Sensitive and comprehensive analysis of O-glycosylation in biotherapeutics: a case study of novel erythropoiesis stimulating protein
Abstract
Aim: Glycosylation of recombinant human erythropoietins (rhEPOs) is significantly associated with drug's quality and potency. Thus, comprehensive characterization of glycosylation is vital to assess the biotherapeutic quality and establish the equivalency of biosimilar rhEPOs. However, current glycan analysis mainly focuses on the N-glycans due to the absence of analytical tools to liberate O-glycans with high sensitivity. We developed selective and sensitive method to profile native O-glycans on rhEPOs. Results:O-glycosylation on rhEPO including O-acetylation on a sialic acid was comprehensively characterized. Details such as O-glycan structure and O-acetyl-modification site were obtained from tandem MS. Conclusion: This method may be applied to QC and batch analysis of not only rhEPOs but also other biotherapeutics bearing multiple O-glycosylations.
Papers of special note have been highlighted as: • of interest; •• of considerable interest
References
- 1 . Erythropoietin; a review on current knowledge and new concepts. J. Renal Inj. Prev. 2(4), 119–121 (2013).
- 2 . Physiology and pharmacology of erythropoietin. Transfus. Med. Hemother. 40(5), 302–309 (2013).
- 3 Erythropoietin to treat head and neck cancer patients with anaemia undergoing radiotherapy: randomised, double-blind, placebo-controlled trial. Lancet 362(9392), 1255–1260 (2003).
- 4 Role of glycosylation on the secretion and biological activity of erythropoietin. Biochemistry 31(41), 9871–9876 (1992).
- 5 Epoetin alfa and beta differ in their erythropoietin isoform compositions and biological properties. Br. J. Haematol. 100, 11 (1998).
- 6 . Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin. Blood 98(13), 3626–3634 (2001).
- 7 . The enigma of the metabolic fate of circulating erythropoietin (Epo) in view of the pharmacokinetics of the recombinant drugs rhEpo and NESP. Eur. J. Haematol. 69(5-6), 265–274 (2002).
- 8 . Darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin. Exp. Hematol. 31(4), 290–299 (2003).
- 9 . Optimisation of the cellular metabolism of glycosylation for recombinant proteins produced by mammalian cell systems. Cytotechnology 50(1–3), 57–76 (2006).
- 10 . Glycosylation-modified erythropoietin with improved half-life and biological activity. Int. J. Hematol. 91(2), 238–244 (2010).
- 11 . Role of sugar chains in the expression of the biological-activity of human erythropoietin. J. Biol. Chem. 267(11), 7703–7709 (1992).
- 12 . Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation. Biotechnol. Bioeng. 68(4), 370–380 (2000).
- 13 . Technologies for glycomic characterization of biopharmaceutical erythropoietins. Trends Anal. Chem. 68, 18–27 (2015). •• Provides an overview of current analytical tools for qualitative and quantitative analysis of erythropoietin glycosylation.
- 14 . Glycan labeling strategies and their use in identification and quantification. Anal. Bioanal. Chem. 397(8), 3457–3481 (2010).
- 15 . Oligosaccharide analysis by mass spectrometry: a review of recent developments. Anal. Chem. 86(1), 196–212 (2014). •• Covers developments in the application of mass spectrometry to the analysis of carbohydrates.
- 16 . Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis – electrospray – time-of-flight mass spectrometry. Electrophoresis 27(13), 2638–2650 (2006).
- 17 . Mass spectrometric glycoform profiling of the innovator and biosimilar erythropoietin and darbepoetin by LC/ESI-MS. J. Pharm. Biomed. Anal. 83, 65–74 (2013).
- 18 . Advances in LC–MS/MS-based glycoproteomics: getting closer to system-wide site-specific mapping of the N- and O-glycoproteome. Biochim. Biophys. Acta 1844(9), 1437–1452 (2014).
- 19 . Analytical glycobiology at high sensitivity: current approaches and directions. Glycoconj. J. 30(2), 89–117 (2013). • Summarizes the analytical advances made during the last several years in the structural and quantitative determinations of glycoproteins in complex biological mixtures.
- 20 . Structural glycomic analyses at high sensitivity: a decade of progress. Annu. Rev. Anal. Chem. (Palo Alto Calif.) 6, 237–265 (2013). • Summarizes recent methodological developments and technical innovations toward structural characterization of glycans and their quantification at high sensitivity.
- 21 . Protein O-glycosylation analysis. Biol. Chem. 393(8), 687–708 (2012). • Provides an overview on the methods available for analysis of O-glycosylation.
- 22 . Advances in mass spectrometry driven O-glycoproteomics. Biochim. Biophys. Acta 1850(1), 33–42 (2015).
- 23 Glycoblotting-assisted O-glycomics: ammonium carbamate allows for highly efficient O-glycan release from glycoproteins. Anal. Chem. 82, 9 (2010).
- 24 . Improved nonreductive O-glycan release by hydrazinolysis with ethylenediaminetetraacetic acid addition. Anal. Biochem. 453, 29–37 (2014).
- 25 . One-pot nonreductive O-glycan release and labeling with 1-phenyl-3-methyl-5-pyrazolone followed by ESI-MS analysis. Proteomics 11(21), 4229–4242 (2011).
- 26 . “One-pot” access to alpha-D-mannopyranosides from glycals employing ruthenium catalysis. RSC Adv. 4(86), 46327–46331 (2014).
- 27 . Separation of one-pot procedure released O-glycans as 1-phenyl-3-methyl-5-pyrazolone derivatives by hydrophilic interaction and reversed-phase liquid chromatography followed by identification using electrospray mass spectrometry and tandem mass spectrometry. J. Chromatogr. A 1274, 107–117 (2013).
- 28 . Simultaneous quantification of N- and O-glycans using a solid-phase method. Nat. Protocols 12(6), 1229–1244 (2017).
- 29 . Chemical Glycoproteomics. Chem. Rev. 116(23), 14277–14306 (2016). •• Provides a foundation on which continued technological advancements can be made to promote further explorations of protein glycosylation.
- 30 . MS platform for erythropoietin glycome characterization. Mass Spectrom. Lett. 6(3), 53–58 (2015).
- 31 . Determination of glycosylation sites and site-specific heterogeneity in glycoproteins. Curr. Opin. Chem. Biol. 13(4), 421–426 (2009).
- 32 Characterization of novel O-glycans isolated from tear and saliva of ocular rosacea patients. J. Proteome Res. 12(3), 1090–1100 (2013).
- 33 Analytical platform for glycomic characterization of recombinant erythropoietin biotherapeutics and biosimilars by MS. Bioanalysis 5(5), 545–559 (2013).
- 34 Site-specific protein glycosylation analysis with glycan isomer differentiation. Anal. Bioanal. Chem. 403(5), 1291–1302 (2012).
- 35 Glyco-analytical multispecific proteolysis (Glyco-AMP): a simple method for detailed and quantitative glycoproteomic characterization. J. Proteome Res. 12(10), 4414–4423 (2013).
- 36 Automated assignments of N- and O-site specific glycosylation with extensive glycan heterogeneity of glycoprotein mixtures. Anal. Chem. 85(12), 5666–5675 (2013).
- 37 . The development of retrosynthetic glycan libraries to profile and classify the human serum N-linked glycome. Proteomics 9(11), 2986–2994 (2009).
- 38 . Detailed characterization of the O-linked glycosylation of the neuropilin-1 c/MAM-domain. Glycoconj. J. 33(3), 387–397 (2016).
- 39 Simultaneous and extensive site-specific N- and O-glycosylation analysis in protein mixtures. J. Proteome Res. 10(5), 2612–2624 (2011).
- 40 Site-specific glycan microheterogeneity of inter-alpha-trypsin inhibitor heavy chain H4. J. Proteome Res. 13(7), 3314–3329 (2014).
- 41 . Characterization of N-glycan heterogeneities of erythropoietin products by liquid chromatography/mass spectrometry and multivariate analysis. Rapid Commun. Mass Spectrom. 28(8), 921–932 (2014).
- 42 . Mass spectrometry in characterising biopharmaceuticals. Chim. Ogi. 32, 4–7 (2014).
- 43 . The sweet tooth of biopharmaceuticals: importance of recombinant protein glycosylation analysis. Biotechnol. J. 7(12), 1462–1472 (2012).