Christian Reichel

SDS-PAGE of recombinant and endogenous erythropoietins: benefits and limitations of the method for application in doping control.

Doping of athletes with recombinant and genetically modified erythropoietins (EPO) is currently detected by isoelectric focusing (IEF). The application of these drugs leads to a significant change in the isoform profile of endogenous urinary erythropoietin (uhEPO). Dynepo, MIRCERA, biosimilars with variable IEF-profiles as well as active urines and effort urines have made additional testing strategies necessary. The new generation of small molecule EPO-receptor stimulating agents like Hematide will also challenge the analytical concept of detecting the abuse of erythropoiesis stimulating agents (ESA). By determining their apparent molecular masses with SDS-PAGE a clear differentiation between endogenous and exogenous substances also concerning new EPO modifications is possible. Due to the orthogonal character of IEF- and SDS-PAGE both methods complement each other. The additional benefits of SDS-PAGE especially in relation to active and effort urines as well as the detection of Dynepo were investigated. Due to significant differences between the apparent molecular masses of uhEPO/serum EPO (shEPO) and recombinant, genetically or chemically modified erythropoietins the presence of active or effort urines was easily revealed. The characteristic band shape and apparent molecular mass of Dynepo on SDS-PAGE additionally evidenced the presence of this substance in urine. A protocol for the detection of EPO-doping in serum and plasma by SDS-PAGE was developed. Blood appears to be the ideal matrix for detecting all forms ESA-doping in the future.

SARCOSYL‐PAGE: a new method for the detection of MIRCERA‐ and EPO‐doping in blood

The detection of doping with MIRCERA (the brand name for Continuous Erythropoietin Receptor Activator, or CERA) is hampered by the limited excretion of the rather large molecule (approximately 60 kDa) in urine. Blood (serum, plasma) in combination with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) appears to be the ideal matrix for detecting all forms of doping with erythropoiesis-stimulating agents (ESAs) because the apparent molecular masses of ESAs are different from the mass of human serum erythropoietin (shEPO). While SDS-PAGE has proven the most sensitive method for the detection of doping with Dynepo, the sensitivity of SDS-PAGE for MIRCERA is drastically decreased. By exchanging the SDS for SARCOSYL (SAR) in the sample and running buffers the sensitivity problem was solved. SARCOSYL, a methyl glycine-based anionic surfactant, is only binding to the protein-part of MIRCERA but not to its polyethylene glycol (PEG)-chain, while SDS binds to both parts. In consequence, the monoclonal anti-EPO antibody (clone AE7A5) no longer interacts with the fully SDS-solubilized MIRCERA molecules. Only those molecules that contain SDS bound to the protein-chain are detected. Due to the inability of SARCOSYL to solubilize PEG-molecules, MIRCERA can be detected on SARCOSYL-PAGE with the same sensitivity as non-PEGylated epoetins. In a typical SAR-PAGE experiment, 200 microL of serum are used, which allows the direct detection of MIRCERA, recombinant epoetins (such as NeoRecormon, Dynepo, NESP), and shEPO in a single experiment and with high (i.e. femtogram) sensitivity.

Erythropoietin and Analogs

Erythropoietin (EPO), a glycoprotein hormone, stimulates the growth of red blood cells and as a consequence it increases tissue oxygenation. This performance enhancing effect is responsible for the ban of erythropioetin in sports since 1990. Especially its recombinant synthesis led to the abuse of this hormone, predominatly in endurance sports. The analytical differentiation of endogenously produced erythropoietin from its recombinant counterpart by using isoelectric focusing and double blotting is a milestone in the detection of doping with recombinant erythropoietin. However, various analogous of the initial recombinant products, not always easily detectable by the standard IEF-method, necessitate the development of analytical alternatives for the detection of EPO doping. The following chapter summarizes its mode of action, the various forms of recombinant erythropoietin, the main analytical procedures and strategies for the detection of EPO doping as well as a typical case report.

Detection of EPO‐Fc fusion protein in human blood: Screening and confirmation protocols for sports drug testing

The neonatal Fc receptor (FcRn) has been under investigation for several years as a pharmaceutical drug target. Clinical studies have shown that fusion proteins consisting of human recombinant erythropoietin (rhEPO) and the Fc-part of IgG can be transported after pulmonary administration via FcRn across the airway epithelium to the blood stream. So far, no clinically approved pharmaceutical formulation of EPO-Fc is available. Since various forms of recombinant erythropoietins have been frequently misused by athletes as performance-enhancing agents, EPO-Fc might play a similar role in sports in the future. In order to investigate the detectability of EPO-Fc in human blood, different strategies were tested and developed. Only two of them fulfilled the necessary requirements regarding sensitivity and specificity. A rapid protocol useful for screening purposes first enriches EPO-Fc from human serum via high capacity protein A beads and subsequently detects EPO-Fc in the eluate with a commercial EPO ELISA kit. The limit of detection (LOD) of the method is about 5 pg (45 amol) EPO-Fc and is independent of the serum volume used. For screening and/or confirmation purposes a second protocol was evaluated, which consists of a fast EPO immunopurification step followed by sodium dodecyl sulfate or sarcosyl polyacrylamide gel electrophoresis (SDS-PAGE, SAR-PAGE) and Western double-blotting with chemiluminescence detection - a method already established in routine EPO anti-doping control. The latter strategy allows the detection of EPO-Fc in serum together with all other recombinant erythropoietins and with an identical LOD (5 pg/45 amol) as for the rapid screening protocol.

The overlooked difference between human endogenous and recombinant erythropoietins and its implication for sports drug testing and pharmaceutical drug design

Sequential deglycosylation by exoglycosidase treatment (Reagent Array Analysis Method, RAAM) and subsequent sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a profound structural difference between human endogenous and recombinant erythropoietins. While both proteins behaved similarly upon digestion with Arthrobacter ureafaciens α-sialidase and Steptococcus pneumoniae β-D-galactosidase, the action of N-acetyl-β-D-glucosaminidase from Steptococcus pneumoniae was partly blocked by endogenous but not recombinant erythropoietins. Consequently, further treatment with Jack bean α-D-mannosidase and Helix pomatia β-D-mannosidase led to only very limited additional deglycosylation of endogenous EPO, while rhEPO glycans continued to be degraded. The behaviour was visualized by SDS-PAGE combined with Western blotting. While the apparent molecular masses of most endogenous glycoforms did not further decrease after treatment with the first three enzymes, masses of most rhEPO glycoforms continued to drop after digestion with the two mannosidases. Both, human urinary and serum EPO showed this blocking effect, and all of the tested 28 recombinant epoetins were accessible to further degradation by exo-mannosidases. The majority of EPO pharmaceuticals is produced in Chinese hamster ovary (CHO) cell lines, few in other ones (i.e. baby hamster kidney (BHK) or human fibrosarcoma (HT-1080) cells). Since human endogenous EPO is primarily produced by the kidneys, tissue specific glycosylation might explain the altered deglycosylation behaviour. This difference was overlooked since EPO was first isolated from human urine in 1977. The results might prove useful for anti-doping testing and future EPO drug development.

Gel electrophoretic methods for the analysis of biosimilar pharmaceuticals using the example of recombinant erythropoietin

Due to their versatility and cost-effectiveness, gel electrophoretic methods provide an important set of tools for the analysis of therapeutic proteins. As an increasing number of biosimilar pharmaceuticals are entering the market, techniques are required that allow reliable demonstration of comparability of these products with the reference products. Isoelectric focusing, SDS-PAGE, native PAGE and 2D electrophoresis (2D-PAGE) have been frequently applied for this purpose. Supplementary techniques are fluorophore-assisted carbohydrate electrophoresis and sarcosyl-PAGE. Of additional importance is the comparison of recombinant with endogenously synthesized glycoproteins. Reagent array analysis combined with SDS-PAGE and western blotting proved especially useful for this purpose. As an example for the application of these methods, the analysis of recombinant originator erythropoietins and some of their biosimilar counterparts is described.

Detection of erythropoiesis-stimulating agents in human anti-doping control: past, present and future

Stimulation of erythropoiesis is one of the most efficient ways of doping. This type of doping is advantageous for aerobic physical exercise and of particular interest to endurance athletes. Erythropoiesis, which takes place in bone marrow, is under the control of EPO, a hormone secreted primarily by the kidneys when the arterial oxygen tension decreases. In certain pathological disorders, such as chronic renal failure, the production of EPO is insufficient and results in anemia. The pharmaceutical industry has, thus, been very interested in developing drugs that stimulate erythropoiesis. With this aim, various strategies have been, and continue to be, envisaged, giving rise to an expanding range of drugs that are good candidates for doping. Anti-doping control has had to deal with this situation by developing appropriate methods for their detection. This article presents an overview of both the drugs and the corresponding methods of detection, and thus follows a roughly chronological order.

Practicing IEF-PAGE of EPO: the impact of detergents and sample application methods on analytical performance in doping control.

Electrophoretic techniques, namely isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are key techniques used for confirming the doping-related abuse of recombinant erythropoietins and analogs. IEF-PAGE is performed on horizontal slab-gels with samples applied to the surface of the gel. Different sample application techniques can be employed, but application pieces and applicator strips are most frequently used. However, defective application pieces cause lane streaking during IEF of erythropoietin (EPO), which is especially pronounced in the acidic region of the gel. The effect is due to an incompatibility of the substance used for enhancing the wettability of the cellulose-based commercial product and is batch-dependent. A detailed mass spectrometric study was performed, which revealed that defective sample application pieces (bought between 2007 and 2010) contained a complex mixture of alcohol ethoxylates, alcohol ethoxysulfates, and alkyl sulfates (e.g. SDS). Anionic detergents, like the sulfates contained in these application pieces, are in general incompatible with IEF. Alternative application techniques proved partly useful. While homemade pieces made of blotting paper are a good alternative, the usage of applicator strips or shims is hampered by the risk of leaking wells, which lead to laterally diffused samples. Casting IEF-gels with wells appears to be the best solution, since sustained release of retained proteins from the application pieces can be avoided. Edge effects do not occur if wells are correctly filled with the samples. The evaluation of EPO-profiles with defects is prohibited by the technical document on EPO-analytics (TD2009EPO) of the World Anti-Doping Agency (WADA).

Isolation, enrichment, and analysis of erythropoietins in anti-doping analysis by receptor-coated magnetic beads and liquid chromatography-mass spectrometry.

Human erythropoietin (hEPO) is an erythropoiesis stimulating hormone frequently employed in antianemia therapy. Its capability to increase the amount of red blood cells however makes hEPO and its derivatives also attractive to dishonest athletes aiming at an artificial and illicit enhancement of their endurance performance. A major objective of the international antidoping fight is the elimination of drug misuse and prevention of severe adverse effects caused by nontherapeutic administrations of highly potent drugs. The emergence of novel and innovative erythropoietin-mimetic agents (EMAs) has been continuously growing in the last years, and the option of using dedicated monoclonal antibodies (mAb) for analytical and sample preparation approaches is gradually reaching limits. In the present study the common ability and property of all EMAs, to bind on the human erythropoietin receptor (hEPOR), is therefore exploited. An alternative methodology to isolate and analyze EMAs, in particular endogenous EPO and the recombinant forms EPOzeta, darbepoetin alfa, and C.E.R.A., from human urine is described, employing conventional ultrafiltration for preconcentration of the target analytes followed by EMA-specific isolation via hEPOR-bound magnetic beads. Analytical data were generated by means of gel-based electrophoretic analysis and nanoliquid chromatography/high resolution/high accuracy tandem mass spectrometry. Limits of detection enabled by the established sample preparation protocols were approximately 20 pg/mL for EPOzeta, 30 pg/mL for darbepoetin alfa, and 80 pg/mL for C.E.R.A.

Time for change: a roadmap to guide the implementation of the World Anti-Doping Code 2015

A medical and scientific multidisciplinary consensus meeting was held from 29 to 30 November 2013 on Anti-Doping in Sport at the Home of FIFA in Zurich, Switzerland, to create a roadmap for the implementation of the 2015 World Anti-Doping Code. The consensus statement and accompanying papers set out the priorities for the antidoping community in research, science and medicine. The participants achieved consensus on a strategy for the implementation of the 2015 World Anti-Doping Code. Key components of this strategy include: (1) sport-specific risk assessment, (2) prevalence measurement, (3) sport-specific test distribution plans, (4) storage and reanalysis, (5) analytical challenges, (6) forensic intelligence, (7) psychological approach to optimise the most deterrent effect, (8) the Athlete Biological Passport (ABP) and confounding factors, (9) data management system (Anti-Doping Administration & Management System (ADAMS), (10) education, (11) research needs and necessary advances, (12) inadvertent doping and (13) management and ethics: biological data. True implementation of the 2015 World Anti-Doping Code will depend largely on the ability to align thinking around these core concepts and strategies. FIFA, jointly with all other engaged International Federations of sports (Ifs), the International Olympic Committee (IOC) and World Anti-Doping Agency (WADA), are ideally placed to lead transformational change with the unwavering support of the wider antidoping community. The outcome of the consensus meeting was the creation of the ad hoc Working Group charged with the responsibility of moving this agenda forward.