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

SARCOSYL-PAGE: Optimized Protocols for the Separation and Immunological Detection of PEGylated Proteins / Christian Reichel, Günter Gmeiner, Philipp Reihlen, Mario Thevis, Wilhelm Schänzer. - (Methods in Molecular Biology (2019) 1855; p. 131-149).
- PMID: 30426415.
- DOI: 10.1007/978-1-4939-8793-1_14
Published in: In: Kurien B., Scofield R. (eds) Electrophoretic Separation of Proteins. Methods in Molecular Biology, vol 1855. Humana Press, New York, NY
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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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Science
Study
Date:
14 November 2018
People
Gmeiner, Günter
Reichel, Christian
Reihlen, Philipp
Schänzer, Wilhelm
Thevis, Mario
Country
Austria
Language
English
Other organisations
Deutsche Sporthochschule Köln (DSHS) - German Sport University Cologne
Seibersdorf Labor Doping Control Laboratory
Laboratories
Cologne, Germany: Institute of Biochemistry - German Sport University Cologne
Seibersdorf, Austria: Seibersdorf Labor GmbH Doping Control Laboratory
Analytical aspects
Testing method development
Doping classes
S2. Peptide Hormones, Growth Factors
S4. Hormone And Metabolic Modulators
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Erythropoietin (EPO)
Methoxy polyethylene glycol-epoetin beta (CERA)
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