Quantitative N-GIycan Mapping of Glycoprotein Therapeutics by HPAEC-PAD: Glycosylation Characteristics of Different Recombinant Human EPQ Products

摘要

The consistent and appropriate control of the quality of N-linked oligosaccharides of recombinant glycoproteins during fermentation is a major issue in successful commercial protein manufacture, and regulatory authorities (FDA, EMEA) put high requirements on the quality and reproducibility of biopharmaceuti-cal products. The glycosylation pattern of a therapeutic glycoprotein is influenced by cell type and cell culture environment (e.g. age of batch, glucose limitation, oxygen starvation, intracellular ammonium ion accumulation, pH changes). Glycosylation is the most complex post-translational modification made by animal cells, which makes a vital contribution to the biological activity and therapeutic efficacy of pharmaceutical glycoproteins (Grabenhorst and Conradt, 1999; Nimtz et al., 1993). GlycoThera has developed quantitative oligosaccharide mapping procedures (HPAEC-PAD) that are highly specific, sensitive (LOQ < 10 pmol), precise (CV < 2%, n = 6), accurate (recovery of di-, tri- and tetrasialylated structures 97-103%) and robust in routine use (under cGMP regulations) for the quality control of recombinant glycoprotein therapeutics (drug substance and drug product batch release; assessment of lot-to-lot consistency, safety and long-term stability), requiring only low |xg quantities of protein (e.g. 1mug of EPO). HPAEC-PAD mapping of glycans is the only method for oligosaccharide analysis without requiring prior chemical derivatisation and will be described in the forthcoming edition of the European Pharmacopoeia in a separate chapter. Additionally, on-line desalted fractions obtained after HPAEC-PAD equipped with a carbohydrate membrane desalter (Dionex Corporation, Sunnyvale, CA, USA) are analysed by mass spectrometric methods; typically 1-5 pmol of oligosaccharide material is used for MS analysis.