Rapid quantitation of erythropoietin and identification of its glycans using membranes for capture and digestion

Erythropoietin (EPO) is a glycoprotein hormone used to treat chronic anemia. Glycans at the three N-glycosylation sites strongly influence EPO's stability and bioactivity, so accurate quantitation of EPO and analysis of its glycosylation patterns are critical during production and subsequent processing. This work demonstrates a rapid workflow for EPO capture, quantitation, and glycan profiling. Newly developed porous membranes containing affinity peptides selectively capture EPO from cell culture supernatants. In a 96-well-plate format, captured EPO was labeled with a fluorescent antibody to enable quantitation in a 10-min assay with an average coefficient of variation of 12 %. Elution of captured EPO from a 2-cm glass-fiber membrane combined with tryptic membranes enabled efficient purification and digestion for liquid chromatography-tandem mass spectrometry (LC-MS/MS) glycan analysis. Without purification, conventional LC-MS/MS identification of EPO N-glycans in Chinese hamster ovary (CHO) cell supernatant was not possible. The rapid in-membrane digestion yielded glycan profiles comparable to those obtained from overnight in-solution digestion. Thus, the membrane-based assays provide a novel approach for rapid EPO quantitation and facilitating glycan identification.