September 15, 2016 (Vol. 36, No. 16)

Angelo DePalma Ph.D. Writer GEN

The Baker’s Secret? Glycan Analysis. With Glycomic Instrumentation, Kits, and Workflows, Glycosylation Is a Piece of Cake.

Post-translational modifications (PTMs), particularly glycosylation, attract attention because they may favor health or disease. Also, since proteins subject to PTMs may tip biomolecular interactions down one pathway or another, PTMs are of intense interest in biopharmaceutical development. 

Glycans on glycoproteins participate in a wide range of physiological processes, including recognition and regulatory functions, cellular communication, gene expression, cellular immunity, growth, development, and cancer progression.

What is of interest for one class of biopharmaceuticals may not be relevant for others. At one time, Synthon Biopharmaceuticals boasted extensive experience in glycoengineering as part of its development program for secretory immunoglobulin A antibodies. “We used several tools to control glycosylation patterns,” recalls Marco Timmers, Ph.D., CSO, Synthon. “Such control was necessary to produce secretory immunoglobulin A antibodies.” Synthon eventually abandoned that program to pursue antibody-drug conjugates (ADCs).

One goal of glycoengineering is to empower antibodies, through increased antibody-dependent cell-mediated cytotoxicity, to recruit immune system cells to assemble, attack, and kill more tumor cells. Next-generation antibodies like obinutuzumab, which are active against CD20 surface proteins, work this way. But immune cell recruitment is undesirable in ADCs for several reasons.

First, the mechanism of tumor cell death with ADCs is internalization of cytotoxic payloads by cancer cells, not immune cell recruitment. Cancer cells are therefore killed directly, not through immune mechanisms.

Second, recruiting immune cells to the tumor site would expose them to the ADC’s cytotoxic effects, thereby killing them and reducing the potential of the body to fight the cancer.

“So there’s little reason to glycoengineer ADCs,” Dr. Timmers concludes.

MS Key for Analytics

Mass spectrometry (MS) methods are revolutionizing glycomics. Moreover, these methods are becoming more widely available because they are reflected in the design of new systems and software.

Sciex, for example, offers the TripleTOF® 6600, a high-resolution, accurate-mass system for biologics characterization, along with BioPharmaView™ 2.0 software for interrogating and examining PTMs. The TripleTOF system’s built-in workflow is able to dig deeply into samples and collect information on low-level PTMs that are of interest to the biopharmaceutical industry.

For example, deamidations in the complementarity-determining region of a protein can affect protein binding. Both oxidation and deamidation might alter tertiary structure and therefore binding and efficacy. “An emerging interest is examining very low-level immunogenic glycan epitopes such as alpha-galactose and high-mannose species that could either change the drug’s clearance level or elicit an undesirable immune response,” says Sean McCarthy, Ph.D., senior market manager for biologics at Sciex.

Glycopeptide sample preparation typically involves proteolytic digests that leave glycans intact, or methods that de-glycosylate but entail greater complexity.

Antibodies normally feature two glycosylation sites, although some have up to four. De-glycosylation indicates where sugars are located. Also, this approach creates sites that are completely deamidated. “This is more of a confirmatory approach, particularly for sites are not within the molecules conserved region,” Dr. McCarthy explains.

Reverse-phase peptide maps provide a first-pass view of the major glycan species. More detailed analysis requires separation of the digested sample, with the preferred method being hydrophilic interaction liquid chromatography (HILIC). In the methods that enable more detailed analysis, nonglycosylated peptides are retained much less on the column, while the glycosylated peptides separate quite well. This kind of separation allows investigators to characterize glycans on specific peptides and recognize thereby site-specific heterogeneity.

Sciex develops solutions for analyzing biologics glycosylation and post-translational modifications, as this biologics characterization workflow suggests. Notice that the workflow incorporates the TripleTOF 6600, a high-resolution accurate-mass system, and BioPharmaView, software designed to accelerate characterization and comparability studies. Together, TripleTOF 6600 and BioPharmaView can deliver accurate assay information for protein digests. Other functions include the profiling of intact protein glycoforms and the identification and quantification of specific glycopeptides.

Exponential Growth

According to Julian Saba, Ph.D., glycoproteomics specialist for mass spectrometry at Thermo Fisher Scientific, interest in instruments for studying glycosylation has grown exponentially; glycosylation and phosphorylation were the two most popular PTM topics at the most recent meeting of the American Society for Mass Spectrometry.

“Glycans are more challenging analytically because we are not dealing with a single structure as we do with PTMs such as acetylation, methylation, or phosphorylation,” explains Dr. Saba. “Multiple glycans can attach at a single site. So we not only have to localize the site, we also have to determine all the different glycoforms. This is something that other PTM analyses do not deal with.” Further complicating analysis is the low abundance of glycosylated peptides and multiple isomers, known as glycoforms.

Glycoprotein characterization has historically been impeded because of the fragmentation methods employed by commercial mass spectrometers. One technique, collisional activated dissociation, was unsuitable for glycosylation analysis because it fragments glycan moieties preferentially rather than the peptide backbone. “Collisional activated dissociation,” notes Dr. Saba, “made it very tough to localize glycosylation sites and obtain information on the peptide backbone.”

Dr. Saba also suggests that the study of glycans has been given the short shrift because researchers have been focused on PTMs that are more easily accessible. This disparity, however, is being lessened through the emergence of fragmentation methods such as electron-transfer dissociation that enable in-depth glycan study.

Characterizing glycosylation involves combining both glycoproteomics and glycomics studies. The glycomics portion involves cleaving and analyzing glycans without the protein, then determining where glycosylations occurred. With reference to glycan databases, researchers can correlate glycan structures to actual locations on the protein.

Glycomics workflows, based on octadecyl carbon chain (C18) reverse-phase liquid chromatography separations, resemble those of proteomics. Dr. Saba recommends that for this step academic researchers stick with their preferred proteomics setup employing similar separations.

“This enables glycan analysis without overhauling LC-MS setup,” he says, with the exception that glycans should be permethylated for the separation step because they otherwise do not ionize or fragment well in MS. Permethylation also enables generation of useful fragment ions for structural identifications.

“Now you can obtain meaningful information,” asserts Dr. Saba. “Not only can you learn about glycan composition, you can also explore glycan links and branches, which is difficult to do when glycans are in their native forms.”

Dr. Saba’s approach allows researchers to perform LC-MS glycan characterization through an LC-MS setup with which they’re already familiar. “We’re trying to get make it easy to do glycomics,” insists Dr. Saba. “It should not be complicated.”

Protein glycosylation affects the behavior of cellular receptors as well as biotherapeutic molecules interacting with them. [Thermo Fisher Scientific]

Non-MS Methods

The emergence of ultra-high-performance liquid chromatography and MS in glycomics workflows has been welcome, but sometimes researchers need only answer a yes/no question: Does a particular PTM exist or not? This is where anti-PTM antibodies play a significant role.

Abcam, a company that produces reagents and kits for biology, offers monoclonal and polyclonal antibodies against post-translationally modified proteins. Targets include glycosylated proteins as well as those that have undergone methylation, acetylation, and phosphorylation—proteins that are structurally nearly indistinguishable from their original forms. Methylation of histone proteins, in particular, is indicative of epigenetic changes, whereas changes in phosphorylation suggest protein activation or deactivation through the activity of kinases.

“A mouse’s immune system has proved to be fairly unsuccessful in giving rise to antibodies against the smaller PTMs,” says Alejandra Solache, Ph.D., head, reagents product development and manufacturing, Abcam. “But the rabbit’s more diverse immune system can produce antibodies that recognize very small modifications, mutations, or even conformational changes. Abcam’s RabMAb antibodies benefit from this superior antigen recognition.”

Applications of anti-PTM antibodies include immunohistochemistry (which can differentiate healthy from tumor tissues), identifying signaling cascades in systems biology, or (for developers of therapeutic biologics) the elucidation of post-translational changes associated with treatment or disease progression. Anti-PTM antibody binding events are suitable for analysis through flow cytometry, immunoprecipitation, Western blot, and high-throughput drug-screening assays. Dr. Solache stresses that it is critical that the antibody “be specific enough to recognize the PTM.”

Abcam goes to great lengths to validate its antibodies to ensure specificity. One technique compares modified and nonmodified peptides that mimic the modified or unmodified protein to assay antibody specificity. The company also uses a peptide array containing 501 modified and unmodified histone peptides, which has been used to validate new anti-PTM histone RabMAb and other antibodies, and is developing an even more extensive peptide array.

ProZyme is a reagents company whose main products are sample preparation kits for N-glycans that release glycans with PNGase F and subsequently label them with fluorescent tags. The company has also recently begun developing instruments. “Glycans don’t work well with ultraviolet detectors, so fluorescence is a good alternative to MS,” says Aled Jones, Ph.D., senior product manager.

The company’s kits prepare samples for HILIC, which separates most Fc N-glycans well. An alternative separation method, involving capillary electrophoresis (CE), uses a different, negatively charged fluorescent dye that assists with the separation. “Neutral glycans coupled with neutral dyes won’t move in CE,” Dr. Jones adds. Specific detection modes are laser- or LED-induced fluorescence for CE and simple fluorescence for LC. ProZyme is developing a CE instrument which, according to Dr. Jones, is easier to use than either LC or existing CE instruments. The instrument is a repurposed DNA fragment analyzer that in a previous iteration used capillary gel electrophoresis for DNA separation.

ProZyme’s CE is advantaged by its very short run time, about two minutes per sample. This kind of speed is a huge bonus for users who screen many samples during early-stage culture media and condition development.

MS provides much deeper characterization than fluorescence alone, but the two methods together provide added flexibility for glycoanalysis.

The major glycans customers look for are IgG Fc-domain N-glycans at the asparagine 297 site. “Depending on the molecule,” says Dr. Jones, “such glycans can be a critical quality attribute.” If the mechanism of action includes antibody-dependent, cell-mediated cytotoxicity, the presence of core fucose N-glycans might matter.

“Having fucose or not can affect activity by around two orders of magnitude,” notes Dr. Jones. “That’s why Seattle Genetics and Genentech have developed technologies for limiting fucose in their antibody products.”

Biosynthesis pathway of N-linked glycoproteins [Wikimedia Commons/Dna 621]

What You Don’t Know

Does glycan analysis open up a can of worms, or perhaps raise regulatory questions that would not be asked if detailed analysis tools were either unavailable or not applied? The short answer, according to Sciex’ Dr. McCarthy, is that regulators are quite advanced in what they expect in a submission package.

“Understanding a molecule thoroughly provides the ability to have a much deeper conversation with regulators,” explains Dr. McCarthy. “The more difficult question is what the impact of different glycans might be. Are they clinically meaningful or not? If they are, they become critical quality attributes, so the more you characterize them the better.”

More-detailed information becomes part of the submission package and may even protect originator companies from competition through biosimilars. A “set” glycsolyation pattern would then be something competitors would have to figure out on their own.

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