The Role of NMR and Mass Spectroscopy in Glycan Analysis

In biological systems, most peptides and proteins are glycosylated. Differences in glycosylation can affect protein structure, stability, immunogenicity,

in-vivo

half-life, and function. Glycans, in fact, are important to normal cellular function, mediating interactions within cells and between cells and pathogens, enabling immune responses, and influencing tumor progression. “In mammals, sugars are most commonly found as glycoconjugates, the most abundant being the glycoproteins, proteoglycans, and glycolipids. These important biological substances are predominantly located on cell membranes, but are also found in secreted fluids where they modulate or mediate a host of events in cell-cell and cell-matrix interactions,” notes Brady Clark, founder and CEO of Sussex Research Laboratories, a Canadian CRO focused on the synthesis of glycans and glycoconjugates, including glycans on functionalized linker/spacers, glycoamino acids, glycopeptides, glycolipids, and glycans on carrier proteins. In addition, Sussex Research is also active in the determination of free (or non-conjugated) reducing sugars in biological samples.The glycosylation of biologic drugs affects their mechanism of action, pharmacokinetics, and efficacy. Glycoconjugation of a biotherapeutic (peptide, protein, or antibody) may, therefore, confer increased therapeutic efficacy by increasing the stability, aqueous solubility, and/or bioavailability; enhancing the target resolution; and/or extending the

in-vivo

half-life. “Glycans are involved in nearly all aspects of the biochemistry of life, and proteins and peptides in the body can’t be evaluated without looking at glycans,” states Garnet McRae, director of bioanalytical services with Sussex Research Laboratories in an interview with

Pharmaceutical Technology.

“Recently, people have begun to realize the importance of glycosylation, and thus there has been more interest in the synthesis and analysis of glycans,” he adds.

Clark, McRae, and Dennis Whitfield, who sits on the scientific advisory board at Sussex Research, spoke with Pharmaceutical Technology, about the analytical methods used at Sussex Research for both the structural determination of glycans during complex synthetic processes and the quantitative evaluation of free sugars involved in biological processes.

Structural determinationPharmTech: Why is structural determination so important during glycan synthesis?

Clark (Sussex): It is as important to be able to characterize the actual structures that we create as it is to know how to synthesize them. Unlike peptides, which are linear structures composed of amino acids with just two reactive sites, glycans are comprised of monosaccharides with up to five hydroxyl groups per molecule that can be reactive. They can, therefore, be linear or highly branched, contain multiple sites of glycosylation, and be linked in either an alpha or beta fashion. It is imperative to know exactly how each saccharide has been linked, which means confirming that the reaction occurred at the correct hydroxyl group and with the right stereochemistry.

We use highly effective and, in general, well-understood chemical, enzymatic, and chemoenzymatic methods for the synthesis of complex glycans that are often drug candidates. These methods may also be used as standards at biotechnology and biopharmaceutical companies as well as research laboratories. While we have extensive experience in synthesizing glycans and generally obtain the desired products, no process is 100% fool proof. Therefore, we comprehensively confirm the structure of each intermediate throughout a synthesis.

PharmTech: What are the major analytical instruments that you use for structural glycan determinations and why?

Clark (Sussex): We use methods such as high-resolution proton and carbon-13 nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry, including both electrospray ionization and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF).

NMR analysis is the key to analyzing the structure of complex glycans. By evaluating the chemical shifts and coupling details, it is possible to confirm the site of attachment and the stereochemistry of the saccharide linkage, which are the two crucial factors in glycan synthesis. With the advanced two-dimensional (2D) NMR techniques available today, it is possible to decipher the structures of very complex glycans.

Because mass spectrometry does not provide real structural information, it is mainly used to confirm that we have synthesized the expected compound since we use well-established methods to prepare the glycans.

Other methods that were previously commonly used for structure determination, such as optical rotation and elemental analysis, are now only used on very rare occasions and only when requested by a client. Much more useful and specific information can be obtained via NMR analysis in combination with mass-spec evaluation.

PharmTech: Which advanced 2D NMR analyses do you use and what do you learn from each of them?

Whitfield (Sussex): For some of the most complex glycan structures we synthesize, it wouldn’t be possible to determine the structures without 2D NMR techniques. The methods we use for nearly every sample are 2D ¹H-¹H-gCOSY (gradient correlation spectroscopy), which takes approximately 15-30 minutes, and ¹³C-¹H-gHSQC (gradient heteronuclear single quantum coherence) spectroscopy, typically the edited version, which distinguishes CH and CH₃ groups from CH₂ groups, and takes about 2-3 hours.

There are several additional methods that are used frequently, but not for every sample. These techniques include ¹³C-¹H-gHSQC with proton coupling for measurement of ¹³C-¹H 1-bond couplings (3-4 hours), ¹³C-¹H-gHMBC (gradient heteronuclear multiple-bond correlation) spectroscopy and ¹H-¹H-NOESY (nuclear overhauser effect spectroscopy) (or ROESY [rotating frame effect spectroscopy]) for determination of the connectivities across linkages and positions of protecting groups (3-4 hours each), and ¹H-¹H-TOCSY (total correlation spectroscopy) for the determination of inter-ring connectivities if the COSY analysis is incomplete (3-4 hours).

We have found, however, that the higher digitization of selective 1D (1Dsel) experiments, such as 1Dsel-¹H-TOCSY and 1Dsel-¹H-NOESY (or ROESY) allow for the determination of coupling constants and work better then 2D for polymer-bound and other complicated samples. They are thus used to determine inter-ring connectivities if the COSY analysis is incomplete (2 hours) and are particularly applicable if the anomericity (alpha versus beta) cannot be determined from the coupling constants, and most commonly for derivatives of L-iduronic acid and D-mannose. In addition, for phosphates or other similar phosphorous-containing species, ³¹P-¹H correlation can be useful.

It is important to note that there are many variants of these spectra, and each is applicable to certain circumstances. In addition, there are many so-called fast NMR techniques that greatly reduce the time to acquire the data and complete these types of experiments, which will have a significant impact on turnaround times.

PharmTech: What are some of the challenges that you face when carrying out structural determinations of complex glycans?

Clark (Sussex): Challenges are mostly presented by the compounds themselves. Larger, more complex glycans, such as those that contain numerous glucosamine groups, can have poor solubility. Glycans with charged structures and a large number of heteroatoms, such as iduronic acids, glucosamines, O-sulfates, and amine-sulfates, can also be difficult to analyze. Often, in these cases, various solvents must be investigated to find a suitable one for NMR analysis. In some cases, the NMR spectrum is so complex that individual peaks cannot be discerned. In many of these situations, smaller sections of the molecule are analyzed independently, and the NMR spectra of the smaller segments are then used to assist in obtaining the complete spectrum of the glycan.

Quantitative analysis of sugars
PharmTech: What information can be gained from the quantitative analysis of sugars in biological systems?

McRae (Sussex): While determining the structure of glycans is crucial for confirming the synthesis of appropriately glycosylated compounds, the quantitative determination of free sugars in biological systems, as well as the total sugars (bound plus free, which is obtained after cleaving the bound sugars), can also provide valuable information about biological processes. In cellular and fermentation systems, various sugars are used as feeds, and other sugars are produced as by-products. By monitoring the consumption and production of these various sugars over time during reactions run under various conditions, it is possible to optimize a biological process in order to obtain the maximum amount of desired product.

PharmTech: What instrumentation do you use for the quantitative determination of sugars and why?

McRae (Sussex): Liquid chromatography-mass spectrometry (LC–MS); in particular LC-MS/MS, is ideally suited for this type of analysis. Mass spectrometry is crucial in this application because it is highly specific and sensitive in the detection of sugars at very low levels. Because the compounds are already known and standards are used for quantitation, LC–MS/MS is a very powerful targeted technique for quantitation.

PharmTech: What recent advances in technology have been of importance for carbohydrate analysis?

McRae (Sussex): The increasing resolution and sensitivity of mass spectrometers and the growing choice of instrument types has had a real impact on glycan and sugar analysis. The development of some new derivatizing agents has also been important.

PharmTech: Why is derivatization required for the quantitative determination of sugars?

McRae (Sussex): Derivatization is a key step in the quantitation of free reducing sugars in solution when using reversed-phase LC–MS/MS. There are many different isomeric sugars with the same molecular weight and only slight differences in structure. There are, for example, three common six-carbon sugars that occur naturally in biological systems—galactose, mannose, and glucose. All have five hydroxyl groups, and it is only the stereochemistry at the hydroxyl groups (axial or equatorial) that differentiates these compounds. While this slight difference can lead to very different reactivity for each molecule, it is not easy to separate them from one another on a reversed-phase column unless they are derivatized.

A common derivatizing reagent for carbohydrates is 1-phenyl-3-methyl-5-pyrazolone (PMP). Not only are the derivatives of isomers prepared using this reagent separable by reversed-phase HPLC, but also up to 100 times more sensitivity during mass-spec analysis can be achieved. The presence of the nitrogen groups in the derivatizing reagent are important for increased ionization and fragmentation efficiency while the added phenyl and aliphatic character provides increased reversed-phase HPLC retention.

Two additional compounds, 2-aminobenzoic acid (2-AA) and 2-aminobenzamide (2-AB), are also classically used as derivatizing agents for free reducing sugars, particularly for quantitative glycoprofiling of biopharmaceuticals such as therapeutic proteins and monoclonal antibodies.