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PubMed2002

Simultaneous microanalysis of N-linked oligosaccharides in a glycoprotein using microbore graphitized carbon column liquid chromatography-mass spectrometry.

We previously reported that graphitized carbon column liquid chromatography-mass spectrometry (GCC-LC-MS) is very useful for the structural analysis of carbohydrates in a glycoprotein. In this study, GCC-LC-MS was adapted for the simultaneous microanalysis of oligosaccharides. A variety of oligosaccharide alditols prepared from fetuin, ribonuclease B, and recombinant human erythropoietin were used as model oligosaccharides. The use of microbore GCC-LC-MS was found to be successful for rapid, sensitive, and simultaneous analysis of high-mannose-type, desialylated fucosyl complex-type, sialylated complex-type, and sialylated fucosyl complex-type oligosaccharide alditols. Furthermore, we demonstrate that this method is applicable to the analysis of carbohydrate heterogeneity in a glycoprotein that possesses diverse oligosaccharides. Microbore GCC-LC-MS was able to characterize high-mannose-type, hybrid-type, and complex-type oligosaccharides in tissue plasminogen activator produced from human melanoma cells in a single analysis.

Carbohydrate Sequence↗
PubMed1999

Liquid chromatography-electrospray mass spectrometry as a tool for the analysis of sulfated oligosaccharides from mucin glycoproteins.

An approach for analyzing sulfated oligosaccharide alditol mixtures by liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) is described. Two columns, an amino-bonded column and a porous graphitized carbon column (PGC) were used. Oligosaccharides were eluted with linear gradients of acetonitrile and water, with 5 mM ammonium hydrogencarbonate or formate buffers at a basic pH. The methods were evaluated on a mixture of sulfated oligosaccharide alditols prepared from mucin glycoproteins from pig stomach. Results from LC-ESI-MS of the mixture were compared with the structural information obtained by high energy collision fragmentation using fast atom bombardment tandem mass spectrometry (FAB-MS-MS). The separation ability of the two columns was also tested using a more complex mixture of sulfated oligosaccharides from pig colon, where several isomers were detected. The potential use of in-source collision-induced dissociation (CID) to gain sequence information of sulfated oligosaccharides was also evaluated. The major fragment ions obtained by in-source CID of the trisaccharide Hex-3HexNAcol6-HexNAc6-SO3 were sufficient for assigning the oligosaccharide sequence and the position of the sulfate group within the monosaccharide moiety. The LC-ESI-MS approach should be a valuable tool for characterization of mucin glycosylation and alterations during pathological conditions.

Animals↗
PubMed1985

Structural characterization of murine Ia antigen N-linked oligosaccharides and localization of specific structures to two unique alpha-chain glycosylation sites.

The sequence of N-linked oligosaccharides of differentially glycosylated murine I-Ak alpha-(alpha 2- and alpha 3-) and beta-chains was determined. I-Ak beta-chains predominantly bear a biantennary complex oligosaccharide with a core fucose, and with the peripheral sequence SA----Gal----GlcNAc----Man. The I-Ak alpha-chain has two N-linked glycosylation sites at Asn-82 and Asn-122. When Lubrol-insoluble alpha 3-chains are examined they are found to bear high-mannose oligosaccharides of either the Man9GlcNAc2 or Man8GlcNAc2 type at both sites. When Lubrol-soluble alpha 2-chains are examined, in about 85% of the molecules the Asn-82 site bears a biantennary complex oligosaccharide with core fucose, and with the peripheral sequence SA----Gal----GlcNAc----Man. Interestingly, the Asn-122 site bears a variety of structures. In about 50% of the molecules, the structure at Asn-122 is a biantennary complex oligosaccharide without core fucose and with the peripheral sequence SA----Gal----GlcNAc----Man. In addition, it can bear other complex structures which we did not define further. The apparently restricted addition of fucose to the oligosaccharide at the alpha-Asn-82 site, even when both alpha-sites bear biantennary complex structures with the same peripheral sequence, is a feature unique to this system. The unusual variety of structures present at the alpha-Asn-122 site may indicate differential processing in different cell types.

Animals↗
PubMed1976

[Study of the fucose-rich oligosaccharides in the urine of healthy and melituric subjects with A, B and O blood groups].

The application of adsorption chromatography on charcoal-Celite leads the authors to characterize in normal urines a class of fucose-rich oligosaccharides which possess blood group activities and are related to the phenotypes ABH, Le and secretor. Most of these oligosaccharides have a glucose residue in reducing terminal positions. Excretion of some oligosaccharides increases in the urine of diabetic and lactosuric subjects. In spontaneous or induced galactosurias, the elimination of oligosaccharides with a glucose residue in reducing terminal position decreases while appears a large amount of new oligosaccharides which all possess a galactose residue in reducing terminal position. These results lead to the conclusion that urinary oligosaccharides do not originate from glycosphingolipids, but from transglycosylation on carbohydrates which exist free in the organism: glucose for normal and diabetic subjects, lactose or galactose for lactosuric and galactosuric subjects, respectively.

ABO Blood-Group System↗
PubMed1997

Extensive features of tight oligosaccharide binding revealed in high-resolution structures of the maltodextrin transport/chemosensory receptor.

BACKGROUND: Active-transport processes perform a vital function in the life of a cell, maintaining cell homeostasis and allowing access of nutrients. Maltodextrin/maltose-binding protein (MBP; M(r) = 40k) is a receptor protein which serves as an initial high-affinity binding component of the active-transport system of maltooligosaccharides in bacteria. MBP also participates in chemotaxis towards maltooligosaccharides. The interaction between MBP and specific cytoplasmic membrane proteins initiates either active transport or chemotaxis. In order to gain new understanding of the function of MBP, especially its versatility in binding different linear and cyclic oligosaccharides with similar affinities, we have undertaken high-resolution X-ray analysis of three oligosaccharide-bound structures. RESULTS: The structures of MBP complexed with maltose, maltotriose and maltotetraose have been refined to high resolutions (1.67 to 1.8 A). These structures provide details at the atomic level of many features of oligosaccharide binding. The structures reveal differences between buried and surface binding sites and show the importance of hydrogen bonds and van der Waals interactions, especially those resulting from aromatic residue stacking. Insights are provided into the structural plasticity of the protein, the binding affinity and the binding specificity with respect to alpha/beta anomeric preference and oligosaccharide length. In addition, the structures demonstrate the different conformations that can be adopted by the oligosaccharide within the complex. CONCLUSIONS: MBP has a two-domain structure joined by a hinge-bending region which contains the substrate-binding groove. The bound maltooligosaccharides have a ribbon-like structure: the edges of the ribbon are occupied by polar hydroxyl groups and the flat surfaces are composed of nonpolar patches of the sugar ring faces. The polar groups and nonpolar patches are heavily involved in forming hydrogen bonds and van der Waals contacts, respectively, with complimentary residues in the groove. Hinge-bending between the two domains enables the participation of both domains in the binding and sequestering of the oligosaccharides. Changes in the subtle contours of the binding site allow binding of maltodextrins of varying length with similarly high affinities. The fact that the three bound structures are essentially identical ensures productive interaction with the oligomeric membrane proteins, which are distinct for transport and chemotaxis.

ATP-Binding Cassette Transporters↗
PubMed2002

Determination of linkages of linear and branched oligosaccharides using closed-ring chromophore labeling and negative ion trap mass spectrometry.

Linear as well as branched oligosaccharides were labeled with p-aminobenzoic ethyl ester (ABEE) using the glycosylamine closed-ring labeling approach and analyzed by negative-ion electrospray ionization mass spectrometry (ESI-MS). Linkage specific fragment ions of ABEE labeled linear oligosaccharides were proposed based on the MS2 and MS3 data for several ABEE labeled linear oligosaccharides with known linkage configurations. Fragmentation at the reducing end was similar to that observed for ABEE disaccharides whereas the fragmentation pattern not involving the reducing end was similar to underivatized disaccharides. Based on these ions, all the linkages of linear oligosaccharides could be unambiguously determined. The fragmentation pattern at the branched sugar was in general not quite the same as the linear one. However, many linkage specific fragment ions were also observed for linkages at the branched sugar. These ions along with the ions proposed for linear oligosaccharides were found to be quite useful for the determination of all the linkages of branched oligosaccharides.

Animals↗
PubMed2002

Chemical characterization of the oligosaccharides in beluga (Delphinapterus leucas) and Minke whale (Balaenoptera acutorostrata) milk.

Carbohydrates were extracted from the milk of a beluga, Delphinopterus leucas (family Odontoceti), and two Minke whales, Balaenoptera acutorostrata (Family Mysticeti), sampled late in their respective lactation periods. Free oligosaccharides were separated by gel filtration and then neutral oligosaccharides were purified by preparative thin layer chromatography and gel filtration, while acidic oligosaccharides were purified by ion-exchange chromatography, gel filtration and high performance liquid chromatography (HPLC). Their structures were determined by 1H-NMR. In one of the Minke whale milk samples, lactose was a dominant saccharide, with Fuc(alpha1-2)Gal(beta1-4)Glc(2'-fucosyllactose), Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc(lacto-N-neotetraose), GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)Glc(A-tetrasaccharide), Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (para lacto-N-neohexaose), Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (sialyl lacto-N-neotetraose), Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (LST c) and Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (sialyl para lacto-N-neohexaose) also being found in the milk. The second Minke whale sample contained similar amounts of lactose, 2'-fucosyllactose and A-tetrasaccharide, but no free sialyl oligosaccharides. Sialyl lacto-N-neotetraose and sialyl para lacto-N-neohexaose are novel oligosaccharides which have not been previously reported from any mammalian milk or colostrum. These and other oligosaccharides of Minke whale milk may have biological significance as anti-infection factors, protecting the suckling young against bacteria and viruses. The lactose of Minke whale milk could be a source of energy for them. The beluga whale milk contained trace amounts of Neu5Ac(alpha2-3)Gal(beta1-4)Glc(3'-N-acetylneuraminyllactose), but the question of whether it contained free lactose could not be clarified. Therefore, lactose may not be a source of energy for suckling beluga whales.

Animals↗
PubMed1999

Neutral oligosaccharide content of preterm human milk.

Human milk oligosaccharides are known to play a role in protection against certain infectious diseases. Previous reports indicate that the content of human milk oligosaccharides varies widely among individuals at term but such information on preterm milk is lacking. After removal of the fat, protein and most of the lactose from non-pooled human milk samples, a total neutral oligosaccharide fraction was isolated by ion-exchange chromatography followed by gel filtration. A Dionex high-performance anion-exchange chromatography system equipped with a pulsed electrometric detector was then employed to measure the levels of ten neutral oligosaccharides in the individual milk samples. Twenty-three milk samples from thirteen mothers who delivered at a mean gestational age of 29.5 (SD 3.1) weeks were collected between days 0 and 33 of lactation, and compared with three samples of term milk from two mothers. The ranges of the total and individual levels of the ten neutral oligosaccharides in preterm milk were similar to those in term milk. Further, as previously described in term milk, preterm milk exhibited a quantitative individual variation. This variation was independent of the gestational age, day of lactation, and postconceptional age. In conclusion, levels of ten neutral oligosaccharides did not differ between preterm and term human milk.

ABO Blood-Group System↗
PubMed1999

Lactose-derived oligosaccharides in the milk of elephants: comparison with human milk.

Human milk is commonly considered to be unique when compared with the milk of other species with regard to its high content of complex fucosylated and sialylated lactose-derived oligosaccharides. We describe the application of high-pH anion-exchange chromatography with pulsed amperometric detection and TLC to characterize and quantitate neutral and sialylated lactose-derived oligosaccharides in milk from three Asian elephants and human milk. The lactose contents of elephant and human milks were 25-30 g/l and about 66 g/l respectively, whereas total oligosaccharide concentration was about three times higher in elephant milk and comprised up to 40% (10% in human milk) of the carbohydrate content. The ratio neutral: acidic components was different in the milk of the two species; in elephant milk, the N-acetylneuraminic acid-containing oligosaccharides made up almost half of the total amount v. 30% in human milk. Most oligosaccharides in elephant milk were more fucosylated and/or sialylated compared with human milk components. By mild acid hydrolysis, fucose and N-acetylneuraminic acid were cleaved off from complex components, and this resulted in increased amounts of fucose, galactose, N-acetylneuraminic acid, lactose and lacto-N-neo-tetraose. Unique to elephant milk are the high levels of 3'-galactosyllactose (up to 4 g/l) and lacto-N-neo-tetraose which are present in human milk only in trace amounts. Elephant and human milks have high levels and unique patterns of oligosaccharides which may reflect the relative importance of these components in neonatal host defence, in endothelial leucocyte interactions or in brain development.

Animals↗
PubMed2005

Analysis of keratan sulfate oligosaccharides by electrospray ionization tandem mass spectrometry.

Keratan sulfate (KS) is a glycosaminoglycan consisting of repeating disaccharide units composed of alternating residues of d-galactose and N-acetyl-d-glucosamine linked beta-(1-4) and beta-(1-3), respectively. In this study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) was employed to identify keratan sulfate oligosaccharides. Two nonsulfated disaccharide isomers and two monosulfated disaccharide isomers were distinguished through MS/MS. In MS(1) spectra of multiply sulfated KS oligosaccharides, the charge state of the most abundant molecular ion equals the number of sulfates. Subsequent MS(2) and MS(3) spectra of mono-, di-, tri-, and tetrasulfated KS oligosaccharides and sialylated tetrasaccharides reveal diagnostic ions that can be used as fingerprint maps to identify unknown KS oligosaccharides. Based on the pattern of fragment ions, the compositions of an oligosaccharide mixture from shark cartilage KS and of two enzyme digests of bovine corneal KS were determined directly, without prior isolation of individual oligosaccharides by HPLC or other methods.

Acetylglucosamine↗
PubMed2005

Infrared multiphoton dissociation of O-linked mucin-type oligosaccharides.

Oligosaccharides are known to play important roles in many biological processes. In the study of oligosaccharides, collision-induced dissociation (CID) is the most common dissociation method to elucidate the sequence and connectivity. However, a disadvantage of CID is the decrease in both the degree and efficiency of dissociation with increasing mass. In the present study, we have successfully performed infrared multiphoton dissociation (IRMPD) on 39 O-linked mucin-type oligosaccharide alditols (both neutral and anionic). CID and IRMPD spectra of several oligosaccharides were also compared. They yielded nearly identical fragment ions corresponding to the lowest energy fragmentation pathways. The characteristic fragmentations of structural motifs, which can provide the linkage information, were similarly presented in both CID and IRMPD spectra. Multistage of CID (MS(3) or MS(4)) is commonly needed to completely sequence the oligosaccharides, while IRMPD of the same compounds yielded the fragment ions corresponding to the loss of the first residue to the last residue during a single-stage tandem MS (MS(2)). Finally, it is shown that the fragmentation efficiency of IRMPD increases with the increasing size of oligosaccharides.

Mucins↗
PubMed2004

Strategy for profiling and structure elucidation of mucin-type oligosaccharides by mass spectrometry.

A strategy combining accurate mass determination, tandem mass spectrometry, structure homology, and exoglycosidases is described that allows the structural characterization of mucin-type O-linked oligosaccharides. The method is used to profile with quantitation the O-linked oligosaccharide (both neutral and anionic) components of the only diploid Xenopus frog, Xenopus tropicalis. Collision-induced dissociation was used to determine connectivity, to identify previously characterized oligosaccharides, and to determine the presence of structural motifs in unknown oligosaccharides. Exoglycosidase digestion was used to identify the individual residues along with the linkages. The enzymes were also used to cleave larger oligosaccharides to smaller units that are similar to previously elucidated components. By using CID, isomeric structures were compared to determine whether they were identical. In this way, the exoglycosidases were more effectively used, and their use was minimized. A total of 35 oligosaccharides including neutral, sialylated, and sulfated were characterized in this way. The relative abundances of all components were also determined based on HPLC.

Animals↗
PubMed1992

Novel sulfated ligands for the cell adhesion molecule E-selectin revealed by the neoglycolipid technology among O-linked oligosaccharides on an ovarian cystadenoma glycoprotein.

E-selectin is the inducible adhesion protein on the surface of endothelial cells which has a crucial role in the initial stages of recruitment of leucocytes to sites of inflammation. In addition, it is almost certainly involved in tumor cell adhesion and metastasis. This report is concerned with identification of a new class of oligosaccharide ligand--sulfate-containing--for the human E-selectin molecule from among oligosaccharides on an ovarian cystadenoma glycoprotein. This has been achieved by application of the neoglycolipid technology to oligosaccharides released from the glycoprotein by mild alkaline beta-elimination. Oligosaccharides were conjugated to lipid, resolved by thin-layer chromatography, and tested for binding by Chinese hamster ovary cells which had been transfected to express the full-length E-selectin molecule. Several components with strong E-selectin binding activity were revealed among acidic oligosaccharides. The smallest among these was identified by liquid secondary ion mass spectrometric analysis of the neoglycolipid, in conjunction with methylation analysis of the purified oligosaccharide preparation as an equimolar mixture of the Le(a)- and Le(x)/SSEA-1-type fucotetrasaccharides sulfated at position 3 of outer galactose: [formula: see text] To our knowledge this is the first report of a sulfofucooligosaccharide ligand for E-selectin. The binding activity is substantially greater than those of lipid-linked Le(a) and Le(x)/SSEA-1 sequences and is at least equal to that of the 3'-sialyl-Le(x)/SSEA-1 glycolipid analogue.

Carbohydrate Conformation↗
PubMed1990

Control of N-linked oligosaccharide synthesis: cellular levels of dolichyl phosphate are not the only regulatory factor.

When MDCK cells were incubated in the presence of the protein synthesis inhibitor puromycin or cycloheximide, there was a rapid and concentration-dependent inhibition in the incorporation of [2-3H]mannose into lipid-linked oligosaccharide and into protein. However, mannose incorporation into dolichyl-P-mannose was not affected. Interestingly, these inhibitors did block [6-3H]glucosamine incorporation into dolichyl-PP-GlcNAc as well as into lipid-linked oligosaccharides. Similar results were obtained when other cell lines were used and also when inhibitors of protein glycosylation such as beta-hydroxynorvaline and beta-fluoroasparagine were used. Cells incubated in puromycin did not show any changes in the levels of sugar nucleotides, GDP-mannose or UDP-GlcNAc, or in the in vitro activities of the glycosyltransferases that add mannose to the lipid-linked oligosaccharides. The inhibition of mannose incorporation into lipid-linked oligosaccharides could not be overcome by addition of dolichyl-P to the inhibited cells, even though the addition of dolichyl-P to control cells stimulated mannose incorporation into dolichyl-P-mannose, lipid-linked oligosaccharides, and protein from 3- to 5-fold. Thus, limitations in the levels of dolichyl-P do not appear to be a major factor in this inhibition. On the other hand, addition of the tripeptide acceptor N-acyl-Asn-Try-Thr did overcome the puromycin inhibition to some extent, suggesting that accumulation of some intermediate such as lipid-linked oligosaccharides might be involved in the inhibition.

Amino Acid Sequence↗
PubMed1980

Linkage and sequence analysis of mannose-rich glycoprotein core oligosaccharides by proton nuclear magnetic resonance spectroscopy.

The anomeric proton (H-1) chemical shifts of D-mannopyranosides in aqueous solution are affected both by the aglycon and by substitution of the ring [Lee, Y. C., & Ballou, C. E. (1965) Biochemistry 4, 257]. We have examined the 1H NMR spectra for a variety of linear and branched mannooligosaccharides and have assigned the H-1 resonances to the component sugars. The chemical shifts, which range from delta 4.76 to 5.36, provide information regarding the linkages, sequences, and anomeric configurations of mannose residues in an oligomer. Thus, 1H NMR spectroscopy can complement enzymatic hydrolysis, methylation analysis, and acetolysis for the structural characterization of oligosaccharides. Furthermore, small structural differences between otherwise identical oligosaccharides are often accompanied by long-range chemical shift changes for the anomeric protons. Because sugars three or more residues away from the structural alteration can be affected, the changes must reflect conformational differences. We have placed emphasis on the mannose-rich oligosaccharides from glycoproteins, particularly those produced by endo-beta-N-acetylglucosaminidase digestion. Two mannose-rich glycopeptides were isolated from a monoclonal human IgM and their positions of origin on the polypeptide chain were determined. The oligosaccharides were released with endo-beta-N-acetylglucosaminidase and fractionated into several size classes. Our structural studies show that each glycopeptide possessed a unique set of oligosaccharides, in agreement with a recent report [Chapman, A. & Kornfeld, R. (1979) J. Biol. Chem. 254, 816]. The NMR spectra were particularly valuable in detecting and quantitating isomeric fragments not observed previously, and our results suggest a modification of the scheme presented by Chapman and Kornfeld for the processing of mannose-rich IgM oligosaccharides.

Carbohydrate Conformation↗
PubMed1976

Characterization of the oligosaccharide units of the bovine erythrocyte membrane glycoprotein.

The major glycoprotein of the bovine erythrocyte membrane was purified by extraction of the ghosts with lithium 3,5-diiodosalicylate followed by phenol-water extraction and acidification. The glycoprotein contains 20% protein and 80% carbohydrate by weight and gives a single band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular weight of 230000 daltons. The carbohydrate composition of the glycoprotein was determined to be (in residues relative to sialic acid): sialic acid, 1.0; fucose, less than 0.01; mannose, 0.1; galactose, 3.3; N-acetylgalactosamine, 0.9; and N-acetylglucosamine, 2.4. Pronase digestion of the isolated glycoprotein followed by Sephadex G-75 gel filtration resulted in the separation of a small pool of glycopeptides (pool III), which included all of the mannose-containing glycopeptides, from the bulk of the glycopeptide material which was in the void fractions of the column (pool I). Alkaline borohydride treatment released over 95% of the oligosaccharide units in pool I and approximately 30% of the oligosaccharide units in pool III. These oligosaccharides were isolated by gel filtration and ion-exchange chromatography. The oligosaccharides released from pool I had molecular weights of 1100-1400 daltons and contained sialic acid, galactose, and N-acetylglucosamine in molar ratios of 0.5-1:3:2 as well as a partial residue of N-acetylgalactosaminitol. The oligosaccharides released from pool III by alkali had molecular weights of 1300-1600 daltons and contained sialic acid, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-ACETYLgalactosaminitol in molar ratios of 1-2:2:1:1:1. These data indicate that the majority of the oligosaccharide units of the bovine erythrocyte glycoprotein are linked O-glycosidically to the peptide backbone of the molecule.

Acetylgalactosamine↗
PubMedDate not supplied

Extraction of hemicellulosic oligosaccharides from spruce using microwave oven or steam treatment.

This paper describes the extraction of hemicellulosic oligosaccharides from spruce, using microwave or steam treatment that can be used for the production of polymers, replacing fossil-based polymers, e.g., hydrogels. The highest yield of oligosaccharides, measured as mannan, was 70% obtained with treatment in the microwave oven at 200 degrees C for 5 min. The amount of oligosaccharides extracted was 12.5 g per 100 g of dry wood. The molecular weights of some selected samples were analyzed using fast protein liquid chromatography and size exclusion chromatography and time-of-flight matrix-assisted laser desorption ionization. Recovered oligosaccharides following steam treatment at 200 degrees C for 2 min had a mean molecular weight of 3400 g/mol with a maximum weight of 12000 g/mol. Higher severity, i.e., increased temperature (>200 degrees C) and residence time, resulted in lower mean molecular weights and yield. Oligosaccharides with higher mean molecular weights were obtained at lower severity, but the yield was considerably lower. The feasibility of using the extracted hemicellulosic oligosaccharides from spruce for the synthesis of hydrogels was demonstrated.

Microwaves↗
PubMedDate not supplied

Comparison of N-linked oligosaccharides of recombinant human tissue kallikrein produced by Chinese hamster ovary cells on microcarrier beads and in serum-free suspension culture.

Glycosylation heterogeneity in recombinant human tissue kallikrein (r-HuTK) produced by Chinese hamster ovary (CHO) cells from microcarrier culture and from a serum-free suspension cell recycle process has been compared. Significant differences in the degree of sialylation were observed in glycoform distribution and oligosaccharide heterogeneity. High-performance liquid chromatography with a pellicular anion-exchange column under low pH eluant conditions was used to characterize the number and types of N-linked complex type oligosaccharides present. The oligosaccharides were released by N-glycanase and, after reduction, were resolved into a number of peaks containing one, two, three, and four sialic acids with an additional subfractionation based on the nature of the antennary structure. The microcarrier process resulted in a reduced amount of sialylated oligosaccharide species as compared to the suspension cell process. Removal of sialic acid followed by chromatography of the asialooligosaccharides under high pH anion-exchange conditions indicated that the same antennary structures were present but in slightly different relative amounts. The oligosaccharide profiles are indicative of a highly complex array of microheterogeneity present, encompassing mono-, di-, tri-, and tetrasialylated complex type oligosaccharides.

Animals↗