control that can be achieved in the manufacturing of complex recombinant therapeutic glycoproteins, which is a prerequisite to the delivering of a guaranteed dose of drug from vial to vial, and in turn to ensuring the clinical efficacy of the product.

Key words: batch-to-batch consistency / glycoprotein / mass spectrometry / N-glycan structure / O-glycan structure


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Conclusions
 Materials and methods
 References
 
Gonadotrophins are a family of four closely related glycoproteins comprising follicle stimulating hormone (FSH), luteinizing hormone (LH), chorionic gonadotrophin (CG), and thyroid-stimulating hormone. FSH, LH, and CG are involved in the regulation of reproductive functions of the organism. They are composed of two noncovalently bound {alpha}- and ß-subunits. Both subunits are glycosylated. Within an animal species, the {alpha}-subunit is common to the four gonadotrophins, whereas the ß-subunit is different and confers to the hormone its biological specificity (Pierce and Parsons, 1981Go). The {alpha}-subunit of the three hormones has two N-glycosylation sites. The ß-subunit of FSH and CG has also two N-glycosylation sites, whereas the ß-subunit of LH has only one N-glycosylation site. The ß-subunit of CG has four supplementary O-glycosylation sites.

The oligosaccharide moieties of gonadotrophins have already been extensively studied (Pierce and Parsons, 1981Go; Sairam, 1983Go; Ryan et al., 1987Go; Baenziger and Green, 1988Go; Hard et al., 1990Go; Kobata, 1992Go; Olijve et al., 1996Go; Amoresano et al., 1996Go; Liu and Bowers, 1997Go; Jacoby et al., 2000Go). 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., 1996Go; Liu and Bowers, 1997Go). 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, 1995Go; Varki, 1993Go; Calvo et al., 1986Go; Cumming, 1991Go; Mulders et al., 1997Go). However, the relationship between charge and in vivo bioactivity is complex (D'Antonio et al., 1999Go; Horsman et al., 2000Go), the most sialic acid-rich glycoforms having a prolonged plasma half-life (Thotakura and Blithe, 1995Go; Varki, 1993Go; Calvo et al., 1986Go; Cumming, 1991Go; Mulders et al., 1997Go) and the less acidic species having in vitro a greater receptor binding affinity, as shown in a recent study (Vitt et al., 1998Go). 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, 1991Go; Hermentin et al., 1996Go; Hermentin and Witzel, 1999Go; Viseux et al., 2001Go). 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., 1996Go; Hermentin and Witzel, 1999Go) 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.


    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Conclusions
 Materials and methods
 References
 
There are different possible strategies to map the oligosaccharides of a glycoprotein (Harvey, 1996Go, 2001Go): analysis of the entire glycoprotein by MS, digestion of the glycoprotein into glycopeptides by endoproteases, or release of the glycans from the peptide part. Due to the high heterogeneity of gonadotrophins, the analysis of the entire glycoprotein by MS is very difficult. In the case of beta-chain of r-hFSH (Figure 1) and of r-hCG, it is impossible without any reduction of the heterogeneity. The digestion of the peptide part into glycopeptides gives information on the occupancy of the glycosylation sites, but the digestion of the peptide part into monoglycopeptides can be difficult depending on the amino acid sequence of the molecule. Therefore the strategy involving the release of the glycans has been selected. N-glycans can be released chemically by hydrazinolysis (Patel et al., 1993Go) or enzymatically with PNGase F (Tarentino et al., 1985Go). O-glycans can only be recovered by hydrazinolysis because no O-glycanase can remove O-glycans without prior desialylation (Fukuda and Kobata, 1993) and chemical de-O-glycosylation is difficult (Rademaker et al., 1998Go).



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Fig. 1. MALDI-TOF mass spectrum of r-hFSH. Due to the heterogeneity of the molecule, the beta-chain could not be resolved by the instrument without any reduction of the heterogeneity linked to the glycans.

 
After release by hydrazinolysis, the intact and unreduced glycans were fluorescently labeled with 2-aminobenzamide (2-AB) in a nonselective manner as previously described (Bigge et al., 1995Go). Figure 2 gives the flow chart of the glycan mapping method applied to gonadotrophins. After 2-AB labeling, the glycans were either separated by anion exchange chromatography according to their charge, linked to the number of sialic acids they bear, or separated, after desialylation, by reverse-phase chromatography according to their antennarity. For both chromatographic separations, MS has been used as a tool to identify the glycan species eluted.



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Fig. 2. Flow chart of the glycan mapping method applied to gonadotrophins.

 
Separation of the N-glycans according to their charge
The N-glycans of r-hFSH and r-hLH were separated by anion exchange chromatography on a weak anion exchanger column. The eluted peaks (Figure 3 for r-hFSH) have been unambiguously identified by MALDI-time-of-flight (TOF) MS. Because very little fragmentation of oligosaccharides is observed with MALDI-TOF MS in the linear mode, it is assumed with confidence that each peak in the spectrum is due to a single component or to several isobaric components rather than to a fragment ion (Harvey, 1996Go, 2001Go). Moreover, because N-glycans of gonadotrophins have already been extensively studied and shown to be of complex type with only four types of monosaccharides—namely, hexose (mannose, galactose), deoxyhexose (fucose), N-acetylaminohexose (N-acetylgalactosamine, N-acetylglucosamine), and sialic acids—a mass measurement can lead directly to a proposed structure.



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Fig. 3. Separation of the N-glycans of r-hFSH by HPAEC. Evidences of differences in sialylation levels for two r-hFSH batches with different biospecific activities: (A) r-hFSH batch with a high biospecific activity; (B) r-hFSH batch with a low biospecific activity. Triangles=sialic acid; closed circles=galactose; closed squares=N-acetylgalactosamine; open squares=N-acetylglucosamine; open circles=mannose. The attribution of theglycan structures to the peaks is deduced from MALDI-TOF mass spectra.

 
All fractions of the chromatogram have been analyzed by MALDI-TOF MS to demonstrate that within a group of peaks, only glycans having the same charge, that is, the same number of sialic acids, were present. MALDI-TOF MS has also been applied to fractions eluted each 30 s on the entire chromatographic run to determine the elution order within a group of charged species (Figure 3). The interpretation of the results was complicated by the presence of artifactual peaks resulting from side reactions during the hydrazinolysis process (re-N-acetylation step) as previously described (Harvey, 1996Go, 2001Go; Naven and Harvey, 1996Go): additional (+42 or +84 Da) or missing (-42 or -84 Da) acetylations.

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., 1996Go) 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:

(1)

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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Fig. 4. Separation of the N-glycans of r-hFSH by reverse phase HPLC after desialylation. triangles=sialic acid; closed circles=galactose;closed squares=N-acetylgalactosamine; open squares=N-acetylglucosamine; open circles=mannose.

 
Hypothetical antennarity index A
A good separation between the different antennarity structures as well as the presence of a single fluorescent label on each glycan allow to define a number representing the overall degree of antennarity of the N-glycans of a given glycoprotein. This number, which we propose to name the antennarity index, A, is defined as the sum of the products of the respective areas (A) of the di-, tri-, tetra-, and pentaantennarity species, each multiplied by the corresponding antennae number:

(2)
The data collected so far have indicated that the A index is an accurate parameter allowing to characterize the antennarity level of both r-hFSH and r-hLH molecules, with a precision around 1%.

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)Go 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., 1995Go).


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Table I. Published O-glycan structures for r-hCG

 
Separation of the N-glycans according to their charge. The pool of hydrazinolyzed N- and O-glycans has been labeled with the 2-AB fluorophore and separated by anion exchange chromatography. The conditions developed for r-hFSH and r-hLH were optimized to separate O- from N-glycans. This separation was achieved on a CarboPac PA-100 column with 250 mM ammonium acetate, pH 4.5, as elution buffer (Figure 5).



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Fig. 5. Separation of the N- and O-glycans of r-hCG by HPAEC. Triangles=sialic acid; closed circles=galactose; closed squares=N-acetylgalactosamine; open squares=N-acetylglucosamine; open circles=mannose.

 
MALDI-TOF MS was applied to the fractions containing N-glycans with the same sialylation level and to the fractions containing O-glycans as well as to the fractions collected each 30 s on the chromatogram. This analysis demonstrated that N-glycans and O-glycans eluted separately, without overlap. This statement was also confirmed by comparing the chromatogram of the N- and O-glycans with the chromatogram of O-glycans after selective release by hydrazinolysis (O-mode, 65°C, 5 h) (data not shown).

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, 1996Go, 2001Go; Naven and Harvey, 1996Go) 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)Go 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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Table II. O-glycan structure of r-hCG deduced from molecular weight determination by MALDI-TOF mass spectra–comparison withpublished data

 
The hypothetical charge Z can be calculated both because N- and O-glycans elute under different peaks on the chromatogram and N-glycans elute as a function of their charge. This Z number takes only into account N-glycans because the sialylation of O-glycans is less important for the activity of the molecule (Nemansky et al., 1995Go).

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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Fig. 6. Separation of the N- and O-glycans of r-hCG by reverse phase HPLC after desialylation. triangles=sialic acid; closed circles=galactose; closed squares=N-acetylgalactosamine; open squares=N-acetylglucosamine; open circles=mannose.

 
There is a good separation between the different antennarity structures as well as no overlap between N- and O-glycan structures. Moreover, there is a single fluorescent label on each glycan. These characteristics allow definition of the antennarity index A for the N-glycans of r-hCG, as defined previously.

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{alpha}/5ß, Y5{alpha}/4ß, Y3{alpha}, Y according to Domon and Costello (1988)Go 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{alpha}/4ß, Y4{alpha}/3ß, contained the fucose, the fragmentation of Y3{alpha}/4ß, Y4{alpha}/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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Fig. 7. IT mass spectra of NA2F-2AB N-glycan. (A) MS2 on the doubly charged ion at m/z 965.3; (B) MS4 of fragment ion at m/z 1037.4.

 
This example of glycan structure elucidation demonstrated, as in previous studies (Bahr et al., 1997Go; Weiskopf et al., 1997Go; Creaser et al., 2002Go; Gennaro et al., 2002Go), the ability of the MSn by IT to fully characterize the structure of complex glycosylation and their labile groups, such as fucose.

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, 1997Go), 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, 1997Go) (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 Y, Y2{alpha} at m/z 793.3 (Table III), which revealed the presence of a sialic acid.


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Table III. Masses and interpretations of O-glycan fragments obtained with MS2, MS3 experiments

 
MS3 analysis of fragments at m/z 793.3 attributed to Y (structure 3b in Table III) and Y2{alpha} (structure 3c in Table III) (Figure 8A), was compared to the MS2 fragmentation of the O-glycan at m/z 793.3 (structure 1a) present in the first fraction (Figure 8B). These structures (structure 1a, 3b, 3c) had the same monosaccharide composition, but they differed by their branching. The fragmentations of these compounds allowed us to distinguish between the different O-glycans. Indeed, the MS3 fragment ion at m/z 631.1 attributed to Y1{alpha} elucidated the localization of the other sialic acid and confirmed the structure proposed for the O-glycan at m/z 1084.4.



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Fig. 8. IT mass spectra of O-glycans of r-hCG. (A) MS3 of fragment ions at m/z 793.3; (B) MS2 of parent ion at m/z 793.3.

 
Comparison of the three gonadotrophins
The three gonadotrophins r-hFSH, r-hLH, and r-hCG have been compared with respect to their N-glycosylation (Table IV). All three molecules bear complex type N-glycans with di-, tri-, tetra-, and minor pentaantennary species with various levels of sialylation. r-hFSH displays a larger heterogeneity in glycosylations with a higher proportion of tri- and tetraantennary species. All three molecules have both fucosylated and nonfucosylated species. Minor species lacking one hexose/hexosamine residue(s) have also been identified by MS in all three molecules.


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Table IV. N-glycans: comparison between the three gonadotrophins

 
The level of sialylation of r-hFSH is higher than for r-hLH and r-hCG with a mean hypothetical charge number Z of 184, versus 150 for both r-hLH and r-hCG. The antennarity index of r-hFSH is also higher than for r-hLH and r-hCG with an A index around 255, versus 216 and 228 for r-hCG and r-hLH, respectively.

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 (4–6% 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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Fig. 9. Batch-to-batch consistency monitoring of the sialylation of r-hFSH: Z number for more than 120 batches over 3 years of production.

 

    Conclusions
 Top
 Abstract
 Introduction
 Results and discussion
 Conclusions
 Materials and methods
 References
 
We have demonstrated that the glycosylation pattern of the three recombinant gonadotrophins r-hFSH, r-hLH, and r-hCG can be characterized using a number of related glycan mapping methods. This approach has been validated by combining MALDI MS and ESMS, allowing the structural identification of the different glycan species of N- or O-type.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results and discussion
 Conclusions
 Materials and methods
 References
 
Materials
Recombinant gonadotrophins (r-hFSH, r-hLH, and r-hCG) were produced by Serono (Switzerland). The chemicals used for the automated hydrazinolysis were purchased from Oxford GlycoSciences (Abingdon, UK). The 2-AB labeling kits, sialidase from Vibrio cholerae, GlycoSep C, and GlycoSep R columns were purchased from Oxford GlycoSciences or Glyko (Novato, CA). All reagents were purchased from Sigma (St. Louis, MO) or Fluka (Bucho, Switzerland).

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)Go.

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 ({lambda}excitation: 330 nm; {lambda}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 ({lambda}excitation: 330 nm; {lambda}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., 1997Go) 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 5x10–3 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 0.8–2.5 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 ({Delta}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)Go in vivo bioassay.

1 To whom correspondence should be addressed; e-mail: annick.gervais{at}serono.com Back


    Abbreviations
 
2-AB, 2-aminobenzamide; CG, chorionic gonadotrophin; CHO, Chinese hamster ovary; ES, electrospray; FSH, follicle stimulating hormone; HPAEC, high-performance anion exchange chromatography; HPLC, high-performance liquid chromatography; IT, ion trap; LH, luteinizing hormone; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; NA2F, neutral biantennary fucosylated complex type glycan; r-h, recombinant human.


    References
 Top
 Abstract
 Introduction
 Results and discussion
 Conclusions
 Materials and methods
 References
 
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