Erythropoiesis stimulating agents and techniques: a challenge for doping analysts

Area of Science:

• Biotechnology and Pharmacology
• Sports Science and Anti-Doping
• Analytical Chemistry

Background:

• Recombinant human erythropoietin (rHuEPO) products like Epoetin alfa, Epoetin beta, and Darbepoetin alfa are misused in sports.
• Detection relies on isoelectric focusing and immunoblotting due to isoform and N-glycan differences between recombinant and endogenous EPO.
• The market now includes rHuEPO biosimilars and diverse novel erythropoiesis-stimulating agents (ESAs).

Purpose of the Study:

• To review the evolving landscape of erythropoiesis-stimulating agents (ESAs) and their detection in anti-doping.
• To highlight the challenges posed by new biosimilars, modified EPO structures, and emerging EPO-mimicking drugs.
• To discuss future doping threats, including orally active agents and gene doping.

Main Methods:

• Isoelectric focusing (IEF) and immunoblotting of urine samples to detect EPO isoforms and glycan variations.
• Analysis of amino acid sequences and glycan structures of various recombinant EPOs and ESAs.
• Review of ongoing research and development in novel ESAs and EPO-stimulating drugs.

Main Results:

• Recombinant EPOs (Epoetin alfa, beta, delta) and analogues (Darbepoetin alfa) exhibit distinct isoform profiles and glycan structures compared to native EPO.
• Biosimilars and international copied rHuEPOs present challenges due to differing glycan profiles.
• Emerging ESAs include fusion proteins, synthetic agents, EPO mimetics, and orally active drugs targeting EPO production.

Conclusions:

• The diversity of ESAs necessitates continuous development of sophisticated doping control methods.
• Distinguishing between different recombinant EPOs and novel agents is crucial for effective anti-doping.
• Future doping strategies may involve gene doping and orally administered EPO-stimulating drugs.