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SARCOSYL—PAGE: Optimized Protocols for the Separation and Immunological Detection of PEGylated Proteins

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Book cover Electrophoretic Separation of Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1855))

Abstract

PEGylation of recombinant proteins and synthetic peptides aims to generate biopharmaceuticals with altered physical properties. The modification may lead to a prolonged serum half-life caused by decreased receptor-mediated endocytosis and/or delay in renal clearance caused by the increased hydrodynamic volume of the pharmaceutical. MIRCERA, a PEGylated recombinant erythropoietin (rhEPO) used in the treatment of anemia due to chronic kidney disease, has also been abused by athletes as performance-enhancing drug. While it can be detected by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, the sensitivity of the test is significantly lower compared to other epoetins. By replacing SDS with sarcosyl in the sample and running buffers, the interaction between SDS and the PEG group of the protein no longer reduces the affinity of the monoclonal anti-EPO antibody (clone AE7A5) to the protein chain. Contrary to SDS, sarcosyl only binds to the amino acid chain of the PEGylated protein and thus leads to a sharper electrophoretic band and enhanced antibody binding. While the method was originally developed for anti-doping purposes, it may also be useful for the electrophoretic separation and immunological detection of other PEGylated proteins. Protocols for urine and serum are presented. They are also applicable for the general detection of EPO-based erythropoiesis-stimulating agents (ESA) in these matrices.

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References

  1. Dehnes Y, Hemmersbach P (2011) Effect of single doses of methoxypolyethylene glycol-epoetin beta (CERA, Mircera) and epoetin delta (Dynepo) on isoelectric erythropoietin profiles and haematological parameters. Drug Test Anal 3(5):291–299

    Article  CAS  Google Scholar 

  2. European Medicines Agency (EMEA) (2007) MIRCERA scientific discussion, http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Scientific_Discussion/human/000739/WC500033669.pdf. Accessed 29 Aug 2017, pp 1–4.

  3. World Anti-Doping Agency (2016) The world anti-doping code. International Standard. Prohibited list January 2017. Montreal. https://www.wada-ama.org/sites/default/files/resources/files/2016-09-29_-_wada_prohibited_list_2017_eng_final.pdf. Accessed 29 Aug 2017.

  4. World Anti-Doping Agency (2014) Technical document TD2014EPO. Harmonization of analysis and reporting of ESAs by electrophoretic techniques. Montreal. https://www.wada-ama.org/sites/default/files/resources/files/WADA-TD2014EPO-v1-Harmonization-of-Analysis-and-Reporting-of-ESAs-by-Electrophoretic-Techniques-EN.pdf. Accessed 29 Aug 2017.

  5. Reichel C, Abzieher F, Geisendorfer T (2009) SARCOSYL-PAGE: a new method for the detection of MIRCERA- and EPO-doping in blood. Drug Test Anal 1(11–12):494–504

    Article  CAS  Google Scholar 

  6. Reihlen P, Blobel M, Kempkes R, Reichel C, Völker Schänzer E, Majer B, Schänzer W (2015) Optimizing SAR-PAGE. Drug Test Anal 7(11–12):1014–1016

    Article  CAS  Google Scholar 

  7. Invitrogen (2003). In: NuPAGE Technical Guide Version E (IM-1001), Carlsbad, CA, p 49.

    Google Scholar 

  8. Bjerrum OJ, Schafer-Nielsen C (1986) Buffer systems and transfer parameters for semidry electroblotting with horizontal apparatus. In: Dunn MJ (ed) Electrophoresis, 86th edn. Wiley-VCH, Weinheim, pp 315–327

    Google Scholar 

  9. Bajla I, Holländer I, Minichmayr M, Gmeiner G, Reichel C (2005) GASepo—a software solution for quantitative analysis of digital images in EPO doping control. Comput Methods Programs Biomed 80:246–270

    Article  CAS  Google Scholar 

  10. Lasne F, Martin L, Martin JA, de Ceaurriz J (2007) Isoelectric profiles of human erythropoietin are different in serum and urine. Int J Biol Macromol 41:354–357

    Article  CAS  Google Scholar 

  11. Berglund B, Wide L (2002) Erythropoietin concentrations and isoforms in urine of anonymous Olympic athletes during the Nagano Olympic games. Scand J Med Sci Sports 12(6):354–357

    Article  CAS  Google Scholar 

  12. MAIIA Diagnostics (2016) EPO Purification Kit, Uppsala. http://maiiadiagnostics.com/gammal/files/Directions%20for%20Use%20-%20EPO%20Purification%20Kit%20%28new%21%29.pdf. Accessed 29 Aug 2017.

  13. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  14. Neville DM Jr (1971) Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J Biol Chem 246:6328–6334

    CAS  PubMed  Google Scholar 

  15. Allen RC, Moore DJ (1971) Electrophoretic system. U.S. Patent 3620947.

    Google Scholar 

  16. Zheng C, Ma G, Su Z (2007) Native PAGE eliminates the problem of PEG-SDS interaction in SDS-PAGE and provides an alternative to HPLC in characterization of protein PEGylation. Electrophoresis 28:2801–2807

    Article  CAS  Google Scholar 

  17. Lönnberg M, Dehnes Y, Drevin M, Garle M, Lamon S, Leuenberger N, Quach T, Carlsson J (2010) Rapid affinity purification of erythropoietin from biological samples using disposable monoliths. J Chromatogr A 1217:7031–7037

    Article  Google Scholar 

  18. Dehnes Y, Lamon S, Lönnberg M (2010) Erythropoietin (EPO) immunoaffinity columns—a powerful tool for purifying EPO and its recombinant analogues. J Pharm Biomed Anal 53:1028–1032

    Article  CAS  Google Scholar 

  19. Reichel C, Thevis M (2012) Detection of EPO-Fc fusion protein in human blood: screening and confirmation protocols for sports drug testing. Drug Test Anal 4(11):818–829

    Article  CAS  Google Scholar 

  20. Lasne F (2001) Double-blotting: a solution to the problem of non-specific binding of secondary antibodies in immunoblotting procedures. J Immunol Methods 253:125–131

    Article  CAS  Google Scholar 

  21. Lasne F (2003) Double-blotting: a solution to the problem of nonspecific binding of secondary antibodies in immunoblotting procedures. J Immunol Methods 276:223–226

    Article  CAS  Google Scholar 

  22. Lasne F (2009) Double-Blotting: a solution to the problem of nonspecific binding of secondary antibodies in immunoblotting procedures. In: Kurien BT, Scofield RH (eds) Methods in molecular biology, protein blotting and detection, vol 536. Humana Press, New York, p 217

    Google Scholar 

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Acknowledgment

This project has been carried out with the financial support of WADA (grant 08B11CR).

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Authors and Affiliations

  1. Doping Control Laboratory, Seibersdorf Labor GmbH, Seibersdorf, Austria

    Christian Reichel & Günter Gmeiner

  2. Institute of Biochemistry, German Sport University Cologne, Cologne, Germany

    Philipp Reihlen, Mario Thevis & Wilhelm Schänzer

Authors
  1. Christian Reichel
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  2. Günter Gmeiner
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  3. Philipp Reihlen
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  4. Mario Thevis
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  5. Wilhelm Schänzer
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Corresponding author

Correspondence to Christian Reichel .

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Editors and Affiliations

  1. Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

    Biji T. Kurien

  2. Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

    R. Hal Scofield

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Reichel, C., Gmeiner, G., Reihlen, P., Thevis, M., Schänzer, W. (2019). SARCOSYL—PAGE: Optimized Protocols for the Separation and Immunological Detection of PEGylated Proteins. In: Kurien, B., Scofield, R. (eds) Electrophoretic Separation of Proteins. Methods in Molecular Biology, vol 1855. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8793-1_14

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  • DOI: https://doi.org/10.1007/978-1-4939-8793-1_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8792-4

  • Online ISBN: 978-1-4939-8793-1

  • eBook Packages: Springer Protocols

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