Detecting ESA (EPO) doping in sports

Jake Young, the MD/PhD student blogging at Pure Pedantry, has a great post this week on the detection of a novel formulation of the erythropoiesis stimulating agent (ESA) erythropoietin in Riccardo Riccó, the Italian cyclist who was thrown out of the Tour de France. Jake's post is a superb primer on the use of this peptide hormone as a therapeutic agent (in the anemia caused by kidney failure and in cancer chemotherapy).

His essay also reminds me that I commented on this issue at DrugMonkey's a post exactly a week ago (and from this same couch at my local coffee shop while waiting for PharmKid to finish her Saturday morning dance lesson.). Drug thought it would be a good post but the week got away from me. So go read Jake's comprehensive post first and then consider my comments (also below) on how this novel formulation of Epo might have been detected:

Micera is simply a pegylated (actually, methoxy polyethylene glycol) EPO-beta. Polyethylene glycol has long been used as an excipient to aid in drug dissolution. But covalent conjugation of it to protein drugs improves half-life and decreases immunogenicity. In my field, pegylated E. coli L-asparaginase (pegaspargase) is used in some leukemia regimens to counter the high asparagine requirements of such hematologic malignancies. I think that pegylation was used even earlier for another drug product.

Although PEG conjugation is meant to disguise the protein drug from immune detection, one can still generate antibodies to PEG in the lab. In fact, anti-PEG monoclonal antibodies are available commercially so my guess is that an ELISA was designed with anti-EPO antibodies for capture and biotinylated anti-PEG antibodies for detection. Anti-EPO should detect any EPO product whereas the anti-PEG will distinguish between EPO or darbopoeitin vs. pegylated-EPO. Alternatively, one could immunoprecipate from plasma with anti-EPO or anti-PEG, then run a Western and probe with the reciprocal antibody. I don't know a lot about this area but I'd also guess that pegylated-EPO runs on a gel with reduced mobility relative to native EPO.

Even though Micera was approved by the EU last August and in the US shortly thereafter, anti-PEG antibodies were already available. So, no surprise to me that anti-doping lab rats were well ahead of the curve on this one.

btw, pharmaceutically-speaking, Roche is in a pissing match with EPO giant Amgen over whether Micera represents a true novel composition of matter relative to Amgen's products. This may be one case where Amgen won't win because of the precedent with other pegylated proteins (and small molecules) represent significant advances over the prior art.

The more I've thought about it since, Roche must've already had a clinical diagnostic test for pegylated-Epo in order to conduct the human pharmacokinetic studies required for drug approval.

Any other thoughts from people work in the Epo field?

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Here's a human PK/PD study of Mircera (aka C.E.R.A.). The methods section states:

Serum C.E.R.A. concentrations were determined by ELISA using a primary mAb that was specific for C.E.R.A. and that did not cross-react with endogenous erythropoietin, and using a secondary polyclonal anti-Ig antibody coupled to horseradish peroxidase.

Many thanks, qetzal. I know that I could've dialed up the ol' PubMed but the little one's dance class was over.