SARCOSYL-PAGE: a new electrophoretic method for the separation and immunological detection of PEGylated proteins

Methods in Molecular Biology
Christian Reichel

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 a decreased receptor-mediated endocytosis and/or a delay in renal clearance caused by the increased hydrodynamic volume of the pharmaceutical. MIRCERA, a PEGylated recombinant erythropoietin (rhEpo) frequently used in the treatment of anemia due to chronic kidney disease, has been also abused by athletes as a performance-enhancing drug. While it can be detected by sodium dodecyl sulfate 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, thus leading to enhanced antibody binding and a sharper electrophoretic band. While the method was originally developed for anti-doping purposes, it may be also useful for other PEGylated proteins and...Continue Reading

Citations

Jan 26, 2017·Molecular and Cellular Endocrinology·Olivier SalaminNicolas Leuenberger
Jun 8, 2018·Drug Testing and Analysis·Gemma Reverter-BranchatJordi Segura
Dec 12, 2012·Drug Testing and Analysis·Mario ThevisWilhelm Schänzer
Oct 15, 2014·Drug Testing and Analysis·Tim SobolevskyGrigory Rodchenkov
Oct 22, 2014·Drug Testing and Analysis·Grigory KrotovGrigory Rodchenkov
Sep 20, 2012·Endocrine·Leonidas H Duntas, Vera Popovic
Jun 25, 2019·Drug Testing and Analysis·Jules A A C HeubergerAdam F Cohen
Feb 16, 2020·The Journal of Pharmacology and Experimental Therapeutics·Weimin Conrad Hong
Nov 11, 2014·Expert Review of Proteomics·Mario ThevisWilhelm Schänzer

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