Glycan Characterization for Protein Drug Development

Glycosylation, or the attachment of sugar moieties to target macromolecules (ex: proteins), is an important aspect of protein drug development. A large fraction of protein therapeutics, which include monoclonal antibodies, enzymes, hormones, and vaccines, are glycoproteins. Monoclonal antibodies (mAbs) alone represent just over half of all drug approvals between 2019-2022.

In this article, we discuss glycan characterization in the context of drug development including the importance of glycan characterization, how glycosylation impacts the drug development process, and glycan analysis methods.

Why is glycan characterization important for protein drug development?

“Glycan characterization plays a pivotal role in protein drug development, and its importance stems from the profound impact that glycans exert on the properties, efficacy, and safety of therapeutic proteins,” says Davy Petit, Senior Director and Portfolio Owner, Biologics at Waters Corporation. “Glycans can significantly influence the biological activity of therapeutic proteins by modulating their interactions with receptors and other molecules. As an example, mAbs are highly sensitive to glycosylation changes.”

Glycosylation can also enhance the stability of the protein therapeutic against proteolytic degradation, oxidation, precipitation, and denaturation. As glycosylation can shield hydrophobic regions on the glycoprotein’s surface, it can prevent protein aggregation, which can help decrease immunogenicity. Glycosylation can also hide immunogenetic protein epitopes on the glycoprotein’s surface.

While glycosylation has many effects on glycoprotein drugs, it’s challenging to ensure that the final drug product has desired glycosylation patterns. The glycosylation patterns on a protein can be heterogeneous in terms of where the modification occurs and the modification itself. “As the modality of therapeutics becomes more and more complex, the heterogeneity of glycoforms increases as well,” says Roxana McCloskey, Senior Global Marketing Manager Protein Therapeutics at SCIEX. Therefore, the ability to control glycosylation and to characterize it is important throughout drug development.

Considering glycosylation throughout the entire drug development process

Glycosylation impacts various stages of the drug development process and therefore it’s important for glycan analysis and control to happen throughout the bioprocess. “Glycan profiling starts from early discovery cell-line optimization and occurs through the entire development,” says Zoe Zhang Ph.D., Senior Manager Biopharma Applications at SCIEX.

Cell-line development

Because protein therapeutics are generated from mammalian cell lines or bacterial expression systems, it’s important to consider how the host cell choice (ex: some cell lines have well-characterized glycosylation profiles), the protein itself, and the culturing conditions can affect the glycosylation profile. “By manipulating the genetic makeup of the cells used to produce therapeutic proteins, scientists can steer the glycosylation process in a more controlled manner. This helps in achieving a more uniform and predictable glycan profile,” says Petit. Elements of the expression vector, including promoters and DNA regulatory elements, can all affect glycosylation patterns.

Upstream bioprocess

During the upstream process, bioreactor conditions can alter the glycosylation of proteins. “Adjusting factors such as nutrient levels, temperature, and pH can influence how these sugars are added to the protein,” says Petit. The impact of these factors on glycosylation has been studied extensively.

Downstream bioprocess

Many methods can be used during the downstream process to purify the drug product from cell culture. Lectin-based purification and boronate-based methods can be used to specifically capture glycoproteins from the cell lysate. However, the heterogenous mixture of various glycoforms poses a challenge for regulatory processes. “Purification techniques are carefully selected to separate different glycoforms, ensuring that the final product is not just pure but also consistent in its glycan composition,” says Petit.

Regulatory compliance

Regulatory bodies, such as the U.S. FDA and the European Medicines Agency, have their own guidelines regarding protein therapeutics glycosylation. “The FDA often mandates comprehensive glycan characterization as part of the approval process for new biologics, ensuring compliance with safety and efficacy standards,” says Petit. While each regulatory agency has specific requirements, glycan analysis is used to ensure consistency across batches, assess how changes in the manufacturing process affect protein glycosylation, or characterize glycoform heterogeneity.

Methods for glycan characterization

There are many methods used to characterize the glycan profile of protein therapeutics. “It’s important to formulate characterization strategies with a tool box or orthogonal approach, with no one technology or method answering every possible need, especially with the increasing complexity of biotherapeutics,” says Zhang. Oftentimes, this involves analyzing released glycans, glycopeptides, and/or intact glycoproteins with methods such as liquid chromatography, capillary electrophoresis, and/or mass spectrometry.

“Usually at early discovery, intact protein analysis is used to get a general glycan profile for screening,” explains McCloskey. On the other hand, glycopeptide analysis maps out the glycosylation sites on a protein and where the glycans are attached while released glycans can provide information on the glycan structure itself.

Liquid chromatography

Liquid chromatography (LC) separates glycans from a mixture by size, charge, or polarity. It can quantify glycan levels in a sample relative to one another. “Liquid chromatography affords deeper characterization and profiling of glycosylation, creating glycoprofiles, and can be used to set or define CQAs for a particular molecule,” says Zhang. While there are many types of LC, hydrophilic interaction liquid chromatography (HILIC) is most often used, according to Zhang. However, LC has its drawbacks in some situations. “Although suited for deep characterization, it could be considered too time consuming and cumbersome for rapid high-throughput screening assays needed early in development or in release assays further downstream,” adds Zhang.

Pros

• Deep characterization of glycans
• Compatibility with mass spectrometry

Cons

• Labor intensive with multiple parameters needing to be optimized
• Long run times
• Some glycans may co-elute (ex: isomeric glycans (same weight but different sugar residue arrangements), glycans with similar masses)

Capillary electrophoresis

Capillary electrophoresis (CE) separates based on electrophoretic mobility (charge and size). This method allows the separation of isomers and different glycan structures and can be useful to resolve complex glycan mixtures. “If high resolution, compatibility with mass spectrometry, and a wide range of column options are critical, LC might be the preferred method,” notes Petit. “On the other hand, if rapid analysis, high separation efficiency, and sensitivity are more important, CE could be the method of choice, especially in situations where speed is essential.”

Pros

• High separation efficiency
• Rapid characterization

Con

• Limited sample capacity

Conclusions

Glycan analysis is an important part of the drug development process and has roles throughout the bioprocess. As glycosylation is a complex attribute of protein therapeutics, new innovations in glycan analysis methods will propel the development of protein drugs. “Glycan characterization is a cornerstone in the development of protein drugs, providing insights that enable researchers and manufacturers to fine-tune therapeutic proteins for optimal performance, adhere to regulatory requirements, and ultimately enhance patient outcomes,” says Petit.