Online 2D-LC Analysis of Complex N-Glycans in Biopharmaceuticals Using the Agilent 1290 Infinity 2D-LC Solution
12 February 2016

Erythropoietin (EPO) is a 30,400 dalton (Da) glycoprotein hormone that regulates the production of red blood cells (erythropoiesis). The molecule consists of a 165 amino acid single polypeptide chain and a complex carbohydrate addition that amounts to 40 % of the total molecular weight. Due to the flexible molecular structure of the glycans, they cover almost all of the surface of EPO. The glycosylation of EPO is highly variable because it contains multiple glycosylation sites, each of which can have a wide variety of glycan structures. This results in a huge complexity of glycan structures that is referred to as microheterogeneity. This application note presents different online 2D-LC concepts for the high-resolution analysis of complex N-linked glycans in erythropoietin (EPO) using the Agilent 1290 Infinity 2D-LC Solution with fluorescence detection.