Glycan Analysis Becomes a QC Specialty

Glycosylation, a significant post-translational modification of many therapeutic proteins, in particular monoclonal antibodies (mAbs), has become big business. Due to the limitless combination of glycan building blocks, chain lengths, and arrangements—including positional and structural isomers—and the fact that sugars are nearly indistinguishable analytically, glycan analysis has become an analytical specialty that is increasingly outsourced. With glycosylation a major “critical quality attribute” for monoclonal antibodies, glycan analysis comprises a significant approach to process monitoring, quality control, and for drugs so designated, for establishing biosimilarity. Glycans and their expression in proteins similarly serve as selection criteria for cell lines expressing mAbs and other biotherapeutics.

Move over, LC-MS

Liquid chromatography-mass spectrometry (LC-MS) has become the standard analytical platform for glycan analysis but baseline resolution of key structures can take several hours. Even then, isomeric glycans and glycopeptides or those with similar or identical masses, do not always separate. In June 2021, MOBILion Systems introduced MOBIE, the company’s first commercial high-resolution ion mobility mass spectrometry (HRIM-MS) system, based on a novel separation technology developed at Pacific Northwest Laboratory in 2015. The driving force behind MOBIE is “structures for lossless ion manipulation” (SLIM), which takes a very long ion mobility analysis path and folds it back onto itself many times, which reduces its footprint. A linear 13-meter flow path, for example, which is impractical for routine laboratory analyses, may be reduced to the size of a briefcase. Species entering the analysis path are guided by electric fields around the path’s turns in a way that avoids collisions between the analyte and the device, hence the term “lossless” to describe the molecules’ paths.

HRIM-MS differs from LC in several ways. First, it occurs in the gas phase vs. solution for LC. Where LC separates molecules based on charge or molecular weight, HRIM-MS exploits analytes’ size, charge, shape, and structure. HRIM-MS also reduces analysis times from the typical 15-minute to 3-hours to around two minutes, thus essentially eliminating the throughput bottleneck. The technique is applicable to monoclonal antibodies, intact and subunit proteins, peptides, and glycans, from the research stage through production.

“LC separation of highly similar biomolecules—like many glycans—requires extended run times, creating a potential bottleneck in analytical workflows,” says Melissa Sherman, Ph.D., CEO of MOBILion Systems. “Even then, some glycans behave so similarly that LC coelution cannot be avoided. By contrast, two-minute runs with a MOBIE-enabled MS system provide a high-throughput and -resolution option for separation and analysis of proteins, peptides, glycans, and PTMs in research settings. We believe the method potentially decreases barriers to adopting more complex MS-based characterization assays in process-monitoring and manufacturing.”

Many if not all analysts working with glycans struggle against the “isomer” problem—same molecular weight but different arrangement of sugar residues. “HRIM-MS can really help with identifying isomer profiles with enhanced resolution and throughput,” Sherman tells Biocompare. “Glycosylation profiles requiring a six-hour separation by LC resolve with HRIM in about two minutes.”

Multi-level possibilities

Glycan analysis on therapeutic proteins, aka “glycoproteomics,” occurs at roughly four levels: On the intact protein, through partly digested protein, on individual glycosylated peptides, and after first releasing glycans from the protein.

Glycan analysis level depends on the aim of investigation and the specific analyte structure, says Xiangkun “Shawn” Yang, Ph.D., Sr. Scientist at Prime Medicine, a gene-editing therapy company. “For example, when there are multiple glycan sites in one protein, you may need to digest the protein to glycopeptides to study each individual site.” Several “levels” may also be addressed during molecule characterization, in preparation for a regulatory submission, “where multiple orthogonal methods are used to characterize glycans at released glycan, glycopeptide, and intact glycoprotein levels.”

Analysts turn to released glycan for accurate, comprehensive glycan profiling on individual N- and O-glycosylation sites on glycoproteins expressed by cells, tissues, or plasma/serum.

Glycopeptide analysis, on the other hand, is used to study glycans on one or more proteins. “This method's key feature is its ability to link glycosylation information to glycosylation sites on proteins,” says Linda Brown, Research Scientist at CD BioGlyco. “However, analysis of intact glycoprotein therapeutics is required for quantifying N-/O-glycosylation of purified glycoprotein therapeutics, including calculating each glycan’s percentage on glycosylation sites and estimating the structure of each N/O-glycan.”

BioGlyco specializes in improving biotherapeutics through glycan analysis, a generally under-utilized strategy with significant potential.

We tend to think of glycan analysis as related primarily to quality functions, but glycoproteomics also has a role to play in drug discovery and creating improved versions of existing biopharmaceuticals.

“Glycosylation is a highly promising target with humongous potential for personalized medicine,” Brown continues. “Glycans play important roles in biological processes, including cell adhesion, macromolecular interactions, and pathogen invasion. Glycomics complements genomics, metabolomics, and proteomics, and shows promise for personalized medicine. For example, glycobiomarkers for certain diseases may be specific to individuals, so targeting the specific protein glycosylation is a promising strategy for clinical treatment.” These strategies may be applied to existing biotherapeutics for “personalization,” as well as to newer compounds specifically targeting glycosylation.

An all-stage activity?

Because of the expertise and time involved, developers of biotherapeutics tend to reserve glycan analysis for the bookends of a product’s lifecycle—during the very earliest characterization stages of discovery and development, and in QC. That is changing as the role of glycosylation becomes better understood.

“Glycoproteins play key roles in molecular and cellular functions,” says Jing Zhang, Ph.D., Chief Technology Officer at Creative Biolabs. “The stability, serum half-life, immunogenicity, and biological activity of therapeutic glycoproteins are regulated by their glycosylation profiles. Thus, there is increasing demand for characterization of glycosylation at all development stages for biologics.”

Increasingly, developers perform glycan analysis during production, as well as for quality testing. Creative Biolabs specializes in strategies for high-throughput glycan screening on antibodies, Fc fusion proteins, non-Fc fusion proteins, and in glycoengineered cell lines. “Most importantly, analysis is performed in real time, to monitor production processes.” One strategy, involving high-throughput glycan screening through UHPLC with fluorescence detection, screens up to 100 samples in under two weeks and works with crude clarified culture media. The second screens in high throughput through a fluorescence-labeled lectin-based method and takes about two hours, according to Zhang.

As the significance of glycosylation is better appreciated, glycan analysis is becoming a specialist activity requiring time, instrumentation, and expertise that drug developers increasingly lack or prefer not to acquire. Since QC of glycosylation modifications occurs only at specific times (albeit with increasing frequency) enlisting a third-party service provider may be more convenient and cost-effective than maintaining this capability in house.

More than 130 organizations worldwide specialize in glycan analysis. According to a study by U.K.-based research firm Roots Analysis, annual growth in this segment, which serves the needs of the $130 billion annual market for glycosylated protein drugs, will be 15% over the next several years.