Glycosylation is one of the core features that make monoclonal antibody (mAb) drugs successful, to the tune of a $100 billion per year market. Since the extent, locations, and composition of glycans influence both pharmacology and pharmacokinetics, glycosylation is a universally recognized key quality attribute. Drug developers are required by law both to understand fully how their expression systems add glycans to protein backbones, and to characterize them in finished products.
Glycan characterization involves complementary analytical methods operating at four levels: intact glycosylated protein, glycosylated subunits, glycopeptides, and released glycans. Liquid chromatography-mass spectrometry (LC/MS), particularly high-resolution accurate mass MS, offers the simplest, most comprehensive coverage at all four levels but no single method provides all we need to know about glycan composition, structure, and location.
Ensuring quality
According to Philip J. Widdowson, Ph.D., senior applications scientist at Thermo Fisher Scientific, the purpose of glycan analysis “is to ensure the quality of the manufactured product, to confirm its safety and efficacy, and to reliably inform of batch-to-batch consistency in accordance with regulatory guidelines.”
The International Council for Harmonisation (ICH), the nexus between pharmaceutical regulators and manufacturers, has not surprisingly weighed in on structural characterization of carbohydrates bound to therapeutic molecules. ICH Q6B states that:
For glycoproteins, the carbohydrate content (neutral sugars, amino sugars, and sialic acids) is determined. In addition, the structure of the carbohydrate chains, the oligosaccharide pattern (antennary profile) and the glycosylation site(s) of the polypeptide chain is analysed, to the extent possible.
Glycan analysis to ensure the quality, safety, and efficacy of mAb drugs is therefore not an option, but a requirement.
Recombinant glycoproteins are expressed in cell lines that produce predominantly human-like glycan structures. Some glycans, for example the galactose-α-1,3-galactose (α-Gal) moiety found in mice, are potentially immunogenic. So, since the anticancer mAb cetuximab is produced in an Sp2/0 murine cell line, manufacturers must monitor and control glycan expression during production and fully characterize it to ensure safety and batch-to-batch consistency.
The effect of glycans on therapeutic function are well known, for example the relationship between antibody-dependent cell mediated cytotoxicity (ADCC) and core fucosylation in monoclonal antibodies. ADCC is the primary mechanism of action of some mAbs, and lower core fucosylation levels (or increased afucosylation) leads to an increase in ADCC.
Another area where glycans affect function relates to oligomannose (high-mannose) glycans, which bind to mannose receptors, expediting protein clearance from the bloodstream. “Circulatory half-life is a critical pharmacokinetic criterion contributing to overall function of a therapeutic, and in fact IgGs containing high-mannose glycans are cleared more rapidly than those which do not,” Widdowson explains. “Similarly, glycan sialylation increases circulating half-life by blocking accessibility of terminal galactose.”
Multiple levels
Intact-level analysis is by far the simplest analytical approach to glycan characterization. Although mAbs are quite complex molecularly, some exhibit very simple glycosylation patterns. Most therapeutic mAbs incorporate a single glycosylation site, in the Fc region. The primary advantage of analyzing intact proteins is its simplicity: little to no sample preparation and low sample requirements. Its main limitation is the potential to miss fine details such as low-abundance glycoforms.
Subunit-level analysis breaks mAbs down into more manageable molecular “chunks” before LC/MS analysis. Subunits are less complex and possess lower mass than intact antibodies, details missed in intact analysis are often observed. Subunit analysis requires some sample preparation but several vendors offer enzyme kits for this purpose. Widdowson mentions the FabRICATOR kit from Genovis as an example.
Glycopeptide mapping may not be necessary for glycoproteins containing a single glycosylation site, according to Widdowson. “The primary advantage of glycopeptide mapping over released glycan analysis is that the glycan remains attached to the peptide backbone during analysis, which enables localizing the presence of glycans to individual sites, an approach known as site-specific glycan analysis.”
Specialized MS fragmentation techniques, such as electron transfer dissociation and electron-transfer/higher-energy collision dissociation, enable simultaneous characterization of glycan and peptide moieties, and localization of glycosylation sites. “These analysis modes will become more important as manufactured biomolecules become more complex, with multiple glycosylation sites, which demand greater understanding of site-specific glycosylation,” Widdowson tells Biocompare.
Released glycan analysis, involving the enzymatic or chemical cleavage of glycans from the protein before analysis, is the most common, and information-rich method for analyzing mAb glycosylation. The chemical nature of glycans requires derivatization to enhance ionization efficiency for MS and to enable optical detection. “This makes released glycan analysis the most demanding and complex with respect to sample preparation,” Widdowson says, noting that prep methods “have improved dramatically” in recent years. Glycan release has also improved toward greater robustness and reliability, improving their utility for quality testing laboratories.
Released glycan analysis provides global information on the total glycan population, and reliably detects glycans down to 0.5% relative abundance. “The potential clinical relevance of these glycans is why this approach is favored in biopharma,” Widdowson says.
Ounce of prevention?
Since glycosylation is at least in part under the control of process conditions, mAb manufacturers can save themselves a lot of grief during quality testing if they design processes in ways that favor the expression of desirable glycan modifications and away from undesirable ones. “The expression, ‘the process is the product,’ is as true today as ever,” says Widdowson.
Glycosylation is particularly susceptible to bioreactor and process conditions such as pH, temperature, dissolved oxygen, osmolality, and levels of glucose and glutamine. Cell lines, culture method, the nature of the expressed protein, and culture media ingredients also play roles.
“To this end, there is a growing need for comprehensive process development activities, often involving a design of experiment, or DoE, approach, to modulate glycan product quality,” Widdowson explains.” Robust, high-throughput glycan analysis supports this activity.
Glycoanalysis is therefore critical not only for quality testing, but during process design and development as well. Analytical system providers have met these needs with instruments, systems, and protocols for analyzing glycans efficiently and reproducibly.
“As the biotechnology industry continues to move toward more complex molecules our analytical methodology must evolve as well, particularly analytical instrumentation suited to glycan analysis,” Widdowson says.