In this fourth part of a series of primers with training experts from the National Institute for Bioprocessing Research and Training (NIBRT), Pauline Rudd, PhD, professor of glycobiology at University College Dublin (UCD), discusses glycan analysis. NIBRT provides training, educational, and research solutions for the international bioprocessing industry in state-of-the-art facilities. Located in South Dublin, it is based on an innovative collaboration between UCD, Trinity College Dublin, Dublin City University, and the Institute of Technology Sligo.
KEY DEVELOPMENT AND MANUFACTURING CONSIDERATIONS
BioPharm: Can you provide a brief overview of what exactly glycan analysis targets and its importance in bioprocessing?Rudd: Most glycoproteins, and almost all of the new biological drugs, are proteins with sugars attached to them. These sugars are important for the safety and efficacy of drugs, so it is necessary to be able to control the processing of the sugar structures to make sure that the drug is as effective as possible. There are many aspects of developing and processing a drug that require having analytical technologies for glycosylation. For example, in the beginning of a drug-development process, one needs to understand the role of the sugars on the protein being used. On erythropoietin, for example, the sugars must be multiantennary and fully capped with sialic acid; otherwise, the erythropoietin will only be in the patient for a few minutes, whereas if it is completely sialylated, it will be present in the patient for 3 hours or more, during which time it will be able to be effective in stimulating the production of red blood cells. It is key to monitor the production process to make sure that the erythropoietin has the sugar structures that provide the full benefits of glycosylation. It is always important for biologics developers to understand exactly how the sugars in their product are going to modulate the functions of the drug in the patient. Once this knowledge is obained, drug developers can define an optimal glycan profile.
The next stage of bioprocessing, clonal selection, requires identifying a high-producing clone that has the ability to fully glycosylate the molecules with the optimal sugars. When selecting clones, glycan analysis enables the producer to determine the complement of glycoenzymes that are operating within a particular clone.
The next aspect of glycosylation that must be checked is the potential introduction of an antigenic epitope. If one is making a product in a nonhuman cell line, such as mouse cells, it is necessary to check the levels of alpha-galactose and N-glycolyl-neuraminic acid residues, which may be antigenic to humans. After the candidate clones are selected, the process of producing the protein from the cells must be monitored. Taking samples during the process allows one to assess whether the media composition is optimal for producing the desired glycosylation profile. In process development, optimizing the media is necessary to produce high levels of correctly folded proteins as well as the desired post-translational modifications, including glycosylation.
BioPharm: What role do glycans play in the manufacturing stage?
Rudd: When making a biologic product, one needs to track the glycans, which will help to determine the best time to harvest the product. When one reaches the downstream processing stage, high-performance liquid chromatography (HPLC) is often used to select subfractions of the product. Being able to analyze the glycans to ensure that their subfractions are correctly glycosylated is crucial. This information will be used during conversations with regulatory authorities so that the agency can agree on specifications for the drug product. Glycan analysis will be part of that specification because it will be part of the regulators assessment of the drug's safety and efficacy. It will also be necessary to demonstrate to regulators that the process is robust and can be reproduced for batch and lot release.
Then comes the consideration of long-term storage. Although in general, glycans are quite stable, it is important to ensure that glycans do not change by testing after degradation and stability studies. Functional assays are also important to determine whether a product, IgG, for example, is able to bind to the desired receptor and not to those receptors that can cause side effects in the patient. Some of these questions require an understanding of how glycosylation modulates activity of the drug.
Overall, there are many reasons to perform glycan analysis and many ways to approach it. It is therefore important to understand the question being asked before deciding which method to use.