Fc‐fragment removal allows the EPO‐Fc fusion protein to be detected in blood samples by IEF‐PAGE

Abstract

EPO-Fc proteins have been under investigation as a potential drug for treating anaemia and have shown larger half-life values than other erythropoiesis-stimulating agents (ESAs). Sodium dodecyl sulfate/sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SDS/SAR-PAGE) methods and subsequent immunoblotting are used for routine anti-doping analysis. This paper reports that EPO-Fc fusion proteins can be detected in serum samples by isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) in carrier ampholyte-based gels with a pH 2-6 gradient after removing the Fc part via site-specific IdeS protease cleavage. The IdeS-digested EPO-Fc protein yields three fragments: two Fc fragments and one dimeric EPO-hinge fragment. After IEF-PAGE was followed by double Western blotting with chemiluminescent detection, the dimeric EPO-hinge fragment showed a unique isoelectric pattern, which differed from those of any other currently known analogue of EPO. We observed that the removal of the Fc fragment from EPO-Fc reduced the apparent molecular weight of entire fusion protein and increased its electrophoretic mobility. As a result, the band for the EPO-hinge fragment was located in a region between the rEPO and NESP standards, at which lower amounts of serum proteins are present. Simple and selective protocols for determining the EPO-Fc protein in human serum were developed to extend the methodological anti-doping arsenal. This protocol has been characterized. The limit of detection (LOD) of the IEF-PAGE method was 20 pg, and that of SDS/SAR-PAGE was 15 pg.

Annual banned‐substance review: analytical approaches in human sports drug testing

“…This is particularly relevant for sporting events with large numbers of doping control specimens necessitating fast reporting times. The improved detection of the hybrid between EPO and the crystallizable fragment (Fc) of human immunoglobulin G (IgG) by means of isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) was reported by Postnikov et al [91] The analysis of the EPO-Fc fusion protein by conventional IEF-PAGE was shown to be limited in isoform separation capabilities and, consequently, the effect of the proteolytic elimination of parts of the Fc moiety by means of an immunoglobin-degrading enzyme (IdeS) was studied. Following enzymatic cleavage of the Fc moiety between the hingeregion and the CH2-domain, IEF-PAGE enabled the unequivocal identification of EPO-Fc with a LOD of 20 pg (on gel).…”

Characterization and Detection of Erythropoietin Fc Fusion Proteins Using Liquid Chromatography–Mass Spectrometry

“…In addition to this approach, some new approaches for the determination of EPO-Fc using electrophoretic techniques were developed. 18 The goal of this study is to identify peptides covering unknown fusion breakpoints (later referred to as "spacer" peptides). The identification of "spacer" peptides will allow us to unequivocally confirm the presence of exogenous EPO-Fc in human biological fluids.…”

“…However, the prototyping peptides derived from EPO and IgG are not selective enough because both free proteins are naturally presented in human serum. In addition to this approach, some new approaches for the determination of EPO-Fc using electrophoretic techniques were developed …”

The 33rd Manfred Donike Workshop on Doping Analysis

“…An approach allowing for implementing the fusion protein of erythropoietin and the dimeric Fc region of human IgG into routine doping controls were presented, enabling a limit of detection (LOD) of approximately 20 pg (on gel) of the prohibited compound. [12] An immunoglobulin-degrading enzyme (IdeS) was utilized prior to conventional isoelectric focusing, complementing the portfolio of analytical techniques for the complex task of testing for erythropoietin analogues. Options to facilitate SAR-PAGE-based erythropoietin analyses were reported by Reihlen et al who demonstrated ways of automating time-consuming sample preparation steps thus increasing the efficiency of erythropoietin routine doping controls.…”

“…Analytes included growth hormone releasing hormones (4), insulins (8), synacthen, longR 3 ‐IGF‐I, and mechano growth factors (3), and detection limits of 5–100 pg/mL and 100–200 pg/mL were shown for urine and plasma, respectively. An approach allowing for implementing the fusion protein of erythropoietin and the dimeric Fc region of human IgG into routine doping controls were presented, enabling a limit of detection (LOD) of approximately 20 pg (on gel) of the prohibited compound . An immunoglobulin‐degrading enzyme (IdeS) was utilized prior to conventional isoelectric focusing, complementing the portfolio of analytical techniques for the complex task of testing for erythropoietin analogues.…”