Site Occupancy

Background on Glycoproteins

Many of the valuable commercial biotherapeutic products, such as monoclonal antibodies, growth factors, hormones and clotting factors, are secreted glycoproteins.  These biotherapeutics are often produced in mammalian cell expression systems and are made up of oligosaccharide chains attached to polypeptides at specific amino acid residues.

The addition of these oligosaccharides to the proteins occurs through a post-translational modification called N-glycosylation.  The number, type, and location of the oligosaccharides (glycans) on the protein can affect key biochemical properties of the biotherapeutic, including its clearance rate, immunogenicity, bioactivity, solubility, and stability against proteolysis. 

The Problem:  Variable Site Occupancy

Unfortunately, when these therapeutic products are produced by over-expression in mammalian and non-mammalian hosts, the glycosylation processing can generate products with highly variable glycosylation patterns. This N-glycan variability limits the yield and affects the quality of the target secreted glycoproteins, and therefore can significantly affect the value of these biotherapeutic products.

The Solution:  Metabolic Engineering

In an attempt to overcome the problems associated with variable N-glycosylation, we are investigating the metabolic pathways involved and evaluating potential causes of this phenomenon.

We are using human transferrin (hTf) as a model protein in our research, because it is a common clinical marker used to diagnose clinical disorders of glycosylation (CDG).  We are using the commercially relevant CHO and HEK cell expression systems to investigate the causes for variable site occupancy.  We plan to apply metabolic engineering strategies to overcome bottlenecks in the N-glycosylation pathway in order to enhance processing and eliminate production of under-glycosylated proteins in mammalian cell culture systems.