Research output: Chapter in Book/Report/Conference proceeding › Contributions to collected editions/anthologies › Research › peer-review
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.
Original language | English |
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Title of host publication | Electrophoretic separation of proteins : methods and protocols |
Editors | Biji T. Kurien, R. Hal Scofield |
Number of pages | 19 |
Place of Publication | New York |
Publisher | Humana Press |
Publication date | 2019 |
Pages | 131-149 |
ISBN (Print) | 978-1-4939-8792-4 |
ISBN (Electronic) | 978-1-4939-8793-1 |
DOIs | |
Publication status | Published - 2019 |
ID: 4726351