Glycobiology and Therapeutic Glycoproteins
Glycoproteins are the active ingredients in most biologic products. At the Centre for Biologics Evaluation, our glycobiology research helps provide new information and tools for studying biologics.
- Why we study glycoproteins
- How we study glycobiology and therapeutic glycoproteins
- Concepts and tools we use to study glycobiology and therapeutic glycoproteins
- Research highlight 1: Antibody glycosylation and implications for antibody function
- Research highlight 2: Glycoanalysis of vaccines
Why we study glycoproteins
Most biological therapeutics are glycoproteins, that is, proteins with carbohydrates attached to them. These carbohydrate sugar chains attach to the protein at specific sites and are known as glycans. Glycans range in size from single monosaccharides (simple sugars) to larger oligosaccharides built up from monosaccharides. They are biosynthesized by cells using complex and dynamic processes that can be specific to each protein, and can change depending on cell growth conditions. The result is the potential to form numerous glycan structures that can greatly differ from one another.
This glycosylation heterogeneity leads to different glycoforms of the same protein. Each glycoform potentially possesses different biophysical properties and biological activities. Different glycoforms may also be used as markers of certain physiological and disease states.
Glycosylation of a glycoprotein may affect
- protein folding and final molecular structure
- solubility, stability, resistance to degradation
- transport (inside/outside cell), bio-distribution
- clearance rate / circulation time
- immunogenicity, antigenicity
- specific activity, ligand-receptor binding
How we study glycobiology and therapeutic glycoproteins
In our laboratory, at the Centre for Biologics Evaluation, we tackle questions regarding the glycosylation of therapeutic glycoproteins. We apply our expertise to provide new knowledge and tools that help us analyse therapeutic glycoprotein efficacy and safety.
With our research, we help
- Characterize the glycans attached to a given glycoprotein
- Understand the relationships between the glycoform profile and function for a given glycoprotein
- Assess glycoprotein efficacies based on their glycoform profiles
- Monitor and compare different batches/lots of the same or similar glycoproteins
- Examine methods that may let us control glycan patterns or heterogeneity during production
Concepts and tools we use to study glycobiology and therapeutic glycoproteins
Glycans are polymers of monosaccharides. In mammals, they range in size from a single monosaccharide to oligosaccharides containing more than 20 monosaccharide units. The monosaccharides commonly found in mammalian glycoproteins are the six neutral sugars, plus the family of negatively charged sialic acids.
Monosaccharides in glycans connect to each other through glycosidic bonds between the anomeric or reducing hydroxyl (OH) group on one monosaccharide unit and a hydroxyl group on another monosaccharide. There is an enormous potential for structural diversity in oligosaccharides, which arises from the following:
- Identity and sequence of constituent monosaccharides
- Anomeric configuration of glycosidic bonds (alpha, α or beta, β)
- Linkage positions of the glycosidic bonds (which hydroxyl groups)
- Type and degree of glycan branching (number of antennae)
Glycosylation is the most common modification made to proteins after they are synthesized in the cell, and about 70% of proteins are glycoproteins. The two most common ways in which glycans attach to proteins are through N-linked and O-linked glycosylation. While methods to characterize oligonucleotides and proteins are well established (e.g. using qPCR and peptide sequencing, respectively), glycan characterization is more difficult due to its structural diversity, and because protein glycosylation may not be directly controlled by its genetic information.
In our laboratory, we are characterizing and determining the role of glycans in therapeutic glycoproteins using strategies that combine analytical chemistry, biotechnology and chemical biology. Specifically, separation and analysis of glycans removed from glycoproteins are often performed by various chromatographic methods such as high-pH anion-exchange (HPAE) and high-performance or high-pressure liquid chromatography (HPLC). Native or chemically modified glycans can be detected by electrochemical and fluorescence methods.
Research highlight 1: Antibody glycosylation and implications for antibody function
Antibodies are widely used in the diagnosis, imaging, and therapy of a broad variety of different illnesses. They are glycoproteins with glycans attached at a specific, fully conserved amino acid. Their glycosylation state strongly affects their structure and function, and therefore their efficacy and safety. In our laboratory, we are conducting glyco-analytical surveys of representative commercially available monoclonal antibodies and other immunoglobulin products.
We are working to
- Better appreciate the variation within and between different immunoglobulin products
- Better understand how these variations have implications for functional differences between them
- Expand the glycosylation survey to include comparisons between other therapeutic glycoproteins including antibody related products, hormones, enzymes, and other biologicals
Research highlight 2: Glycoanalysis of vaccines
Many vaccines against bacterial infection consist of purified polysaccharides derived from the bacterial capsules of the target species and strains, and are conjugated to carrier proteins to yield a potent and sustained immune response. In addition, many vaccines against viral infection comprise live attenuated or inactivated viruses, or recombinant viral proteins, all of which may be glycosylated. The efficacy and safety of these vaccines depend on the identity, purity and amount of each carbohydrate component. We are working to develop techniques to analyze and quantitate the polysaccharide components of various vaccines to evaluate their efficacy and safety.