Effect of ammonia on the glycosylation of human recombinant erythropoietin in culture

Recombinant human erythropoietin (EPO) was produced by a stable transfected CHO-K1 cell clone (EPO-81) grown in serum-free medium. Our previous work showed that there was a significant increase in the heterogeneity of the glycoforms of EPO and a reduction of the sialylation at 20 mM NH(4)Cl. In the work presented here, the effects of ammonia on EPO N-linked oligosaccharides were analyzed. EPO was purified from culture supernatants by immunoaffinity chromatography. The N-linked oligosaccharides were released enzymatically and analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and HPLC. The FACE N-linked oligosaccharide profile showed that the sialylated glycans contain one prominent band at a position corresponding to eight glucose units. The density of the major band was greatly diminished and the width was significantly increased in cultures containing added ammonia. The proportion of tetraantennary structures was reduced by 60%, while the tri- and biantennary structures were increased proportionally in the presence of ammonia. Glycan analysis by HPLC using a weak anion exchange column showed that the most significant characteristic effect of ammonia was a reduction of the proportion of glycans with four sialic acids from 46% in control cultures to 29% in ammonia-treated cultures. Analysis of the desialylated glycans by normal phase chromatography indicated a distribution of tetra-, tri-, and biantennary structures similar to that shown by FACE. The N-linked glycan sequence was determined by sequential exoglycosidase digestion followed by FACE. The results indicated a typical N-linked complex oligosaccharide structure. Glycans from ammonia-containing cultures showed the same sequence pattern. In conclusion, we showed that ammonia in the culture medium affected EPO glycosylation, which was observed as a reduction of the tetraantennary and tetrasialylated oligosaccharide structures. However, the presence of ammonia in the cultures did not change the oligosaccharide sequence.     

N-linked oligosaccharide structures in the diamine oxidase from porcine kidney

Structures of the N-linked glycans released from porcine kidney diamine oxidase (DAO) were characterized utilizing various analytical techniques, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), high-performance capillary electrophoresis (HPCE), and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The oligosaccharide sequences present in DAO were conclusively determined using specific exoglycosidases in conjunction with MALDI/TOF-MS. The structures found in the glycoprotein are primarily linear, di-, or tribranched fucosylated complex type. MS analysis of the esterified N-glycan pool derived from DAO indicated the presence of several di- and trisialylated structures.     

Tools for glycomics: relative quantitation of glycans by isotopic permethylation using 13CH3I

Analysis of oligosaccharides by mass spectrometry (MS) has enabled the investigation of the glycan repertoire of organisms with high resolution and sensitivity. It is difficult, however, to correlate the expression of glycosyltransferases with the glycan structures present in a particular cell type or tissue because the use of MS for quantitative purposes has significant limitations. For this reason, in order to develop a technique that would allow relative glycan quantification by MS analysis between two samples, a procedure was developed for the isotopic labeling of oligosaccharides with (13)C-labeled methyl iodide using standard permethylation conditions. Separate aliquots of oligosaccharides from human milk were labeled with (12)C or (13)C methyl iodide; the labeled and non-labeled glycans were mixed in known proportions, and the mixtures analyzed by MS. Results indicated that the isotopic labeling described here was capable of providing relative quantitative data with a dynamic range of at least two orders of magnitude, adequate linearity, and reproducibility with a coefficient of variation that was 13% on average. This procedure was used to analyze N-linked glycans released from various mixtures of glycoproteins, such as alpha-1 acid glycoprotein, human transferrin, and bovine fetuin, using MS techniques that included matrix assisted laser desorption ionization-time of flight MS and electrospray ionization with ion cyclotron resonance-Fourier transformation MS. The measured (12)C:(13)C ratios from mixtures of glycans permethylated with either (12)CH(3)I or (13)CH(3)I were consistent with the theoretical proportions. This technique is an effective procedure for relative quantitative glycan analysis by MS.     

An analytical system for the characterization of highly heterogeneous mixtures of N-linked oligosaccharides

A novel system for characterizing complex N-linked oligosaccharide mixtures that uses a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), capillary electrophoresis (CE), and high-performance liquid chromatography (HPLC) has been developed. In this study, oligosaccharides released from recombinant TNK-tPA (tissue plasminogen activator) were derivatized with 5-amino-2-naphthalenesulfonic acid (ANSA). The negative charge imparted by the ANSA label facilitated the analysis of the oligosaccharides by MALDI-TOF MS by allowing the observation of both neutral and sialylated oligosaccharides in a single negative ion mode spectrum. Labeling with ANSA was also determined to be advantageous in the characterization of oligosaccharides by both HPLC and CE. The ANSA label was demonstrated to provide superior resolution over the commonly used label 8-aminopyrene-1,3,6-trisulfonic acid (APTS) in both the CE and HPLC analysis of oligosaccharides. To date, no other labels that enable the analysis of complex oligosaccharide mixtures in a single mass spectral mode, while also enabling high-resolution chromatographic and electrophoretic separation of the oligosaccharides, have been reported. By integrating the structural information obtained by MALDI-TOF MS analysis with the ability of CE and HPLC to discriminate between structural isomers, the complete characterization of complex oligosaccharide mixtures is possible.     

Analysis of N-glycosylation of phospholipase A2 from venom of individual bees by microbore high-performance liquid chromatography-electrospray mass spectrometry using an ion trap mass spectrometer

The N-linked oligosaccharides were released from the phospholipase A2 (PLA) with glycopeptidases and reductively aminated with the chromophore, p-aminobenzoic acid ethyl ester (ABEE). The ABEE-labeled oligosaccharides were separated by microbore high-performance liquid chromatography (micro-HPLC) using a reversed-phase column and analyzed by electrospray mass spectrometry. Differentiation between alpha-1,3 and alpha-1,6 core-fucosylated glycans was achieved by comparison the glycans released by glycopeptidases peptide-N-glycanase A (PNase A) and peptide-N-glycanase F (PNase F). All N-linked oligosaccharides except 3B and 3C could be identified in this approach. The analysis of PLA oligosaccharides from the venom of individual bees indicated that glycosylation patterns between the younger and the older bees were similar.     

Capillary electrophoresis-electrospray ionization mass spectrometry for rapid and sensitive N-glycan analysis of glycoproteins as 9-fluorenylmethyl derivatives

It is well known that most protein therapeutics such as monoclonal antibody pharmaceuticals and other biopharmaceuticals including cancer biomarkers are glycoproteins, and thus the development of high-throughput and sensitive analytical methods for glycans is essential in terms of their determination and quality control. We previously reported a novel alternative labeling method for glycans involving 9-fluorenylmethyl chloroformate (Fmoc-Cl) instead of the conventional reductive amination procedure. The derivatives were analyzed by high-performance liquid chromatography (HPLC) (Kamoda S, Nakano M, Ishikawa R, Suzuki S, Kakehi K. 2005. Rapid and sensitive screening of N-glycans as 9-fluorenylmethyl derivatives by high-performance liquid chromatography: A method which can recover free oligosaccharides after analysis. J Proteome Res. 4:146-152). This method was rapid and simple; however, it was time-consuming in terms of analysis by HPLC and did not provide so much information such as the detailed structures and mass numbers of glycans. Here we have developed a high-throughput and highly sensitive method. It comprises three steps, i.e., release of glycans, derivatization with Fmoc, and capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI MS) analysis. We analyzed several glycoproteins such as fetuin, alpha1 acid glycoprotein, IgG, and transferrin in order to validate this method. We were able to analyze the above glycoproteins with the three-step procedure within only 5 h, which provided detailed N-glycan patterns. Moreover, the MS/MS analysis allowed identification of the N-glycan structures. As novel applications, the method was employed for the analysis of N-glycans derived from monoclonal antibody pharmaceuticals and also from alpha-fetoprotein; the latter is known as one of the tumor markers of hepatocellular carcinomas. We were able to easily and rapidly determine the detailed structures of the N-glycans. The present method is very useful for the analysis of large numbers of samples such as a routine analysis.     

A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

This report describes a convenient method for the rapid and efficient release of N-linked oligosaccharides from low microgram amounts of glycoproteins. A 96-well MultiScreen assay system containing a polyvinylidene difluoride (PVDF) membrane is employed to immobilize glycoproteins for subsequent enzymatic deglycosylation. Recombinant tissue-type plasminogen activator (rt-PA) is used to demonstrate the deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled the recovery of a sufficient amount of N-linked oligosaccharides released enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5 microgram rt-PA for subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry. The immobilization of rt-PA to the PVDF membrane did not sterically inhibit the PNGaseF-mediated release of oligosaccharides from rt-PA as determined by tryptic mapping experiments. Comparison of the oligosaccharides released from 50 micrograms of rt-PA by either the 96-well plate method or by a standard solution digestion procedure showed no significant differences in the profiles obtained by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both neutral and sialylated oligosaccharide standards spiked into wells were recovered equally as determined by HPAEC-PAD. One advantage of this approach is that reduction and alkylation can be performed on submicrogram amounts of glycoproteins with easy removal of reagents prior to PNGaseF digestion. In addition, this method allows 60 glycoprotein samples to be deglycosylated in 1 day with MALDI-TOF or HPAEC-PAD analysis being performed on the following day.      

Application of liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin

High-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) and liquid chromatography with tandem mass spectrometry (LC/MS/MS) were applied to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin (EPO) used as a model of the sialylated glycoprotein. N-linked oligosaccharides were released from recombinant human EPO expressed in Chinese hamster ovary cells enzymatically and reduced with NaBH(4). Many different sialylated oligosaccharides of EPO were separated and characterized by LC/MS equipped with a graphitized carbon column (GCC). Glycosylation sites and the preliminary glycosylation pattern at each glycosylation site were determined by LC/MS of endoproteinase Glu-C-digested EPO. The detailed site-specific carbohydrate heterogeneity caused by the differences in the molecular weight, branch, linkage, and sequence was elucidated by GCC-LC/MS of the N-linked oligosaccharides released from the isolated glycopeptides. Structural details of the isomers were analyzed by LC/MS/MS, and it was indicated that di- and trisialylated tetraantennary oligosaccharides are attached to Asn24, 38, and 83, whereas their isomers, di- and trisialylated triantennary oligosaccharides containing N-acetyllactosamines, are combined with Asn24. Our method is useful for the determination of glycosylation sites, the site-specific carbohydrate heterogeneity of glycoproteins, and the carbohydrate structure.     

A relative and absolute quantification of neutral N-linked oligosaccharides using modification with carboxymethyl trimethylammonium hydrazide and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Quantification of oligosaccharides is of great importance to investigate variations or changes in the glycans of glycoconjugates. Mass spectrometry (MS) has been widely applied to identification and structural analysis of complex oligosaccharides. However, quantification using MS alone is still quite challenging due to heterogeneous charge states and different ionization efficiency of various types of oligosaccharides. To overcome such shortcomings, derivatization with carboxymethyl trimethylammonium hydrazide (Girard’s reagent T [GT]) was introduced to generate a permanent cationic charge at the reducing end of neutral oligosaccharides, resulting in mainly [M]⁺ ion using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), so that the ambiguities caused by metal adduct peaks such as [M+K]⁺ and [M + Na]⁺ were avoided. To verify our method, the relative and absolute quantification of neutral glycans from human immunoglobulin G (IgG) and ovalbumin with internal standards of dextran ladders using MALDI-TOF MS were compared with those performed by conventional normal-phase high-performance liquid chromatography (NP-HPLC) profiling. The quantification using GT derivatization and MALDI-TOF MS agreed well with the HPLC profiling data and showed excellent reliability and reproducibility with better resolution and sensitivity. This method was further applied to quantify the enzymatically desialylated N-glycans from miniature pig kidney membrane proteins. The results showed that the low-abundance structures that could not be resolved by NP-HPLC were quantified with high sensitivity. Thus, this novel method of using modification of neutral sugars with GT is quite powerful for neutral glycan analysis, especially to quantify minute glycan samples with undetectable levels using HPLC.     

Analysis of folate binding protein N-linked glycans by mass spectrometry

The folate binding protein (FBP), also known as the folate receptor (FR), is a glycoprotein which binds the vitamin folic acid and its analogues. FBP contains multiple N-glycosilation sites, is selectively expressed in tissues and body fluids, and mediates targeted therapies in cancer and inflammatory diseases. Much remains to be understood about the structure, composition, and the tissue specificities of N-glycans bound to FBP. Here, we performed structural characterization of N-linked glycans originating from bovine and human milk FBPs. The N-linked glycans were enzymatically released from FBPs, purified, and permethylated. Native and permethylated glycans were further analyzed by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS), while tandem MS (MS/MS) was used for their structural characterization. The assignment of putative glycan structures from MS and MS/MS data was achieved using Functional Glycomics glycan database and SimGlycan software, respectively. It was found that FBP from human milk contains putative structures that have composition consistent with high-mannose (Hex(5-6)HexNAc(2)) as well as hybrid and complex N-linked glycans (NeuAc(0-1)Fuc(0-3)Hex(3-6)HexNAc(3-5)). The FBP from bovine milk contains putative structures corresponding to high-mannose (Hex(4-9)HexNAc(2)) as well as hybrid and complex N-linked glycans (Hex(3-6)HexNAc(3-6)), but these glycans mostly do not contain fucose and sialic acid. Glycomic characterization of FBP provides valuable insight into the structure of this pharmacologically important glycoprotein and may have utility in tissue-selective drug targeting and as a biomarker.     

The way forward, enhanced characterization of therapeutic antibody glycosylation: comparison of three level mass spectrometry-based strategies

Glycosylation which plays a crucial role in the pharmacological properties of therapeutic monoclonal antibodies (MAbs) is influenced by several factors like production systems, selected clonal population and manufacturing processes. Efficient analytical methods are therefore required in order to characterize glycosylation at different stages of MAbs discovery and production. Three mass spectrometry (MS)-based strategies were compared to analyze N-glycosylation of MAbs either expressed in murine myeloma (NS0) or Chinese Hamster Ovary (CHO) cell lines, the two current main production systems used for therapeutic MAbs. First a top-down approach was used on intact and reduced MAbs by liquid chromatography coupled to an electrospray ionization-time of flight mass spectrometer (LC-ESI-TOF), which provided fast and accurate profiles of MAbs glycosylation patterns for routine controls. Secondly, after digestion of the antibody with the peptide N-glycosidase F (PNGase F) enzyme, released N-linked glycans were directly analyzed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) without any prior derivatization, which gave precise details on the structure of the most abundant glycoforms. Finally, a bottom-up approach on tryptic glycopeptides using a nanoLC-Chip-MS/MS ion trap (IT) system equipped with a graphitized carbon column was investigated. Data were compared to those obtained with a more classical C18 reversed phase column showing that this last method is well suited to detect low abundant glycoforms and to provide in one shot information regarding both the oligosaccharide structure and the amino acid sequence of its peptide moiety.     

Glycan profiling of monoclonal antibodies using zwitterionic-type hydrophilic interaction chromatography coupled with electrospray ionization mass spectrometry detection

We present a new method for the analysis of glycans enzymatically released from monoclonal antibodies (MAbs) employing a zwitterionic-type hydrophilic interaction chromatography (ZIC–HILIC) column coupled with electrospray ionization mass spectrometry (ESI–MS). Both native and reduced glycans were analyzed, and the developed procedure was compared with a standard HILIC procedure used in the pharmaceutical industry whereby fluorescent-labeled glycans are analyzed using a TSK Amide-80 column coupled with fluorescence detection. The separation of isobaric alditol oligosaccharides present in monoclonal antibodies and ribonuclease B is demonstrated, and ZIC–HILIC is shown to have good capability for structural recognition. Glycan profiles obtained with the ZIC–HILIC column and ESI–MS provided detailed information on MAb glycosylation, including identification of some less abundant glycan species, and are consistent with the profiles generated with the standard procedure. This new ZIC–HILIC method offers a simpler and faster approach for glycosylation analysis of therapeutic antibodies.     

Labelling saccharides with phenylhydrazine for electrospray and matrix-assisted laser desorption-ionization mass spectrometry

A well-known reaction of carbonyl compounds with phenylhydrazine has been applied to saccharides, providing increased sensitivity for mass spectrometric (MS) and ultraviolet (UV) detection during high-performance liquid chromatographic (HPLC) separations. After a simple derivatization procedure for 1 h at 70 degrees C and purification of the reaction mixture from excess reagent by extraction, the sugar derivatives were characterized by direct injection or on-line HPLC/electrospray ionization (ESI) and by matrix-assisted laser desorption/ionization (MALDI) MS. Because no salts are used or produced upon reaction, this procedure is very simple and suitable for the tagging of saccharides. The reaction allows for on-target derivatization and products are very stable. The derivatization procedure has been applied to commercially-obtained small saccharides and standard N-linked oligosaccharides. Lastly, hen ovalbumin N-glycans were detached enzymatically and characterized by MALDI-MS as their phenylhydrazone derivatives.     

Plant-derived mouse IgG monoclonal antibody fused to KDEL endoplasmic reticulum-retention signal is N-glycosylated homogeneously throughout the plant with mostly high-mannose-type N-glycans

Plants are potential hosts for the expression of recombinant glycoproteins intended for therapeutic purposes. However, N-glycans of mammalian glycoproteins produced in transgenic plants differ from their natural counterparts. The use of the endoplasmic reticulum (ER)-retention signal has been proposed to restrict glycosylation of plantibodies to only high-mannose-type N-glycans. Furthermore, little is known about the influence of plant development and growth conditions on N-linked glycosylation. Here, we report a detailed N-glycosylation profiling study of CB.Hep1, a mouse IgG2b monoclonal antibody (mAb) against hepatitis B surface antigen (HBsAg) currently expressed in tobacco plants (Nicotiana tabacum L.). The KDEL ER-retention signal was fused to the C-terminal of both light and heavy chains. The structures of the N-linked glycans of this mAb produced in transgenic tobacco plants at various growth stages were analysed by high-performance liquid chromatography (HPLC) profiling techniques and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and compared with those of murine origin. The high-mannose-type oligosaccharides accounted for more than 80% of the total N-glycans, with Man7GlcNAc2 being the most abundant species. Some complex N-glycans bearing xylose and small amounts of oligosaccharides with both xylose and fucose were identified. No appreciable differences were detected when comparing glycosylation at different leaf ages, e.g. from seedling leaves up to 8 weeks old and top or basal leaves of mature plants, or between leaves, stems and whole plants. A strict retention of glycoproteins to ER by the use of the tetrapeptide KDEL was not sufficient, even though the majority of the resulting N-glycosylation was of the high-mannose type. It is highly likely to be dependent on other factors, which are most probably protein specific.     

Impact of variable domain glycosylation on antibody clearance: an LC/MS characterization

Variable (Fv) domain N-glycosylation sites are found in approximately 20% of human immunoglobulin Gs (IgGs) in addition to the conserved N-glycosylation sites in the C(H)2 domains. The carbohydrate structures of the Fv glycans and their impact on in vivo half-life are not well characterized. Oligosaccharide structures in a humanized anti-Abeta IgG1 monoclonal antibody (Mab) with an N-glycosylation site in the complementary determining region (CDR2) of the heavy chain variable region were elucidated by LC/MS analysis following sequential exoglycosidase treatments of the endoproteinase Lys-C digest. Results showed that the major N-linked oligosaccharide structures in the Fv region have three characteristics (core-fucosylated biantennary oligosaccharides with one or two N-glycolylneuraminic acid [NeuGc] residues, zero or one alpha-linked Gal residue, and zero or one beta-linked GalNAc residue), whereas N-linked oligosaccharides in the Fc region contained typical Fc glycans (core-fucosylated, biantennary oligosaccharides with zero to two Gal residues). To elucidate the contribution of Fv glycans to the half-life of the antibody, a method that allows capture of the Mab and determination of its glycan structures at various time points after administration to mice was developed. Anti-Abeta antibody in mouse serum was immunocaptured by immobilized goat anti-human immunoglobulin Fc(gamma) antibody resin, and the captured material was treated with papain to generate Fab and Fc for LC/MS analysis. Different glycans in the Fc region showed the same clearance rate as demonstrated previously. In contrast to many other non-antibody glycosylated therapeutics, there is no strong correlation between oligosaccharide structures in the Fv region and their clearance rates in vivo. Our data indicated that biantennary oligosaccharides lacking galactosylation had slightly faster clearance rates than other structures in the Fv domain.     

Investigation of cationic peanut peroxidase glycans by electrospray ionization mass spectrometry

Cationic peanut peroxidase (CP) was isolated from peanut (Arachis hypogaea) cell suspension culture medium. CP is a glycoprotein with three N-linked glycan sites at Asn60, Asn144, and Asn185. ESI-MS of the intact purified protein reveals the microheterogeneity of the glycans. Tryptic digestion of CP gave a near complete sequence coverage by ESI-MS. The glycopeptides from the tryptic digestion were separated by RP HPLC identified by ESI-MS and the structure of the glycan chains determined by ESI-MS/MS. The glycans are large structures of up to 16 sugars, but most of their non-reducing ends have been modified giving a mixture of shorter chains at each site. Good agreement was found with the one glycan previously analyzed by (1)H NMR. This work is the basis for the future studies on the role of the glycans on stability and folding of CP and is another example of a detailed structural characterization of complex glycoproteins by mass spectrometry.     

Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion

Cetuximab is a novel therapeutic monoclonal antibody with two N-glycosylation sites: a conserved site in the CH2 domain and a second site within the framework 3 of the variable portion of the heavy chain. The detailed structures of these oligosaccharides were successfully characterized using orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight mass spectrometry (oMALDI Qq-TOF MS) and tandem mass spectrometry (MS/MS) in combination with exoglycosidase digestion. The N-linked oligosaccharides were released by treatment with N-glycanase F, reductively aminated with anthranilic acid, and fractionated by normal phase high-performance liquid chromatography (NP-HPLC). The fluorescent-labeled oligosaccharide pool and fractions were analyzed by oMALDI Qq-TOF MS and MS/MS in negative ion mode. Each fraction was further digested with an array of exoglycosidase mixtures, and subsequent MALDI TOF MS analysis of the resulting products yielded information about structural features of the oligosaccharide. The combined data revealed the presence of 21 distinct oligosaccharide structures in cetuximab. These oligosaccharides differ mainly in degree of sialylation with N-glycolyl neuraminic acid and extent of galactosylation (zero-, mono-, di-, and alpha(1-3)-galactosidase). The individual oligosaccharides were further assigned to the specific sites in the Fab and Fc regions of the antibody. This study represents a unique approach in that MS/MS data were used to identify and confirm the oligosaccharide structures of a protein.     

Chemical and enzymatic N-glycan release comparison for N-glycan profiling of monoclonal antibodies expressed in plants

Plants synthesize N-glycans containing the antigenic sugars α(1,3)-fucose and β(1,2)-xylose. Therefore it is important to monitor these N-glycans in monoclonal antibodies produced in plants (plantibodies). We evaluated several techniques to characterize the N-glycosylation of a plantibody produced in tobacco plants with and without the KDEL tetrapeptide endoplasmic reticulum retention signal which should inhibit or drastically reduce the addition of α(1,3)-fucose and β(1,2)-xylose. Ammonium hydroxide/carbonate-based chemical deglycosylation and PNGase A enzymatic release were investigated giving similar 2-aminobenzamide-labeled N-glycan HPLC profiles. The chemical release does not generate peptides which is convenient for MS analysis of unlabeled pool but its main drawback is that it induces degradation of α1,3-fucosylated N-glycan reducing terminal sugar. Three analytical methods for N-glycan characterization were evaluated: (i) MALDI-MS of glycopeptides from tryptic digestion; (ii) negative-ion ESI-MS/MS of released N-glycans; (iii) normal-phase HPLC of fluorescently labeled glycans in combination with exoglycosidase sequencing. The MS methods identified the major glycans, but the HPLC method was best for identification and relative quantitation of N-glycans. Negative-mode ESI-MS/MS permitted also the correct identification of the linkage position of the fucose residue linked to the inner core N-acteylglucosamine (GlcNAc) in complex N-glycans.     

Comparisons of the glycosylation of a monoclonal antibody produced under nominally identical cell culture conditions in two different bioreactors

The murine B-lymphocyte hybridoma cell line, CC9C10, was grown in serum-free continuous culture at steady-state dissolved oxygen (DO) concentrations of 10%, 50%, and 100% of air saturation in both LH Series 210 (LH) and New Brunswick Scientific (NBS) CelliGen bioreactors. All culture parameters were monitored and controlled and were nominally identical at steady state in the two bioreactors. The secreted monoclonal antibody (mAb), an immunoglobulin G(1), was purified and subjected to enzymatic deglycosylation using peptide N-glycosidase F (PNGase F). Asparagine-linked (N-linked) oligosaccharide pools released from mAb samples cultured in each bioreactor at each of the three DO setpoints were analyzed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The predominant N-linked structures were core-fucosylated asialo biantennary chains with varying galactosylation. There were also minor amounts of monosialyl oligosaccharides and trace amounts of afucosyl oligosaccharides. The level of DO affects the glycosylation of this mAb. A definite reduction in the level of galactosylation of N-glycan chains was observed at lower DO in both bioreactors, as evidenced by prominent increases in the relative amounts of agalactosyl chains and decreases in the relative amounts of digalactosyl chains-with the relative amounts of monogalactosyl chains being comparatively constant. However, the quantitative results are not precise matches between the two bioreactors. The effect of DO on galactosylation is less pronounced in the NBS bioreactor than in the LH bioreactor, particularly the shift between the relative amounts of agalactosyl and digalactosyl chains in 10% and 50% DO. There are also perceptibly higher levels of sialylation of the mAb glycans in the NBS bioreactor than in the LH bioreactor at all three DO setpoints. The results indicate that the DO effect is not bioreactor specific and that nominally identical steady-state conditions in different chemostat bioreactors may still lead to some incongruities in glycosylation, possibly due to the particular architectures of the bioreactors and the design of their respective monitoring and control systems. The observed differences in N-linked glycosylation of the mAb secreted by the hybridoma grown in the LH and NBS bioreactors may be explained by the differences in oxygen supply and control strategies between the two bioreactors.     

Structure determination by MALDI-IRMPD mass spectrometry and exoglycosidase digestions of O-linked oligosaccharides from Xenopus borealis egg jelly

Differences in the fertilization behavior of Xenopus borealis from X. laevis and X. tropicalis suggest differences in the glycosylation of the egg jellies. To test this assumption, O-linked glycans were chemically released from the egg jelly coat glycoproteins of X. borealis. Over 50 major neutral glycans were observed, and no anionic glycans were detected from the released O-glycan pool. Preliminary structures of ～30 neutral oligosaccharides were determined using matrix-assisted laser desorption/ionization (MALDI) infrared multiphoton dissociation tandem mass spectrometry (MS). The mass fingerprint of a group of peaks for the core-2 structure of O-glycans was conserved in the tandem mass spectra and was instrumental in rapid and efficient structure determination. Among the 29 O-glycans, 22 glycans contain the typical core-2 structure, 3 glycans have the core-1 structure and 2 glycans contained a previously unobserved core structure with hexose at the reducing end. There were seven pairs of structural isomers observed in the major O-linked oligosaccharides. To further elucidate the structures of a dozen O-linked glycans, specific and targeted exoglycosidase digestions were carried out and the products were monitored with MALDI-MS. Reported here are the elucidated structures of O-linked oligosaccharides from glycoproteins of X. borealis egg jelly coats. The structural differences in O-glycans from jelly coats of X. borealis and its close relatives may provide a better understanding of the structure-function relationships and the role of glycans in the fertilization process within Xenopodinae.