Detection of Mircera in blood plasma using isoelectric focusing combined with magnetic beads purification

C.E.R.A., a continuous erythropoietin receptor activator, is a new third-generation erythropoiesis-stimulating agent (ESA). ESAs are prohibited doping substances. In doping control the current detection method uses concentrated urine samples, which are analysed by isoelectric focusing (IEF) (Lasne et al., 2002). Mircera contains a methoxy polyethylene glycol moiety, which is covalently conjugated to recombinant human EPO (rhEPO) beta. Pharmacokinetic studies revealed slower renal elimination of Mircera compared to rhEPO (Macdougall, 2005). The plasma half-life of Mircera is approximately 15 to 20 times longer. Thus it seems to be more reasonable to use plasma or serum samples for the detection of Mircera instead of urine. Due to the high protein concentration in plasma it is necessary to purify the EPO variants before IEF. Recently, different purification methods for plasma samples by immunoaffinity have been reported (Lasne et al., 2007, Reichel et al., 2009). The methods exploit both possibilities of separating EPO from plasma proteins: either EPO or high abundant proteins are retained on the affinity column. Here we present an alternative purification method that uses magnetic beads and a specific anti-hEPO antibody. Verf.-Referat