Array-Based Analysis of Secreted Glycoproteins for Rapid Selection of a Single Cell Producing a Glycoprotein with Desired Glycosylation

摘要

The therapeutic efficacy and in vivo biological function ofna glycoprotein is significantly affected by its glycosylationnprofile. For the development of glycoproteins with therapeuticnapplications, selection of cell lines producing anglycoprotein with adequate glycoform is crucial. Here, wendemonstrate an array-based analysis of secreted glycoproteinsnfor rapid and efficient selection of a single cellnproducing a glycoprotein with desirable glycosylation. Ournapproach relies on microengraving and interrogation ofnglycoproteins produced by individual cells in a microwellnarray in terms of glycosylation profile as well as thenproduced amount. On the basis of statistical analysis ofnthe interrogation, single cells which are predicted tonproduce a desired glycoprotein are selected, retrieved,nand expanded. We applied the approach to human recombinantnerythropoietin (rhEPO)-producing CHO cellsnand verified the selection of a single CHO cell thatnproduces rhEPO with a high sialylation degree. Humannerythropoietin (hEPO) bearing highly sialylated oligosaccharidenwas shown to display a much longer plasma halflife,nresulting in high therapeutic efficacy. This methodnmay find widespread use in the clonal selection for thenproduction of other glycoproteins with specific glycosylationnas well as analysis of the heterogeneity in cellnpopulations in a high-throughput manner.