Glycan analysis of recombinant proteins produced by Gypsy moth and Chinese hamster ovary cells

Abstract

N-Glycosylation of protein affects not only physical properties but also biological properties of protein, and it is important to evaluate the glycan structures of a glycoprotein. In this study, N-glycan structures of recombinant proteins, human transferrin (hTf), erythropoietin (EPO), and humanized antibody expressed in Gypsy moth and Chinese hamster ovary (CHO) cells were structurally analyzed with 2-D HPLC and MALDI-TOF MS. First, N-glycan structures of recombinant hTf expressed by Gypsy moth cells were determined. Recombinant hTf was overexpressed and purified, and N-glycans were released from the purified recombinant human transferrin by glycoamidase-A digestion and derivatized with 2-aminopyridine, the glycan structures were analyzed by 2-D HPLC and MALDI-TOF MS. Structures of 11 glycans (88.8% of total N-glycans) were elucidated. The glycan analysis revealed that the most abundant glycans were $Man_{1-3}(±Fucα6)GlcNAc_2$ (75.5%) and $GlcNAcMan3(±Fucα6)GlcNAc_2$ (7.4%). There was only ~6% of high-mannose type glycans identified. Nearly half (49.8%) of the total N-glycans contained α1,6-fucosylation on the Asn-linked GlcNAc residue. However, α1,3-fucosylation on the same GlcNAc, often found in N-glycans produced by other insects and insect cells, was not detected. Second, human α2,3-sialyltransferase (ST) and β1,4-galactosyltransferase (GT) were engineered into CHO cells which produce human EPO in order to enhance the sialylation of the protein. ST and GT are responsible for the terminal sialylation and galactosylation, respectively. Recombinant human EPO was purified from the culture supernatant of the engineered CHO cells by an immuno-affinity chromatography. N-Glycans were isolated after digestion with glycoamidase-F, and the sialylation of EPO was evaluated by HPLC. When the α2,3-ST was expressed in CHO cells (EC1-ST2), the sialylation of EPO glycans was significantly increased compared with that of control cells (EC1). In detail, the amounts of di-, t...