Electrophoretic Methods for the Analysis of N-Linked Oligosaccharides

Analytical Biochemistry 283, 125–132 (2000) doi:10.1006/abio.2000.4647, available online at http://www.idealibrary.com on

THEMATIC REVIEW Electrophoretic Methods for the Analysis of N-Linked Oligosaccharides T. Shantha Raju 1 Analytical Chemistry, MS 62, Genentech Inc., One DNA Way, South San Francisco, California 94080

N-linked oligosaccharides, covalently bound to proteins, perform significant biological functions (1). They affect protein structure, bioactivity, and pharmacokinetics (2– 6). Glycosylation of proteins is a cotranslational and posttranslational process mediated by many enzymes including glycosyltransferases and glycosidases (7–9). Some of the glycosyltransferases and glycosidases involved in the biosynthesis and degradation of mammalian N-linked oligosaccharides are listed in Table 1. Glycosyltransferases mediate the transfer of sugar from an activated sugar donor (usually a nucleotide sugar) to an acceptor molecule, whereas glycosidases catalyze the hydrolysis of glycosidically bound sugar residue(s). Since these enzymes compete with each other for the available substrates and/or acceptor molecules within the cellular compartments, the biosynthesized oligosaccharides are, usually, extremely heterogeneous. Because of the biological importance, these heterogeneous oligosaccharides need to be characterized in greater detail to understand structure– function relationships. The analytical technique(s) for characterization must be sufficiently sensitive and discriminating in order to detect subtle as well as significant changes in the oligosaccharide structures of glycoproteins. A tremendous number of oligosaccharide structures can arise from variations in the sequence and arrangement (linear, branched, or cyclic) of the monosaccharide building blocks (see Table 2 for a list of monosaccharides commonly found in mammalian N-linked oligosaccharides). More than 36,000 tetrasaccharide structures can arise from combinations of just four different monosaccharide units. The N-linked oligosaccharides attached to proteins via asparagine residues 1 Address correspondence to author. Fax: (650) 225-3554. E-mail: [email protected].

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are very complex. This complexity and heterogeneity of N-linked oligosaccharide structures present a separation problem which has been addressed by different analytical techniques including high-performance liquid chromatography (HPLC) (10 –13), high-pH anionexchange chromatography with pulsed amperometric detection (HPAEC–PAD) (14 –18), and gas chromatography (GC) (19). Electrophoretic methods, such as paper electrophoresis, gel electrophoresis, and capillary electrophoresis (CE), 2 offer a potentially high separation efficiency due to the application of a high electric field (20). Electrophoresis can be used to separate molecules that have different charge-to-mass ratios like native proteins or to separate molecules that possess the same charge-to-mass ratio but different molecular masses. N-linked oligosaccharides are often mixtures of neutral and charged molecules with different or, in the case of isomers of some oligosaccharides, the same molecular weight, hence producing species with the same and/or different charge-tomass ratios. Electrophoretic methods can be used to separate closely related molecular species like oligosaccharides (21). Two major difficulties must be overcome in analyzing the N-linked oligosaccharides by electrophoretic methods. With the exception of sialylated, phosphorylated, and sulfated oligosaccharides, neutral oligosaccharides, such as high mannose, desialylated hybrid, and complex oligosaccharides, do not contain ionizable charged functions, a condition that excludes their direct separation un2 Abbreviations used: CHO cells, Chinese hamster ovary cells; APTS, 9-aminopyrene-1,4,6-trisulfonic acid; ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid; FACE, fluorophore-assisted carbohydrate electrophoresis; RAAM, reagent array analysis method; CE, capillary electrophoresis; LIF, laser-induced fluorescence; PNGase F, peptide-N-glycosidase F.

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T. SHANTHA RAJU TABLE 1

Enzymes Involved in the Biosynthesis and/or Degradation of N-Linked Oligosaccharides Enzyme category Glycosyltransferases

Exoglycosidases

Endoglycosidases

Enzyme type (with anomeric configuration of the sugar linkage) Glucosyltransferases (␣- and ␤-) Galactosyltransferases (␣- and ␤-) N-Acetylglucosaminyltransferases (␤-) N-Acetylgalactosaminyltransferases (␣- and ␤-) Mannosyltransferases (␣- and ␤-) Xylosyltransferases (␤-) Fucosyltransferases (␣-) Sialyltransferases (␣-) Oligosaccharyltransferases (␤-) Glucosidases (␣- and ␤-) Galactosidases (␣- and ␤-) N-Acetylglucosaminidases (␤-) N-Acetylgalactosaminidases (␣- and ␤-) Mannosidases (␣- and ␤-) Xylosidases (␣- and ␤-) Fucosidases (␣- and ␤-) Sialidases (␣- and ␤-) Endo-␤-galactosidase PNGase F (peptide-N-glycosidase F) PNGase A Endo H/F

Note. Besides glycosyltransferases and glycosidases, other enzymes such as nucleotide sugar synthetases and sugar transporters like nucleotide sugar transporters are also involved in the biosynthesis of N-linked oligosaccharides.

der electric field. Further, most oligosaccharides neither absorb light nor fluoresce, a property that hinders their sensitive detection. However, it is possible to label oligosaccharides containing reducing end groups with chromophoric/fluorophoric molecules by reductive amination. This type of labeling reaction is

SCHEME 1. Structure of fluorescent labels (ANTS and APTS) used for the analysis of N-linked oligosaccharides by FACE and CE–LIF methods.

also useful to introduce ionizable functional groups, hence making the oligosaccharides uniformly charged and able to be analyzed using electrophoretic methods (22–25). This review discusses the electrophoretic methods used to analyze N-linked oligosaccharides released from glycoproteins by chemical or enzymatic means followed by labeling with fluorescent groups. PAPER ELECTROPHORESIS

TABLE 2

Monosaccharides Commonly Found in Mammalian N-Linked Oligosaccharides Monosaccharides D-Glucose D-Galactose L-Fucose D-Mannose D-Xylose

N-Acetyl D-glucosamine N-Acetyl D-galactosamine N-Acetylneuraminic acid N-Glycolyneuraminic acid 2-Keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) (deamidated neuraminic acid)

Anomeric configuration a

␣␣␣␣␤␤␣␣␣-

and ␤and ␤and ␤and ␤-

␣-

a Anomeric configuration of the glycosidically linked monosaccharides is shown.

Paper electrophoretic methods were widely used for a long time to separate oligosaccharides (26). This separation technique was very successful in its early introduction because of good resolution, the possibility of accurate quantitative and qualitative data, and the sample requirement of less than a milligram. In paper electrophoresis, a mixture of analytes was applied at the center of a long strip of paper (such as cellulose acetate paper), the ends of which were placed in close contact with the cathode and anode. The rate of migration of oligosaccharide depends on the magnitude of the applied current. Enhanced resolution of oligosaccharides could be obtained by carrying out electrophoresis at a particular pH in one dimension followed by a second electrophoresis at another pH in a second dimension. Oligosaccharides were also separated by paper chromatography followed by electrophoresis. This was very useful to separate both neutral (such as high

REVIEW: FACE AND CE–LIF METHODS FOR THE ANALYSIS OF N-LINKED OLIGOSACCHARIDES

FIG. 1. FACE analysis of cleavage products of asialoagalactobiantennary oligosaccharide by Streptococcus pneumonia N-acetylglucosaminidase. A standard asialoagalactobiantennary oligosaccharide was treated with recombinant S. pneumonia N-acetylglucosaminidase (ProZyme). Aliquots were withdrawn at 15, 30, and 45 min and analyzed by gel electrophoresis. Lane 1, standard asialoagalactobiantennary oligosaccharide (from Dionex Corp.); lanes 2, 3, and 4, aliquots withdrawn from the enzyme reaction mixture at 15, 30, and 45 min, respectively. R, GlcNAc␤(1,4)GlcNAc-ANTS.

mannose type) and charged oligosaccharides (such as sialylated ones). Long run times, high voltages, complex sample preparation steps, and unpleasant buffer systems are some of the limitations of paper electrophoretic methods. However, advances in glycobiology have led to a renewed interest in applying electrophoretic methods for the analysis of complex oligosaccharides. Gel electrophoresis methods are relatively fast, require little equipment, and are user-friendly. Capillary electrophoresis offers the potential of highly efficient separations, high sensitivity, and short analysis time. GEL ELECTROPHORESIS

In 1990 Jackson introduced the analysis of oligosaccharides by gel electrophoresis (23, 27). The basic

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approach of this method was the use of high-percentage polyacrylamide slab gels for the separation of 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS, see Scheme 1)-labeled oligosaccharides. The main advantage of the method is the introduction of highly charged fluorescent label (ANTS) covalently linked to oligosaccharides by reductive amination at the reducing end group (for N-linked oligosaccharides, the reducing end group is GlcNAc in the core). The charged groups (three sulfonic acid groups) enabled the separation of acidic as well as neutral oligosaccharides in the same run (28). The gel bands were visualized by fluorography and/or CCD gel imagers. Glyko (Novato, CA) and Millipore (Boston, MA) developed commercial systems such as the fluorophoreassisted carbohydrate electrophoresis (FACE) system. FACE technology not only offers a rapid and sensitive method to separate monosaccharides and oligosaccharides, but also has been shown to be quantitative and reproducible (28). The resolving power of FACE for complex mixtures of oligosaccharides and their isomers has been demonstrated (28, 29). Prior to separation by FACE, the N-linked oligosaccharides were released from glycoproteins by a chemical method using hydrozinolysis or by enzymatic methods using endoglycosidases like peptideN-glycosidase F (PNGase F). Using the FACE method, it is possible to analyze multiple samples on a single slab gel, which is helpful to compare, for example, batch to batch variation in the glycosylation of recombinant glycoproteins. The FACE method is also useful to obtain sequence information of Nlinked oligosaccharides. Using a mixture of highly specific exoglycosidases, Masada et al. (30) developed a sequencing strategy similar to RAAM (reagent array analysis method) which had been developed by Oxford GlycoSciences (31). This enzymatic sequencing method has been successfully employed by Kumar et al. (32) to elucidate the structure of N-linked oligosaccharides released from recombinant human factor VIII. One limitation of both RAAM and FACE technologies is that it is difficult to obtain sequence information of unknown oligosaccharides. The possibility of encountering unknown structures cannot be ruled out due to the occurrence of mutations of cells under stressed environmental conditions (33– 40). Nevertheless, the FACE method is very useful to obtain oligosaccharide mapping information from different batches and/or runs of well-characterized recombinant glycoproteins in the quality control and quality assurance divisions of the biopharmaceutical industry. Harvey Miller (personal communications) has used gel electrophoretic methods to elucidate the branch specificity of ␤-N-acetylglucosaminidases. One such example is illustrated in Fig. 1, in which a stepwise

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SCHEME 2. Digestion of asialoagalactobiantennary oligosaccharide with a recombinant S. pneumonia ␤-N-acetylglucosaminidase. An aliquot of the reaction mixture at the 15-min time point was digested with ␣-mannosidase to confirm the structure of the intermediate.

removal of terminal GlcNAc residues of asialoagalactobiantennary oligosaccharide by a recombinant Streptococcus pneumonia ␤-N-acetylglucosaminidase (ProZyme, San Leandro, CA) is shown. The GlcNAc linked to the Man␣(1,3) arm was completely removed by the first time point and the intermediate product was sensitive (data not shown) to ␣-mannosidase specific for mannose in the Man␣(1, 2, or 3) linkage (41) establishing the structure of the intermediate as indicated (see Scheme 2). The GlcNAc linked to the Man␣(1,6) arm was slowly removed by the enzyme ␤-N-acetylglucosaminidase (see lanes 3 and 4 in Fig. 1). This type of application of gel electrophoresis allows one to compare the position of

degradation products from several reactions simultaneously. The method is useful to elucidate the specificity of exoglycosidases with relative ease compared to other methods. CAPILLARY ELECTROPHORESIS

Separation and analysis of oligosaccharides using CE is an area of significant basic and advanced research. The broad acceptance of CE has been largely facilitated by the high intrinsic resolving power and good reproducibility of the capillary format for the separation of closely related oligosaccharide structures coupled with high sensitivity provided by a laser-in-

TABLE 3

CE Methods for Carbohydrate Analysis Sugars

Capillary

Buffer/components

Mono/oligosaccharides

Fused silica, pH 9.4

Phosphate

Oligosaccharides

Fused silica

Mono/oligosaccharides Mono/oligosaccharides Monosaccharides

Fused silica Fused silica Fused silica

Phosphate, pH 6.6, or Tricine pH 8.2, ⫹ putrescine 100 mM NaOH, LiOH, or KOH NaOH Sorbate/hexadimethrine bromide

Detection Reductive amination; laserinduced fluorescence Low UV (200 nm) DC amperometry (Cu electrode) PAD (Au electrode) Indirect UV (254 nm)

REVIEW: FACE AND CE–LIF METHODS FOR THE ANALYSIS OF N-LINKED OLIGOSACCHARIDES

duced fluorescence (LIF) detection system. Separation is based on the electrophoretic mobility of the oligosaccharide species resulting from variations in their hydrodynamic radii. Various approaches have been used to separate and detect oligosaccharides by CE (see Table 3 for a list of CE methods for carbohydrate analysis). In this review, the discussion will be limited to the CE methods with laser-induced fluorescence detection (CE–LIF) for the analysis of N-linked oligosaccharides released from glycoproteins. ANTS-labeled oligosaccharides have been separated in the CE format by a number of groups (42). The two aromatic rings of ANTS allow detection either by UV absorption at 226 nm or by LIF using a helium– cadmium laser with excitation at 325 nm and the three sulfonate groups provide the required charged groups for the separation (see Scheme 1). Chen and Evangelista introduced 9-aminopyrene-1,4,6-trisulfonic acid (APTS) as a derivatizing agent for monosaccharides and oligosaccharides (22, 43, 44). Like ANTS, APTS also contains three sulfonic acid groups but has four conjugated aromatic rings giving a much higher molar absorptivity (see Scheme 1). APTS-labeled oligosaccharides are suitable for detection using an argon-ion laser (488-nm spectral line). The “free APTS” which is added in greater excess in the labeling reaction has a greatly reduced fluorescence compared to the APTS–sugar adducts at 488 nm. CE–LIF separation of APTS-labeled oligosaccharides released from a recombinant humanized monoclonal antibody (rhuMab) produced in Chinese hamster ovary (CHO) cells is shown in Fig. 2. The main problem of derivatization in an aqueous acetic acid medium is that heat-labile sugars, specifically sialic acid, may be destroyed during the reductive amination reaction. Further, labeling efficiencies for different glycoforms can vary, resulting in erroneous quantification. However, introduction of citric acid instead of acetic acid to the labeling reaction seems to minimize the destruction of acid-labile sugar residues like sialic acid (22, 45– 47). From Fig. 2 it is evident that good resolution of different N-linked oligosaccharides of rhuMab can be achieved by CE– LIF. Standards representing the asialoagalactobiantennary oligosaccharide (G0-Fuc), the asialoagalactobiantennary oligosaccharide with core fucose (G0), and the asialobiantennary oligosaccharide with core fucose (G2) species were used to verify the identity of peaks I, II, and V, respectively (see Scheme 3). Peaks III and IV were identified as structural isomers of the species terminating with a single galactose (G1 isomers, see Scheme 3) (48 –52). The resolution of peaks III and IV was useful to identify the species-specific and branch-specific galactosylation of IgGs isolated from the serum of different animal species (48). The relative proportions of peaks III

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FIG. 2. CE–LIF of APTS-labeled oligosaccharides. The PNGase Freleased oligosaccharides were labeled with APTS and subjected to CE–LIF analysis. Separation was performed on a Bio-Rad LPA-coated capillary (40 cm ⫻ 50 ␮m) thermostated through a liquid cooling system to 20°C in 25 mM ammonium acetate, pH 5.5, using a Beckman PACE/ 5000 series instrument. Sample was introduced into the capillary by pressure injection at 0.5 psi for 4 – 8 s. Electrophoresis was performed in the reversed polarity mode at a constant voltage of 12 kV (19 ␮A). Separated peaks were detected using an argon-ion laser-induced fluorescence detector (Beckman) with excitation at 488 nm and emission at 520 nm. The photomultiplier gain (PMT) was set at 10.

and IV were found to be different in IgGs of different animal species (see Fig. 3 for data on bovine, canine, horse, human, mouse, and rhesus IgG). In the case of recombinant monoclonal antibodies produced in CHO cells, the relative proportion of peaks III and IV remains constant despite the change in cell culture conditions. However, the proportion of peaks II–V changes with the culture conditions. These data suggested that the branch specificity of ␤1,4-galactosyltransferase(s) (␤1,4GT) is different in different species. Further, by taking advantage of the separation of peaks II–V, an enzyme assay was developed to measure ␤1,4-galactosyltransferase activity in real time (53). ␤1,4-Galactosyltransferase(s) mediates the transfer of Gal residue from UDP-Gal to the O-4 position of terminal GlcNAc residues of glycoconjugates. The real-time monitoring of glycosyltransferase reactions can be used to study the acceptor specificity. CONCLUSION

The APTS- and ANTS-labeled oligosaccharides have very high molar absorptivities making the CE– LIF and FACE methods sensitive for the analysis of N-linked oligosaccharides. In CE–LIF, the oligosaccharide species derived from IgGs were very well resolved. Even the oligosaccharides that have the same net charge-to-mass ratio were baseline resolved (see Fig. 2). This shows that excellent resolu-

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SCHEME 3.

Structures of N-linked oligosaccharides commonly found in rhuMab’s expressed in CHO cells.

tion can be achieved by CE–LIF solely on the differences in frictional coefficient associated with differences in branching pattern. Similar resolution power was observed with the FACE method (see Fig. 1). Hence, the ideal approach for microscale carbohydrate analysis would be derivatization using a fluorophore with high molar absorptivity such as APTS or ANTS followed by CE–LIF or FACE analysis. Derivatization with APTS has proven to be a suitable approach for the quantitation of different glycoforms derived from rhuMab’s. The relative simplicity in the number of carbohydrate structures present in rhuMab’s facilitated the interpretation of the electropherograms. This method was validated for the routine analysis of N-linked oligosaccharides released from rhuMab’s by PNGase F (52). Further, Guttman

and Ulfeder described a strategy to sequence oligosaccharides by consecutive enzymatic digestion using an array of exoglycosidases followed by capillary electrophoresis separation of the digests (54, 55). In this case, multistructure sequencing of a complex glycan pool was performed without prior isolation of the individual oligosaccharides. High resolving power, good reproducibility, and high sensitivity of CE–LIF enabled the acquisition of complete sequence information from several picomoles of glycoproteins. Furthermore, APTS-labeled oligosaccharides have been shown to be suitable for MALDITOF–MS analysis (56). Hence, with the advancement in CE–MS (57) and CE–NMR methods (58), sensitive structural characterization of novel oligosaccharides is possible in the future.

REVIEW: FACE AND CE–LIF METHODS FOR THE ANALYSIS OF N-LINKED OLIGOSACCHARIDES

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FIG. 3. Analysis of IgG oligosaccharides by CE–LIF. The N-linked oligosaccharides of bovine, canine, horse, human, mouse, and rhesus were released by PNGase F, labeled with APTS, and analyzed by CE–LIF as described in the legend to Fig. 2. Peaks III and IV were identified as G1␣(1,6) and G1␣(1,3), respectively (see Scheme 3).

ACKNOWLEDGMENTS The author greatly acknowledge Drs. Andy Jones, Marjorie Winkler, John O’Connor, and Viswantham Katta for helpful discussions and Dr. Harvey I. Miller (ProZyme, San Leandro, CA) for the gel electrophoresis data on ␤-N-acetylglucosaminidase digestion of the asialoagalactobiantennary oligosaccharide. The author thanks Professor Vernon Reinhold (University of New Hampshire) and others for helping to organize the “Satellite Meeting on Analytical Methods in Glycobiology” on October 30, 1999, in San Francisco.

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