Analysis of recombinant human erythropoietin and darbepoietin in spiked plasma

Darbepoietin (DAR) and recombinant human erythropoietin (rhEPO) stimulate erythropoiesis, leading to an increase in red blood cells. Along with their legitimate clinical use, rhEPO and DAR are also misused in racing horses for performance enhancement. To control the illegal use of DAR and rhEPO, it is important to develop analytical methods for the detection and confirmation of these proteins in plasma. Analysis of rhEPO and DAR in plasma is challenging due to the presence of a number of high abundance proteins including albumin that interferes with their extraction. The present study showed that the extraction of rhEPO or DAR from plasma using anti-EPO-antibody coupled immunoaffinity (IA) extraction yielded low (25-40%) recovery. Albumin-depletion using antialbumin-antibody coupled IA columns also depleted the target proteins and further reduced their recovery. Pre-extraction of spiked plasma using hydroxyapatite (HTP)-ProGel or ConA columns followed by the IA column yielded 65 to 70% recovery. The extracted samples were (i) analyzed directly with or without SDS-PAGE for intact proteins and (ii) analyzed after trypsin hydrolysis, with or without SDS-PAGE, for peptide fingerprinting using MALDI-TOF. Trypsin and enolase were used as internal calibrators for intact protein analysis and a peptide EYEATLEECCAK was used as internal calibrator for fragment analysis. Analysis of extracted sample without SDS-PAGE yielded, along with the target proteins (rhEPO and DAR), albumin and other related proteins. SDS-PAGE separated the target proteins with albumin and yielded clean samples. Inclusion of internal calibrators resulted in a linear dose-response relationship for both intact protein and digested fragments and allowed quantification of the target peptides. Thus, extraction of plasma using a combination of ConA and IA extractions yielded approximately 70% recovery of target proteins with a small amount of albumin and other proteins. SDS-PAGE improved the quality of the MALDI-TOF results. Minimum detection limits for digested fragments were lower than those for intact proteins.