Fig. 1: A Chinese hamster ovary cell stained to show the major organelles. Protein glycosylation is initiated in the endoplasmic reticulum (green) and completed in the Golgi apparatus (red), which are arranged around the nucleus (blue).
© 2010 P. Zhang
Zhiwei Song and co-workers at the A*STAR Bioprocessing Technology Institute in Singapore have developed a genetically modified mammalian cell line that could greatly increase the efficiency of glycoprotein drug production.
Protein glycosylation is the natural process by which many proteins are converted into glycoproteins after their manufacture in cells through the enzymatic attachment of carbohydrate ‘glycans’, which alter their stability and activity. For example, erythropoietin (EPO) is a glycoprotein hormone that controls red blood cell production. Genetically engineered ‘recombinant’ EPO is used to treat anemia caused by kidney disease or cancer therapy, as well as cardiovascular disease. Infamously, it is also abused by ‘blood-doping’ athletes to increase endurance.
Like many other glycoproteins, EPO is sialylated, meaning that its glycans contain sialic acid. When sialic acid is removed, residues of the sugar galactose are exposed. These galactose residues are recognized by receptors in the liver, leading to EPO’s rapid clearance from circulation. Highly sialylated EPO therefore stays in the bloodstream much longer than less well sialylated EPO.
“Proper sialylation of glycoproteins drugs such as recombinant EPO is crucial for their therapeutic efficacy,” says Song. “Unfortunately, much of the recombinant EPO made using present biotechnological methods is not fully sialylated and has to be discarded.”
In a bid to resolve this problem, the researchers studied factors affecting glycoprotein sialylation in lines of Chinese hamster ovary (CHO) cells, which are widely used for the commercial production of glycoprotein drugs (Fig. 1).
Analyzing glycoprotein sialylation using conventional biochemical methods is laborious and costly. Instead, the researchers used a technique called isoelectric-focusing (IEF), which efficiently separates different molecules by their electric charge differences. “IEF allowed rapid assessment of the sialylation patterns of glycoproteins produced under different conditions or by different CHO cell lines,” explains Song.
They found that some of the CHO cell lines were resistant to RCA-I, a sugar-binding protein (lectin) found in castor beans that normally kills CHO cells. DNA sequencing confirmed genetic analyses suggesting that the gene encoding the protein glycosylation enzyme GnT I was dysfunctional in the mutant cell lines. They corrected the defect in protein glycosylation when they genetically engineered the mutant cells to express normal GnT I. Furthermore, the team’s cell line produces recombinant EPO with enhanced sialylation when normal GnT I is reintroduced into the cells.
“We believe that our findings could be applied by the biotechnology industry as an efficient strategy for producing highly sialylated EPO and other therapeutically important glycoprotein drugs,” says Song.
The A*STAR-affiliated researchers contributing to this research are from the Bioprocessing Technology Institute.
