Reverse-normal immunopurification: An effective approach for purifying recombinant erythropoietin from its analogues in doping analysis

Abstract

Background: Recombinant erythropoietin (rEPO) is commonly used in therapy but may be abused in sports to enhance endurance. In doping analysis, rEPO can be detected in human urine or blood samples at picogram (pg) levels based on its slightly higher molecular weight (MW) than that of endogenous EPO using western blotting (WB). However, a type of variant erythropoietin (VAR-EPO) encoded by the EPO c.577del variant has a similar MW to rEPO, and these 2 molecules cannot be distinguished using conventional analytical methods. A fit-for-purpose method needs to be developed immediately.

Methods: In this study, we introduced a reverse-normal immunopurification technique for sample pretreatment to remove VAR-EPO from samples to eliminate its interference with rEPO detection. Firstly, a rabbit monoclonal antibody (mAb) that can specifically recognize trace amounts of VAR-EPO with high affinity was generated. Then, using this antibody to enrich VAR-EPO, we developed reverse-normal immunopurification coupled with WB on the purpose of analyzing rEPO in urine and serum samples. Next, the method was fully validated and evaluated using blank samples, spiked samples and rEPO excreted samples. Finally, the identification criteria of rEPO was established.

Results: A specific anti-VAR mAb with high affinity was developed. Using it, we developed the doping analytical method for rEPO. Our method effectively detects and removes VAR-EPO, enabling accurate rEPO detection.

Conclusion: A method has already been applied for rEPO confirmation in routine doping analyses.

Keywords: Doping analysis; Erythropoietin; Immunopurification; Western blotting.

Graphical abstract

Fig. 1

Production of anti-VAR mAb. To acquire a specific anti-VAR mAb, candidate antigens based on the differential peptide between WT-EPO and VAR-EPO were tested. The peptide sequence CHTLPRHSGGGSHTLPRHS was selected following titre testing after 3 rounds of immunization in mice. The recombinant rabbit mAb was generated using single B-cell antibody technology after immunization. Multiple screening steps were conducted during immunization, single B-cell sorting, and B-cell selection, aiming to achieve high affinity for trace amounts of VAR-EPO and ensuring no cross-reactivity with WT-EPO. anti-VAR mAb = anti VAR-EPO monoclonal antibody; BLI = bio-layer interferometry; ELISA = enzyme-linked immunosorbent assay; FACS = fluorescence-activated cell sorting; mAb = monoclonal antibody; PCR = polymerase chain reaction; rEPO = recombinant erythropoietin; rVAR-EPO = recombinant variant erythropoietin; SPR = surface plasmon resonance; VAR-EPO = variant erythropoietin; VH = heavy chain; VL = light chain; WB = western blotting; WT-EPO = wild-type erythropoietin.

Fig. 2

Development of the reverse–normal immunopurification method coupled with western blotting. (A) Schematic of the reverse–normal immunopurification method. VAR-EPO is designed to be removed and analyzed separately from WT-EPO and rEPO. With this method, rEPO can be detected without interference of VAR-EPO. (B) EC50 curve of antibody. Each antigen, including the differential peptide between WT-EPO and VAR-EPO (DP), rVAR-EPO, and rEPO, was coated in a series of 2-fold dilutions ranging from 1 ng to 7.81 pg per well. Each well contained 1 µg of antibody. The anti-VAR mAb specifically recognizes DP and rVAR-EPO, with no cross-reactivity to rEPO. Anti-VAR mAb: TYJ9-R0016 (in-house developed); anti-EPO pAb: AB-286-NA (R&D systems). (C) Surface plasmon resonance (SPR) assay to determine the affinity between anti-VAR mAb (TYJ9-R0016, developed in-house) and rVAR-EPO. The protein A chip was coupled with anti-VAR mAb (TYJ9-R0016) at 5 µg/mL, while rVAR-EPO concentrations ranged from 100 nM to 6.25 nM in 2-fold dilutions. This anti-VAR mAb shows high affinity for trace amounts of rVAR-EPO. (D) Experimental protocol for reverse–normal immunopurification coupled with western blotting. Eluate from RI and NI were loaded on separate SAR-PAGE gels, followed by western blotting. Ab = antibody; CERA = continuous erythropoietin receptor activator; DP = differential peptide; DYNEPO = epoetin-δ (a type of rEPO); EC50 = 50% effective concentration; EPO = erythropoietin; EPO-Fc = recombinant fusion protein comprising EPO linked to the human immunoglobulin Fc domain; mAb = monoclonal antibody; NESP = darbepoetin-α; NI = normal immunopurification; OD = optical density; pAb = polyclonal antibody; rEPO = recombinant erythropoietin; RI = reverse immunopurification; RT = room temperature; RU = response unit; SAR = sarcosyl; SAR-PAGE = sarcosyl polyacrylamide gel electrophoresis; VAR = variant; VAR-EPO = variant erythropoietin; VAR-PEG = PEGylated recombinant VAR-EPO; WT-EPO = wild-type erythropoietin.

Fig. 3

Inter-lab comparison. The screening method refers to the original process without RI, and the confirmation method refers to our developed approach. Mix: DYNEPO and NESP. (A) Result from Lab 1 A1–A10: samples conducted by Lab 1. (B) Result from Lab 2 B1–B6: samples conducted by Lab 2. (C) Result from Lab 3 C1–C6: samples conducted by Lab 3. DYNEPO = epoetin-δ (a type of rEPO); NESP = darbepoetin-α; NI = normal immunopurification; rEPO = recombinant erythropoietin; RI = reverse immunopurification.

Fig. 4

Analysis of rEPO in urine and blood samples. The screening method refers to the original process without RI, and the confirmation method refers to our developed approach. Blank samples used for QC were collected from variant c.577del non-carriers. (A) Urine samples collected from an rEPO administration study in variant c.577del non-carriers. Samples were collected on the day before administration (D0) and on each subsequent day (D1, D3, D5, D7, and D9). Mix: DYNEPO and NESP. (B) Urine samples collected from an rEPO administration study in variant c.577del carriers analyzed using double rounds of RI followed by NI. Samples were collected the day before administration (D0) and on each subsequent day (D1–D7). Mix: DYNEPO and NESP; QCN: blank urine; QCV: blank urine spiked with 10 pg of rVAR-EPO; QCP: blank urine spiked with 15 mIU of rEPO; 1st RI: eluate from the first round of RI; 2nd RI: eluate from the second round of RI. (C) Urine samples in doping analysis. Urine 1: washout sample from rEPO administration study in a variant c.577del non-carrier; Urine 2: blank sample from a variant c.577del carrier; Urine 3: washout sample from rEPO administration study in a variant c.577del carrier. Mix1: DYNEPO and NESP and continuous erythropoietin receptor activator (CERA); Mix2: DYNEPO and NESP and recombinant fusion protein comprising EPO linked to the human immunoglobulin Fc domain (EPO-Fc); QCN: blank urine; QCV: blank urine spiked with 10 pg of rVAR-EPO; QCP: blank urine spiked with 15 mIU of rEPO. (D) Serum samples in doping analysis. Serum 1: washout sample from an rEPO administration study in a variant c.577del non-carrier; Serum 2: blank sample from a variant c.577del carrier. Mix1: DYNEPO and NESP and CERA; Mix2: DYNEPO and NESP and EPO-Fc; QCN: blank serum; QCV: blank serum spiked with 50 pg of rVAR-EPO; QCP: blank serum spiked with 5 mIU of rEPO. DYNEPO = epoetin-δ (a type of rEPO); EPO = erythropoietin; NESP = darbepoetin-α; NI = normal immunopurification; QC = quality control; QCN = negative control; QCP = positive control; QCV = variant control; rEPO = recombinant erythropoietin; RI = reverse immunopurification; rVAR-EPO = recombinant variant erythropoietin; TSC = test sensitivity control; VAR = recombinant VAR-EPO (rVAR-EPO); VAR-EPO = variant erythropoietin.

Fig. 5

Identification strategy for rEPO. (A) Identification strategy for rEPO in urine samples. (B) Identification strategy for rEPO in blood samples. NI = normal immunopurification; rEPO = recombinant erythropoietin; RI = reverse immunopurification; WT-EPO = wild type erythropoietin; VAR-EPO = variant erythropoietin.