title: Sialic acid-specific affinity chromatography for the separation of erythropoietin glycoforms using serotonin as a ligand.
abstract: Recombinant human erythropoietin (rhEPO) is an important CHO cell-derived glycoprotein and the degree of sialylation of this hormone is crucial for its in vivo bioactivity. In order to improve the purification process serotonin as a potential affinity ligand was tested for preparative chromatographic separation of rhEPO glycoforms into fractions of different degrees of sialylation. Therefore, two chromatographic matrices were prepared by immobilizing serotonin on CNBr- and NHS-Sepharose™. First it was shown both matrices bind rhEPO only in its sialylated form. Results indicate that binding is pH independent between pH 3.5 to 8 suggesting it is not only based on electrostatic interactions. Second, after optimal binding conditions were identified, semi-purified rhEPO was loaded onto both matrices and eluted using a stepwise elution gradient of sodium chloride. For comparison same affinity purification experiments were performed using wheat germ agglutinin-coupled agarose, a lectin known for its affinity towards sialylated glycoproteins. To monitor changes in N-glycan fingerprint, eluate fractions were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence (xCGE-LIF). For the serotonin matrices an increasing degree of sialylation was observed from the first to the third elution fraction while purity of rhEPO could be increased at the same time. The late elution fractions of serotonin-coupled CNBr- and NHS-Sepharose™ also showed an overall sialylation degree exceeding that of the starting material. In contrast, for rhEPO bound to wheat germ agglutinin-coupled agarose, no distinct change in the degree of sialylation could be observed after elution. Overall, these encouraging results highlight the potential of serotonin as a chromatographic ligand for the improvement of pharmaceutical purification processes of rhEPO.
authors: Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany. Electronic address: matthiasmei@ymail.com.
authors: M
authors: M
authors: Meininger
authors: Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany; Flensburg University of Applied Sciences, Flensburg, Germany.
authors: M
authors: M
authors: Stepath
authors: Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany; GlyXera GmbH, Magdeburg, Germany.
authors: R
authors: R
authors: Hennig
authors: Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany; GlyXera GmbH, Magdeburg, Germany.
authors: S
authors: S
authors: Cajic
authors: Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany; GlyXera GmbH, Magdeburg, Germany.
authors: E
authors: E
authors: Rapp
authors: Merckle Biotec GmbH, Ulm, Germany.
authors: H
authors: H
authors: Rotering
authors: Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany; Otto von Guericke University, Magdeburg, Germany. Electronic address: mwolff@mpi-magdeburg.mpg.de.
authors: M W
authors: MW
authors: Wolff
authors: Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany; Otto von Guericke University, Magdeburg, Germany.
authors: U
authors: U
authors: Reichl
mesh_terms: D002236:Carbohydrate Conformation; D002846:Chromatography, Affinity / Q000379:methods*; D004921:Erythropoietin / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D012701:Serotonin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D020033:Protein Isoforms; D011994:Recombinant Proteins; D004921:Erythropoietin; D012701:Serotonin; D019158:N-Acetylneuraminic Acid
keywords: Affinity Chromatography; Erythropoietin Glycoforms; Glycan Analysis; Glycoproteins; Serotonin; Sialic Acid