Skip to main content
SpringerLink
Log in

A LC-MS All-in-One Workflow for Site-Specific Location, Identification and Quantification of N-/O- Glycosylation in Human Chorionic Gonadotropin Drug Products

  • Research Article
  • Published:
The AAPS Journal Aims and scope Submit manuscript

Abstract

Site-specific characterization of the N- and O-linked glycosylation on a set of different human chorionic gonadotropin (hCG) drug products was performed by a LC-MS method combining high resolution (120K at m/z 200) mass spectrometry, multiple dissociation methods, tandem mass tag (TMT 10plex) labeling, and partial least squares-discriminant analysis (PLS-DA). In total, the data provided identification, relative quantification, and comparison of site-specific glycosylation of protein therapeutics with a single experiment. Ten different lots and/or brands of commercial therapeutic hCG were labeled with TMT 10plex reagents after tryptic digestion. The labeled intact glycopeptides were then analyzed by high resolution LC-MS with online alternating HCD/ETD/CID dissociation methods. For digested hCG drugs, 1000 intact N- and O-linked glycopeptides were identified. The relative amount of each glycopeptide from hCG products was determined based on the reporter signal intensities of the TMT labeling reagents. Moreover, with the help of TMT 10plex, through just one LC-MS run, PLS-DA was performed to ascertain the differences in glycosylation among different sources of hCG drug products. The results of PLS-DA showed that 167 glycopeptides were found to be significantly different between the naturally derived and recombinant hCG products. The results demonstrate the suitability of this method for similarity assessments and counterfeit identification of hCG as well as other glycoproteins.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Woo CM, Iavarone AT, Spiciarich DR, Palaniappan KK, Bertozzi CR. Isotope-targeted glycoproteomics (IsoTaG): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis. Nat Methods. 2015;12(6):561–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Pan S, Chen R, Aebersold R, Brentnall TA. Mass spectrometry based glycoproteomics—from a proteomics perspective. Mol Cell Proteomics. 2011;10(1):R110.003251.

  3. Jefferis R. Glycosylation as a strategy to improve antibody-based therapeutics. Nat Rev Drug Discov. 2009;8(3):226–34.

    Article  CAS  PubMed  Google Scholar 

  4. Carter PJ. Potent antibody therapeutics by design. Nat Rev Immunol. 2006;6(5):343–57.

    Article  CAS  PubMed  Google Scholar 

  5. Jefferis R. Glycosylation of recombinant antibody therapeutics. Biotechnol Prog. 2005;21(1):11–6.

    Article  CAS  PubMed  Google Scholar 

  6. Walsh G, Jefferis R. Post-translational modifications in the context of therapeutic proteins. Nat Biotechnol. 2006;24(10):1241–52.

    Article  CAS  PubMed  Google Scholar 

  7. Sinclair AM, Elliott S. Glycoengineering: the effect of glycosylation on the properties of therapeutic proteins. J Pharm Sci. 2005;94(8):1626–35.

    Article  CAS  PubMed  Google Scholar 

  8. Zhang H, Li X-J, Martin DB, Aebersold R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat Biotechnol. 2003;21(6):660–6.

    Article  CAS  PubMed  Google Scholar 

  9. Zhu Z, Su X, Go EP, Desaire H. New glycoproteomics software, GlycoPep evaluator, generates decoy glycopeptides de novo and enables accurate false discovery rate analysis for small data sets. Anal Chem. 2014;86(18):9212–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Scott NE, Parker BL, Connolly AM, et al. Simultaneous glycan-peptide characterization using hydrophilic interaction chromatography and parallel fragmentation by CID, higher energy collisional dissociation, and electron transfer dissociation MS applied to the N-linked glycoproteome of Campylobacter jejuni. Mol Cell Proteomics. 2011;10(2):M000031–MCP201.

    Article  PubMed  Google Scholar 

  11. Windwarder M, Altmann F. Site-specific analysis of the O-glycosylation of bovine fetuin by electron-transfer dissociation mass spectrometry. J Proteome. 2014;108:258–68.

    Article  CAS  Google Scholar 

  12. Ye H, Boyne MT, Buhse LF, Hill J. Direct approach for qualitative and quantitative characterization of glycoproteins using tandem mass tags and an LTQ Orbitrap XL electron transfer dissociation hybrid mass spectrometer. Anal Chem. 2013;85(3):1531–9.

    Article  CAS  PubMed  Google Scholar 

  13. Håkansson K, Cooper HJ, Emmett MR, Costello CE, Marshall AG, Nilsson CL. Electron capture dissociation and infrared multiphoton dissociation MS/MS of an N-glycosylated tryptic peptide to yield complementary sequence information. Anal Chem. 2001;73(18):4530–6.

    Article  PubMed  Google Scholar 

  14. Richards AL, Hebert AS, Ulbrich A, et al. One-hour proteome analysis in yeast. Nat Protoc. 2015;10(5):701–14.

    Article  CAS  PubMed  Google Scholar 

  15. Senko MW, Remes PM, Canterbury JD, et al. Novel parallelized quadrupole/linear ion trap/Orbitrap tribrid mass spectrometer improving proteome coverage and peptide identification rates. Anal Chem. 2013;85(24):11710–4.

    Article  CAS  PubMed  Google Scholar 

  16. Wu S-W, Pu T-H, Viner R, Khoo K-H. Novel LC-MS2 product dependent parallel data acquisition function and data analysis workflow for sequencing and identification of intact glycopeptides. Anal Chem. 2014;86(11):5478–86.

    Article  CAS  PubMed  Google Scholar 

  17. Saba J, Dutta S, Hemenway E, Viner R. Increasing the productivity of glycopeptides analysis by using higher-energy collision dissociation-accurate mass-product-dependent electron transfer dissociation. Int J Proteome. 2012;2012

  18. Frese CK, Altelaar AM, Hennrich ML, et al. Improved peptide identification by targeted fragmentation using CID, HCD and ETD on an LTQ-Orbitrap Velos. J Proteome Res. 2011;10(5):2377–88.

    Article  CAS  PubMed  Google Scholar 

  19. Gaspari M, Verhoeckx KC, Verheij ER, van der Greef J. Integration of two-dimensional LC-MS with multivariate statistics for comparative analysis of proteomic samples. Anal Chem. 2006;78(7):2286–96.

    Article  CAS  PubMed  Google Scholar 

  20. America AH, Cordewener JH, van Geffen MH, et al. Alignment and statistical difference analysis of complex peptide data sets generated by multidimensional LC‐MS. Proteomics. 2006;6(2):641–53.

    Article  CAS  PubMed  Google Scholar 

  21. Pérez-Enciso M, Tenenhaus M. Prediction of clinical outcome with microarray data: a partial least squares discriminant analysis (PLS-DA) approach. Hum Genet. 2003;112(5–6):581–92.

    PubMed  Google Scholar 

  22. Chevallier S, Bertrand D, Kohler A, Courcoux P. Application of PLS‐DA in multivariate image analysis. J Chemom. 2006;20(5):221–9.

    Article  CAS  Google Scholar 

  23. Youssef MA, Abou‐Setta AM, Lam WS. Recombinant versus urinary human chorionic gonadotrophin for final oocyte maturation triggering in IVF and ICSI cycles. Cochrane Libr. 2011.

  24. Chang P, Kenley S, Burns T, et al. Recombinant human chorionic gonadotropin (rhCG) in assisted reproductive technology: results of a clinical trial comparing two doses of rhCG (Ovidrel R) to urinary hCG (Profasi R) for induction of final follicular maturation in in vitro fertilization–embryo transfer. Fertil Steril. 2001;76(1):67–74.

    Article  CAS  PubMed  Google Scholar 

  25. Trinchard-Lugan I, Khan A, Porchet H, Munafo A. Pharmacokinetics and pharmacodynamics of recombinant human chorionic gonadotrophin in healthy male and female volunteers. Reprod Biomed Online. 2002;4(2):106–15.

    Article  CAS  PubMed  Google Scholar 

  26. Sakhel K, Khedr M, Schwark S, Ashraf M, Fakih MH, Abuzeid M. Comparison of urinary and recombinant human chorionic gonadotropin during ovulation induction in intrauterine insemination cycles: a prospective randomized clinical trial. Fertil Steril. 2007;87(6):1357–62.

    Article  CAS  PubMed  Google Scholar 

  27. Madani T, Yeganeh LM, Ezabadi Z, Hasani F, Chehrazi M. Comparing the efficacy of urinary and recombinant hCG on oocyte/follicle ratio to trigger ovulation in women undergoing intracytoplasmic sperm injection cycles: a randomized controlled trial. J Assist Reprod Genet. 2013;30(2):239–45.

    Article  PubMed  Google Scholar 

  28. Papanikolaou EG, Fatemi H, Camus M, et al. Higher birth rate after recombinant hCG triggering compared with urinary-derived hCG in single-blastocyst IVF antagonist cycles: a randomized controlled trial. Fertil Steril. 2010;94(7):2902–4.

    Article  CAS  PubMed  Google Scholar 

  29. Fournier T, Guibourdenche J, Evain-Brion D. Review: hCGs: different sources of production, different glycoforms and functions. Placenta. 2015;36:S60–5.

    Article  CAS  PubMed  Google Scholar 

  30. Group ERHCGS. Induction of final follicular maturation and early luteinization in women undergoing ovulation induction for assisted reproduction treatment—recombinant HCG versus urinary HCG. Hum Reprod. 2000;15(7):1446–51.

    Article  Google Scholar 

  31. Fernández-Tejada A, Vadola PA, Danishefsky SJ. Chemical synthesis of the β-subunit of human luteinizing (hLH) and chorionic gonadotropin (hCG) glycoprotein hormones. J Am Chem Soc. 2014;136(23):8450–8.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Ye H, Hill J, Gucinski AC, Boyne II MT, Buhse LF. Direct site-specific glycoform identification and quantitative comparison of glycoprotein therapeutics: imiglucerase and velaglucerase alfa. AAPS J. 2015;17(2):405–15.

    Article  CAS  PubMed  Google Scholar 

  33. Kelly LS, Birken S, Puett D. Determination of hyperglycosylated human chorionic gonadotropin produced by malignant gestational trophoblastic neoplasias and male germ cell tumors using a lectin-based immunoassay and surface plasmon resonance. Mol Cell Endocrinol. 2007;260:33–9.

    Article  PubMed  Google Scholar 

  34. Toll H, Berger P, Hofmann A, et al. Glycosylation patterns of human chorionic gonadotropin revealed by liquid chromatography‐mass spectrometry and bioinformatics. Electrophoresis. 2006;27(13):2734–46.

    Article  CAS  PubMed  Google Scholar 

  35. Valmu L, Alfthan H, Hotakainen K, Birken S, Stenman U-H. Site-specific glycan analysis of human chorionic gonadotropin β-subunit from malignancies and pregnancy by liquid chromatography—electrospray mass spectrometry. Glycobiology. 2006;16(12):1207–18.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Author notes

  1. Ashley C. Ruth

    Present address: Biotechlogic Inc., Glenview, Illinois, 60025, USA

Authors and Affiliations

  1. Division of Pharmaceutical Analysis, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 645 S. Newstead Ave, St. Louis, Missouri, 63110, USA

    Hongbin Zhu, Chen Qiu, Ashley C. Ruth, David A. Keire & Hongping Ye

Authors
  1. Hongbin Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chen Qiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ashley C. Ruth
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David A. Keire
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hongping Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hongping Ye.

Ethics declarations

Disclaimer

This publication reflects the views of the authors and should not be construed to represent FDA’s views or policies.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 408 kb)

ESM 2

(XLSX 30 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhu, H., Qiu, C., Ruth, A.C. et al. A LC-MS All-in-One Workflow for Site-Specific Location, Identification and Quantification of N-/O- Glycosylation in Human Chorionic Gonadotropin Drug Products. AAPS J 19, 846–855 (2017). https://doi.org/10.1208/s12248-017-0062-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1208/s12248-017-0062-z

KEY WORDS

Search

Navigation