title: Glycan analysis of glycoprotein pharmaceuticals: Evaluation of analytical approaches to Z number determination in pharmaceutical erythropoietin products.
abstract: N-Glycosylation of many glycoprotein drugs is important for biological activity and should therefore be the target of specific and quantitative analytical methods. In this study, we focus on the two N-glycan mapping approaches that are used in pharmacopoeial monograph to analyse N-glycans released from fifteen preparations of recombinant human erythropoietin supplied by ten Chinese manufacturers. Underivatised N-glycans were analysed by high performance anion-exchange chromatography with pulsed amperometric detection and fluorophore-labelled N-glycans were analysed by weak anion-exchange and normal-phase high performance liquid chromatography. N-glycans were also analysed by matrix assisted laser desorption ionisation mass spectrometry. The release of N-glycans by PNGase F was shown to be consistent. Z number, a mathematical expression of the total negatively charged N-glycans composition has provided a convenient way to summarise the complex dataset and it might be suitable for product consistency monitoring. However, this Z number reduces the information of individual acidic N-glycan structure and is also found to be method dependent. Therefore, its use requires clear specification and validation. In this study, we only found weak but positive correlation between the Z number and its bioactivity. Wide range of N-glycans yields were obtained from the fifteen preparations but the significance of their differences is unclear.
authors: National Institute for Biological Standards and Control, TDI/Protein Science, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, UK. ct.yuen@nibsc.hpa.org.uk
authors: Chun-Ting
authors: CT
authors: Yuen
authors: Yong
authors: Y
authors: Zhou
authors: Qing-Zhou
authors: QZ
authors: Wang
authors: Ji-Feng
authors: JF
authors: Hou
authors: Adrian
authors: A
authors: Bristow
authors: Jun-Zhi
authors: JZ
authors: Wang
mesh_terms: D010129:4-Aminobenzoic Acid / Q000737:chemistry; D000818:Animals; D001681:Biological Assay; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D004921:Erythropoietin / Q000032:analysis / Q000235:genetics / Q000494:pharmacology; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D051379:Mice; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006023:Glycoproteins; D004364:Pharmaceutical Preparations; D011134:Polysaccharides; D011994:Recombinant Proteins; D004921:Erythropoietin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010129:4-Aminobenzoic Acid