After the chemical synthesis of glycans, the next important step is their purification and structural characterization, which bridges synthetic chemistry with functional glycobiology. Chromatographic methods are the critical final arbiter of structure and function, as each separation medium (column) reflects unique physicochemical and spatial interactions with different carbohydrate functionalities, imparting the chromatographic fingerprint. The chromatographic approach to glycan purification is distinct from that of proteins or nucleic acids. This is due to unique features such as the extensive structural diversity of glycans, the fact that glycan structural isomers (stereoisomers, regioisomers) are common, and the lack of universal detection methods for glycans. Chromatography has developed from just a tool for separation to an orthogonal method with multiple dimensions, separating by a variety of specific and general physicochemical interactions to allow for the separation of subtle stereoisomers and regioisomers (variants that differ only in linkage position and/or anomeric configuration) for which the identification and structural assignment is not trivial. Stationary phase chemistries for carbohydrate separations are also continuously being developed and optimized, which are often highly specific in their recognition and include hydrophilic interaction liquid chromatography (HILIC), porous graphitic carbon which separates by planar recognition, among many others. State-of-the-art glycan purification can be readily coupled on-line with mass spectrometry and nuclear magnetic resonance structural confirmation during the chromatographic separation. This transition of glycan purification from being a painstaking process to a highly effective means to deliver not only chemically pure glycans but also the necessary structural confirmation within the same time frame is an important tool for biological studies as well as for industrial and regulatory glycobiology.
The purification of chemically or enzymatically synthesized glycans from complex reaction mixtures is a critical step that determines the validity of any subsequent biological assessment and the safety of therapeutic applications. Chromatographic techniques have evolved into a modular toolbox in which selectivity, throughput and scalability can be adjusted by combining orthogonal separation principles–charge, size, polarity, biospecificity–in various combinations. Purification is no longer seen as a single "polishing" step; rather, it is now regarded as a design challenge in which the choice of stationary phase chemistry, mobile phase additives, and detection scheme is co-optimized with the synthetic route itself, ensuring that minor stereoisomers, regioisomers or deletion sequences that escape mass-spectrometric detection are physically separated rather than mathematically deconvoluted.
The low epitope densities with which glycans typically engage in biological recognition events mean that small levels of contaminants may still be tolerated. An impurity α-anomer in an otherwise homogeneously β-linked hexasaccharide, for instance, may cause artefactual lectin binding or off-target immunological read-outs, producing spurious structure–activity correlations that become enshrined in the literature. In the drug development setting, for their part, undefined heterogeneities in the glycoform distribution may give rise to unpredictable pharmacokinetic behaviors: foreign-epitope contaminants may be cleared rapidly by pre-existing antibodies, whereas undersialylated glycans may present as hepatotoxic ligands for asialoglycoprotein receptors. The purification step thus plays a risk-mitigation role in addition to being an analytic imperative, underpinning reproducibility between laboratories, qualified batch-to-batch comparison in the process-validation phase, and, where the glycan portion of the molecule is explicitly part of the active substance definition, the regulatory dossier itself. The increasing clinical translation of glycan-based vaccines, as well as antibody–drug conjugates, in which the glycan is the linker unit to the cytotoxic payload, has only further reinforced the need for chromatographic methodologies capable of ensuring high chemical purity, as well as endotoxin, residual solvent and heavy-metal clearance, in a single workflow.
Preparation of natural glycans for functional glycomics.1,2
The carbohydrate chromatographer is presented with a matrix of physico-chemical challenges that make the glycan landscape distinctly different from that of peptides or oligonucleotides. The presence of multiple hydroxyl groups leads to extreme hydrophilicity. Such molecules often have distribution coefficients near unity on reversed phase media and can only be retained by exploiting their ionic or H-bonding properties. Branching and linkage variability leads to isomeric families that have identical mass/charge ratios but different biological activities. Such species elute together on generic gradients unless the stationary phase is modified to impart subtle shape selectivity. Mixture of anomers, especially those generated by chemical glycosylations, can show only a small difference in retention time. Porous graphitic carbon or cyclodextrin-modified silica is then required to enhance π–π or inclusion interactions, respectively. Labile substituents such as sialic acids, sulfates, and phosphates may be lost on the mild acid conditions typically used to elute the glycans, requiring on-line pH monitoring and post-column neutralization. Low UV absorbance of most glycans relegates detection to refractive index or pulsed amperometric modes that are sensitive to temperature and salt gradients, making method transfer from analytical scouting to preparative scale more challenging.
The result is a chromatographic framework in which multiple separation mechanisms are applied to the same glycan sample, usually in parallel series to achieve a single separation based on two or more physicochemical principles, such as polarity, shape, charge, and biospecific interactions. The relative contributions of the different mechanisms can be readily changed using column-switching techniques, where the effluent from the first column is heart-cut into the second column, which is selected for orthogonality to the first. Mobile phase additives are often employed to enhance detection sensitivity or provide specific reactivity for isomeric differentiation: for example, post-column addition of an ion-pairing agent can improve resolution of structural isomers, or addition of a chiral resolving agent can effect enantiomeric resolution without sample re-injection. As detection methodologies have expanded to include pulsed amperometry for universal carbohydrate detection, high-resolution mass spectrometry for confirmation, and fluorescence tagging with reagents, each of which may generate radical-initiated fragmentation patterns, chromatographic purification no longer just yields mass, but can also provide spectroscopic information during the separation.
State-of-the-art HPLC of glycans balances kinetic and thermodynamic considerations. Highly efficient (fully porous sub-2 µm) hydrophilic phases provide steep gradients with manageable back-pressure, while superficially porous particles with polar (glycan-interacting) shells achieve fast mass-transfer for high-mannose-type species, prone to aggregation in solution. Volatile-buffered (usually ammonium acetate or ammonium bicarbonate) eluents allow fractions to be lyophilized directly after collection, without the dialysis steps that previously led to sample loss. Temperature programming is being used more and more: mild heating (30–50 °C) improves anomeric resolution, while sub-ambient operation stabilizes labile sialylated glycans that otherwise dehydrate in the column. Heart-cutting 2D-LC approaches, for example, transfer a pool of fucosylated glycoforms resolved on a first-dimension amide column, on-the-fly, to a porous graphitic carbon phase where linkage isomers separate according to their π-interaction strength. Derivatization is not obligatory: when sensitivity is a limitation, reductive amination with charge-bearing fluorophores, not only improves fluorescence but also localizes charge for predictable MS fragmentation, delivering both quantitation and sequence information in a single injection.
Affinity separations make use of the co-evolution of lectins, antibodies and synthetic affinity binding proteins which recognize carbohydrate epitopes with high affinity (dissociation constants comparable to antigen–antibody complexes). A series of immobilized lectins in serial cartridges (concavalin A for high-mannose types, wheat-germ agglutinin for terminal GlcNAc and Aleuria aurantia lectin for fucosylated structures) can concentrate and resolve a crude glycome into compositionally different streams, without the need for a preliminary labelling step. The ligand density is adjusted so that the glycans of interest are eluted under isocratic conditions by simply changing the competing sugar (methyl-α-mannoside, chitobiose or fucose) in the mobile phase. This circumvents the need for abrupt changes in pH which can desialylate the analyte. More recently columns of aptamers selected by SELEX against tumor associated glycans have become available. These ligands are nucleic acids, which can be exposed to repeated alkaline cleaning steps, allowing them to be used for preparative GMP workflows. One important, often overlooked factor is the orientation of the affinity surface: monolithic supports allow minimal diffusion limitation and can allow separation of kilogram scale feedstocks without loss of resolution. Coupled with on-line fluorescence detection, affinity chromatography can be used as a real time biomarker discovery tool: a serum glycoprotein fraction that binds to an immobilized sialyl-LewisX specific lectin can be collected, buffer exchanged and submitted to downstream MS characterization without manual intervention.
Charge-based separations take advantage of the non-electroneutral nature of many glycans of biological interest: sialic acids, sulfation and amino-sugars (or other cationic substituents) give rise to negative and positive charges, respectively. Weak anion-exchange resins functionalized with tertiary amines separate mono-, di- and tri-sialylated N-glycans using shallow acetate gradients at neutral pH. The mildness of these conditions is sufficient to retain labile O-acetyl modifications, which are lost in the alkaline conditions necessary to use stronger anion-exchange resins. Strong anion-exchange phases containing quaternary ammonium functionality are used for highly sulfated glycosaminoglycans. In these cases, temperature is increased in parallel with the salt concentration to break micelle-like self-association, which otherwise results in peak broadening. Cation-exchange chromatography is also used to separate glycans but is less common and is particularly useful for amino-sugar isomers, such as glucosamine and galactosamine epimers. The ability of such resins, when functionalized with chelating iminodiacetate groups, to coordinate the reducing end of glycans into a transient metal complex is often used in this context. A versatile hybrid approach is to use mixed-mode ion-exchange/hydrophilic columns, where the electrostatic interaction is supplemented by hydrogen bonding. This permits the resolution of phosphorylated high-mannose species, which co-elute on purely charge-based media. Because elution is performed using volatile salts (ammonium formate, ammonium acetate), the resulting fractions are directly compatible with mass-spectrometric read-out, and the charge state envelope observed in the MS can be correlated with the order of elution in the chromatographic peak to verify that the observed separation can indeed be ascribed to the number and distribution of ionizable substituents along the glycan chain.
In situations where the analyte is too labile for high-pressure pumps or where a gentle, low-shear environment is otherwise preferred, glycan chemists are increasingly turning to footprint-free methods that operate outside of the classical HPLC paradigm. Gel filtration remains the work-horse for buffer exchange, as well as removal of reagent cocktails after reductive amination, while supercritical fluid chromatography (SFC) is emerging as a "green" route to resolve isomers in close proximity, without prolonged exposure of sialic acids to bulk water. The important point is that both approaches can be threaded into multi-stage workflows (size-exclusion cartridges can be placed upstream of affinity columns, or SFC fractions can be collected directly onto graphitic carbon SPE tips) so that the mildness of the first dimension does not preclude the resolving power of the second. Recent hardware miniaturization (3 mm I.D. SEC columns, 1 mL SFC holders) now allows these "alternative" techniques to be deployed on microgram-scale glycomes without sacrificing recovery or reproducibility.
Gel filtration, also known as size-exclusion chromatography, separates glycans by hydrodynamic volume rather than chemical affinity. It is the method of choice for rapid desalting, detergent removal, or fractionation of polydisperse polysaccharides. Available beaded agarose or composite methacrylate supports provide fractionation ranges that straddle the Stokes radii of typical N-glycans (1–15 kDa) while excluding proteases, labeling reagents and excess fluorophores that would otherwise swamp subsequent LC traces. Because the technique is performed under isocratic aqueous conditions, anomeric centers are left unperturbed and acid-labile substituents, such as O-acetyl esters on sialic acids, are retained. Column length can be adjusted to the task: 5 cm cartridges are sufficient for buffer exchange prior to mass spectrometry, whereas 60 cm analytical columns provide size resolution of micro-heterogeneity in high-mannose clusters or chondroitin sulfate oligomers. A recent practical improvement couples in-line refractive-index detection to a miniature fraction collector to make real-time pooling decisions without the need for external UV tags. When SEC is employed as the final "polishing" step, glycans elute in volatile ammonium acetate, permitting direct lyophilization and obviating the dialysis steps that have historically degraded yields.
Supercritical CO2-based separations benefit from the low viscosity and high diffusivity of the supercritical state, which results in faster equilibration between stationary and mobile phase and hence narrower peaks. They also operate at lower temperatures, an advantage for heat-labile glycoconjugates. In addition, because the technique is so powerful at separating native (or permethylated) glycans that lack a chromophore, the high UV transparency of CO2 allows for sensitive detection at short wavelengths without baseline drift. Stationary-phase chemistry is similar to that of normal-phase HPLC (bare silica, diol, amide), but the absence of bulk water suppresses anomerisation during the run, so α- and β-linked isomers that co-elute in reversed-phase systems are often baseline-resolved. Elution strength is tuned by small pressure or temperature ramps, rather than large organic steps, so fractions can be collected directly into aqueous buffers for immediate bio-assay, without solvent evaporation. Recent methodological notes describe the use of SFC for preparative isolation of sialylated glycans: the near-neutral pH of the CO2/methanol modifier prevents acid-catalysed desialylation, and the low surface tension of the supercritical mixture avoids micelle formation that plagues aqueous SEC of amphiphilic gangliosides. Coupling SFC to solid-phase extraction in a "trap-and-elute" format further allows gram-scale processing without the need for high-pressure slurry packing, positioning the technique as a scalable, environmentally benign complement to conventional liquid chromatography.
Purified glycans have long evolved from analytical curiosities to mission-critical reagents that underpin both drug discovery and process analytics/regulatory compliance. In early-stage development, homogeneous oligosaccharides are used as bait to screen fragment libraries for lectin-binding motifs, rapidly accelerating the design of glycomimetic antagonists. Glycan reference standards are also spiked into harvest fluids during cell-line selection to calibrate LC-FLD responses and track batch-to-batch shifts in sialylation or fucosylation before they propagate all the way to manufacturing. Purified glycans are further used as qualification reagents for exoglycosidase arrays, enabling linkage-level deconstruction of candidate biologics without recourse to isotope labels. Perhaps most significantly, clinical-grade glycoforms are now incorporated as surrogate markers in pharmacokinetic modelling: a defined afucosylated mAb glycan can be dosed alongside the therapeutic antibody to provide a real-time read-out of Fc-receptor engagement, de-risking Phase-I trials by flagging exaggerated immune activation earlier than traditional cytokine panels.
Glycan profiling is a standard part of the analytical control strategy for monoclonal antibodies and fusion proteins. The purified glycan pool, released enzymatically and labelled with a fluorescent tag, is analyzed by HILIC-FLD; retention times are correlated against a library of purified standards that collectively represent every major biosynthetic branch, from high-mannose intermediates to tetra-sialylated complex types. Relative quantitation is confirmed by exoglycosidase sequencing, where purified glycans are digested in a stepwise fashion and re-analyzed after each enzyme addition; the disappearance or shift of a peak provides unambiguous linkage assignment without the need for MS instrumentation. The same purified standards are used to spike placebo formulations during forced-degradation studies, allowing investigators to distinguish true glycan degradation products from matrix artefacts. Regulatory submissions now expect a "glycan passport" that traces each observed structure back to a purified reference material, ensuring that clinical lots remain within the quality envelope defined during process validation.
For glycan-based drug candidates, where the glycan is the active pharmaceutical ingredient (API), e.g. a synthetic cancer-vaccine construct or an enzyme-replacement therapeutic, purification processes need to meet both chemical purity and endotoxin requirements. Plant-derived batches of kilograms to multi-ton quantities of complex glycans, purified by initial membrane-based clarification, undergo one or more anion- and cation-exchange polishing steps to remove phenolic pigments and residual protein; this intermediate may then be further purified by size-exclusion chromatography to remove high-molecular-weight impurities that could activate innate immune receptors. Reference glycans purified in-house are added to each column load as internal standards so that step yields can be calculated and to confirm clearance of host-cell DNA or endotoxin to below pharmacopoeial requirements. Final pools are brought up to isotonic buffer, sterile filtered, and packaged under nitrogen to minimize oxidative cleavage of terminal sialic acids. The purified glycan therapeutic is then used to make drug-product placebos for double-blind studies to make sure that excipient glycoforms will not bias the assay read-out. By combining purified analytical standards with clinical grade purification procedures, drug developers establish an uninterrupted quality chain from early discovery to late-stage supply.
Crystallising glycans to an analytical or therapeutic standard of homogeneity is often an iterative process of reducing micro-heterogeneity while maintaining the stability of labile chemical decorations. Differing from peptides or nucleotides, a single mass value can mask dozens of linkage and anomeric isomers whose biological activity can differ greatly, yet whose physicochemical properties differ subtly at best. Lacking a universal chromophore, most derivatization methods are sensitive but inevitably introduce a kinetic bias against large or acid-labile structures. Scaling from analytical micrograms to preparative gram quantities exacerbates the above challenges: longitudinal diffusion in extended columns causes peak broadening of close-eluting species, while over-loading increases yield but decreases resolution. Trace metals, endotoxins, and pigment co-extractives that come along with plant- or tissue-derived glycans poison the enzymes in post-purification digestion steps. This necessitates orthogonal clearance modules, which can be time and sample mass intensive. One of the most challenging issues is the lack of isomerically pure reference standards; without standards, even high-resolution instruments cannot confirm that the "purified" fraction does not contain a minor but bio-active contaminant that co-fragments in the mass spectrometer.
Resolution enhancements now rely on taking advantage of subtle differences in shape rather than prolonging the column. Recycle-HPLC, where the effluent is recycled through the same stationary phase multiple passes, can produce the effective length of a 3-metre bed without suffering the back-pressure penalty, and deliver baseline separation of trisaccharides that differ only by a single α versus β linkage. Detection sensitivity is improved further by next-generation fluorescent tags, that carry multiple sulfonate or phosphonate charges, to supercharge both the fluorescence quantum yield and electrospray ionization efficiency in the same molecule. Combining such chemistry with pulsed-amperometric or charge-transfer detectors can let low-abundance glycans be observed without pre-concentration, minimizing the risk of selective adsorption to glass or plastic surfaces. Temperature programming provides a third selectivity axis: modest cooling to strengthen hydrogen-bonding networks, or slight heating to disrupt hydrophobic collapse, to let the operator fine-tune peak capacity on-the-fly. Taken together, these refinements help turn marginal separation gaps into discrete, collectable bands without prolonging the run, a necessary prerequisite for introducing sensitive downstream assays such as surface-plasmon-resonance or single-cell glycomics.
Three examples, of many, are branched N-glycans, sulfated glycosaminoglycans, and fucosylated human-milk oligosaccharides. Their complexity is not one-, but many-dimensional: size, charge, branching, hydrophobicity, and more can change in parallel. Thus, multiple orthogonal strategies are required. But each additional dimension, each further level of separation, also causes dilution and possible sample loss. By using closed-loop recycle SEC, the first size-based "pre-fractionation" step now routinely processes gram-scale natural mixtures to yield fractions that differ by a single deoxyhexose unit, before the enriched pool is heart-cut onto a porous graphitic carbon column that provides linkage-level resolution. When highly sulfated motifs are targeted, a mixed anion-exchange/hydrophilic interaction column is used in concert with an on-line desalter to eliminate both counter-ion variability and bulk salt before the sample enters the electrospray source, thereby avoiding ion-suppression at the final mass verification step. Importantly, the above workflows make use of isotopically labeled internal standards synthesized in-house; spiking the sample with a known quantity of the per-2H-methylated analogue at the outset allows tracking of its recovery through each orthogonal step, ensuring that minor but bio-active isomers are not being inadvertently lost. The result is a purification cascade that ultimately yields milligram quantities of individual complex glycans whose structures are verified by parallel NMR and ion-mobility MS, thus satisfying the identity requirements of functional glycomics and regulatory guidelines both.
Chromatographic separation of glycans has evolved from an art based on single-column optimization to a multistage approach which integrates size-exclusion pre-fractionation, isomer-resolution recycle-HPLC, ion-exchange chromatography and orthogonal detection modules which provide mass and linkage identity on-line. Carefully sequenced, these multi-step purification procedures turn diverse biological samples or crude synthetic mixtures into homogeneous glyco-species and can document purity by multi-orthogonal analytics. The continuing development of mixed-mode phases, green solvents (supercritical CO2), and magnetic-bead micro-scale platforms will continue to expand the purification toolbox to meet the rapidly growing demand for defined glycans in drug discovery, vaccine design and precision glycomedicine.
Ensure the highest purity and structural accuracy of your synthesized glycans with our advanced glycan purification and analytical services. Using industry-leading chromatographic platforms—including HILIC-HPLC, ion-exchange chromatography, size-exclusion chromatography, and LC-MS-guided fractionation—we isolate complex glycans with exceptional precision, reproducibility, and yield. Our purification workflows are optimized for chemically synthesized, enzymatically synthesized, and chemoenzymatic glycan products, delivering clean, well-resolved fractions suitable for research, assay development, diagnostics, and therapeutic applications. Each purified glycan undergoes comprehensive characterization using LC-MS/MS, MALDI-MS, NMR, monosaccharide composition analysis, and linkage determination. Our solutions help you:
Whether you need purification of complex oligosaccharides, separation of structural isomers, or full analytical characterization, our glycan purification and analysis services deliver the accuracy, clarity, and confidence required to move your project forward.
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