Comparison of the Isoelectric Focusing Patterns of Darbepoetin Alfa, Recombinant Human Erythropoietin, and Endogenous Erythropoietin from Human Urine

Catlin, Don H.; Andreas, Breidbach; Elliott, Steve; Glaspy, John A.

doi:10.1093/clinchem/48.11.2057

Cited by 82 publications

(24 citation statements)

References 10 publications

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“…This latter showed it could easily detect both rhEPOs, but the positivity criteria had to be adapted. [13] In order to clean up the biological samples, an immunopurification tool was set up to improve the overall quality of the results. This tool also had the advantage of excluding all potential cross-reactivity of the anti-EPO antibody.…”

Section: Erythropoiesis-stimulating Agentsdirect Detectionmentioning

confidence: 99%

Detection of EPO doping and blood doping: the haematological module of the Athlete Biological Passport

Schumacher

Saugy²,

Pottgießer

et al. 2012

Drug Testing and Analysis

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The increase of the body's capacity to transport oxygen is a prime target for doping athletes in all endurance sports. For this pupose, blood transfusions or erythropoiesis stimulating agents (ESA), such as erythropoietin, NESP, and CERA are used. As direct detection of such manipulations is difficult, biomarkers that are connected to the haematopoietic system (haemoglobin concentration, reticulocytes) are monitored over time (Athlete Biological Passport (ABP)) and analyzed using mathematical models to identify patterns suspicious of doping. With this information, athletes can either be sanctioned directly based on their profile or targeted with conventional doping tests. Key issues for the appropriate use of the ABP are correct targeting and use of all available information (e.g. whereabouts, cross sectional population data) in a forensic manner. Future developments of the passport include the correction of all concentration‐based variables for shifts in plasma volume, which might considerably increase sensitivity. New passport markers from the genomic, proteomic, and metabolomic level might add further information, but need to be validated before integration into the passport procedure. A first assessment of blood data of federations that have implemented the passport show encouraging signs of a decreased blood‐doping prevalence in their athletes, which adds scientific credibility to this innovative concept in the fight against ESA‐ and blood doping. Copyright © 2012 John Wiley & Sons, Ltd.

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Section: Erythropoiesis-stimulating Agentsdirect Detectionmentioning

confidence: 99%

Detection of EPO doping and blood doping: the haematological module of the Athlete Biological Passport

Schumacher

Saugy²,

Pottgießer

et al. 2012

Drug Testing and Analysis

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“…[18,58] average more basic compared to the majority of uhEPO isoforms, with exception of NESP, a biotechnologically modified epoetin, which shows a more acidic isoform distribution. [59] So far, the reasons for this altered behavior of uhEPO have not been elucidated. Usually, the appearance of up to fourteen EPO isoforms has been ascribed to the presence of up to fourteen sialic acids, and a fifteenth isoform was attributed to desialylated EPO (asialo-EPO).…”

Section: Ief-page Analyses Of Exoglycosidase Digested Endogenous and mentioning

confidence: 99%

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

Reichel

2011

Drug Testing and Analysis

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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.

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“…The pattern (number of bands, positions, relative intensities) allows identification. At the 2002 Olympic Games of Salt Lake City, the IEF test with double blotting was successfully used to detect darbepoetin alpha, a long-acting form of rHuEPO, which had been released in US just 4 months before the Games [34]. Darbepoetin alpha has additional negative charges which cause the bands to migrate to the more negative portion of the IEF gel, therefore darbepoetin alpha can readily be distinguished from rHuEPO based on different isoform band patterns.…”

Section: H Catlin Et Al | Review: Medicine and Science In Anti-dmentioning

confidence: 99%

Medicine and science in the fight against doping in sport

Catlin¹,

Fitch

Ljungqvist

2008

Journal of Internal Medicine

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Comparison of the Isoelectric Focusing Patterns of Darbepoetin Alfa, Recombinant Human Erythropoietin, and Endogenous Erythropoietin from Human Urine

Cited by 82 publications

References 10 publications

Detection of EPO doping and blood doping: the haematological module of the Athlete Biological Passport

Detection of EPO doping and blood doping: the haematological module of the Athlete Biological Passport

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

Medicine and science in the fight against doping in sport

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