Capillary isoelectric focusing of erythropoietin glycoforms and its comparison with flat-bed isoelectric focusing and capillary zone electrophoresis

The influence of several operation conditions on separation of recombinant human erythropoietin glycoforms by capillary isoelectric focusing (cIEF) is explored. From this study it is deduced that in order to separate several glycoforms of erythropoietin, urea has to be added to sample, which should not be completely depleted of the excipients used in its formulation. On-line desalting does not provide separation enhancement for samples with high content of salt. Better resolution is obtained using a mixture of a broad and a narrow pH-range carrier ampholytes than with either one used separately. Under the experimental conditions, focusing voltages of 25 kV improve separation compared to lower and higher electric fields. Focusing times shorter than the time necessary for electric current to reach a minimum provide similar separations than longer focusing times at which a minimum value of the current has already been achieved. The optimized method allows the separation and quantitation in 12 min of at least seven bands containing glycoforms of recombinant erythropoietin with apparent isoelectric points in the range 3.78–4.69. Compared to flat-bed isoelectric focusing, cIEF provides better separation of bands of glycoforms in a shorter time, and allows quantitative determination. Capillary zone electrophoresis (CZE) gives rise to resolution of erythropoietin glycoforms similar to that obtained by cIEF. Although CZE requires a longer analysis time, its reproducibility in terms of peak area of glycoforms is better than in cIEF.     

IEF pattern classification‐derived criteria for the identification of epoetin‐δ in urine

Epoetin‐δ (Dynepo? Shire Pharmaceuticals, Basing stoke, UK) is a synthetic form of erythropoietin (EPO) whose resemblance with endogenous EPO makes it hard to identify using the classical identification criteria. Urine samples collected from six healthy volunteers treated with epoetin‐δ injections and from a control population were immuno‐purified and analyzed with the usual IEF method. On the basis of the EPO profiles integration, a linear multivariate model was computed for discriminant analysis. For each sample, a pattern classification algorithm returned a bands distribution and intensity score (bands intensity score) saying how representative this sample is of one of the two classes, positive or negative. Effort profiles were also integrated in the model. The method yielded a good sensitivity versus specificity relation and was used to determine the detection window of the molecule following multiple injections. The bands intensity score, which can be generalized to epoetin‐α and epoetin‐β, is proposed as an alternative criterion and a supplementary evidence for the identification of EPO abuse.     

Anti-EPO and anti-NESP antibodies raised against synthetic peptides that reproduce the minimal amino acid sequence differences between EPO and NESP

Erythropoietin (EPO) is a hormone that regulates red blood cell production. Recombinant human EPO (rHuEPO) and NESP (novel erythropoiesis stimulating protein) have been produced for therapeutic purposes and also to improve sports performance. The primary sequences of rHuEPO and NESP differ by just five amino acids. Due to the high homology, no antibodies that are able to discriminate between both molecules have been obtained until now. The aim of the present work was to design synthetic peptides corresponding to the sequence that differs between EPO and NESP (87–90aa), that can then be used as immunogens to develop specific rabbit polyclonal antibodies for selectively detecting EPO and NESP. Three peptides were synthesized: EPO (81–95), NESP (81–95), and NESP (86–104), and these were coupled to KLH and OVA for immunization and screening purposes, respectively. The sera obtained were tested by ELISA on synthetic peptide–OVA conjugates and purified by immunoaffinity chromatography against the corresponding synthetic peptide. The specific purified antibodies were characterized by ELISA, SDS-PAGE, and isoelectric focusing, followed by western blot. Antisera raised against EPO (81–95) recognized rHuEPO but not NESP. In contrast, anti-NESP (84–106) sera gave a specific anti-NESP response only after immunoaffinity purification on a NESP (86–91) column. An efficient strategy for generating specific antibodies against EPO and NESP can be achieved by selecting suitable synthetic peptides. The antibodies obtained are able to differentiate between rHuEPO and NESP, and may be particularly useful for screening purposes in both therapeutic and antidoping contexts.     

Assessing the instability of the isoelectric focusing patterns of erythropoietin in urine

IEF can be used to differentiate human urinary erythropoietin (uEPO), recombinant human erythropoietin or epoetin (rEPO) and darbepoetin (novel erythropoiesis stimulating protein (NESP)). This is the basis of the method currently used to detect misuse of rEPO and NESP by elite athletes. Recently, an unknown activity has been attributed to some urine samples (denominated 'unstable' urine by the World Anti-Doping Agency; WADA). This activity has shown to give rise to artefactual profiles for both rEPO and NESP when incubated with such urine and, thus, raised concerns with respect to doping control. We have evaluated which charges produce the characteristic IEF profiles of uEPO, rEPO and NESP and how these profiles respond to distinct enzymatic reactions. From sialidase digestions it became evident that only uEPO contains charges different from sialic acid, and a comparison of all substances after complete de-N-glycosylation localized these charges in the carbohydrate moiety. Partial desialylation, or digestion with arylsulfatase from Helix pomatia yielded profiles for recombinants species similar to those observed for unstable urine samples. The contributions from our studies to the anti-doping problem include: (i) protocols that may corroborate the potential misuse of rEPO or NESP based on the particular enzymatic activity of an arylsulfatase preparation, or a broad-specificity sialidase; (ii) assurance that the instability observed in some urine samples may only result from false-negatives, but not from false-positive testing; and (iii) a simple remedy to prevent an unstable urine from altering the IEF profile by adding selective competitive substrates.     

Profiling of erythropoietin products by capillary electrophoresis with native fluorescence detection

The potential of CE with native fluorescence detection (Flu) for the profiling of the therapeutic protein erythropoietin (EPO) was studied. EPO is a highly heterogeneous glycoprotein comprising a large number of isoforms. CE was applied to induce separation among the various glycoforms. Native Flu of EPO provided high detection selectivity yielding good signal‐to‐noise ratios and stable baselines, particularly when compared to conventional UV absorbance detection. In order to enhance EPO isoform resolution, CE was performed using a capillary with a neutral coating in combination with a simple BGE of 2.0 M acetic acid (pH 2.1). CE‐Flu analysis of the EPO biological reference preparation of the European Pharmacopeia resulted in a highly detailed glycoform profile. Migration time RSDs for selected EPO isoforms were less than 0.22% and 0.80% for intraday and interday repeatability, respectively. RSDs for relative peak intensity of the major EPO isoforms were less than 3%. The achieved resolution, migration time stability, and sensitivity allowed discrimination of different EPO products (EPO‐α and EPO‐β) based on the recorded glycoform pattern. The developed CE‐Flu method is relatively straightforward, and shows potential for quality control in biopharmaceutical production.     

Isolation and characterization of an anti-recombinant erythropoietin single-chain antibody fragment using a phage display antibody library

The production of a large amount of specific antibodies against erythropoietin (EPO) is necessary for both clinical treatment and doping control. However, the weak immunogenicity of EPO and the side effects of excessive injection make the conventional immunological protocol rather inefficient and time-consuming. In this study, a single-chain antibody fragment of variable region (scFv) against recombinant human erythropoietin (rHuEPO) was produced after three rounds of panning a phage display antibody library. The selected scFv-B₂ was expressed in soluble form in Escherichia coli DH5α F′ and purified by His-bond nickel affinity chromatography with a yield of about 1–2 mg of antibody in 1 L of the culture supernatant. The molecular weight of the scFv was estimated to be 29 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the affinity constant was found to be 1.0×10⁸ L mol⁻¹ based on a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). The potential ability of the scFvs for immunopurification of rHuEPO from related sample was demonstrated by using a double-antibody sandwich ELISA. The reported method is a very powerful tool to produce specific antibodies for rHuEPO detection demands.     

Precision and variance components in quantitative gel electrophoresis

The error in quantitative gel electrophoresis/Western blotting was investigated considering the purity testing of erythropoietin. The overall error was over 35% relative standard deviation. However, an analysis of variance elucidated that the interoperator variability was the dominant error source, which already explained almost 80% of the total variance. Careful compilation and investigation of the possible error sources strongly indicates that the immunoreaction after blotting and the subsequent color reaction are the major error sources in this case.     

促红细胞生成素后处理减轻大鼠再灌注损伤肺细胞凋亡中的作用及机制

目的探讨促红细胞生成素后处理在减轻肺缺血/再灌注损伤（LIRI）大鼠肺细胞凋亡中的作用及其机制。方法健康雄性成年SD大鼠40只,按随机数字表法分成5组,每组8只,即假手术对照组（C组）、缺血/再灌注(IR)组、促红细胞生成素（EPO）组、EPO+溶剂对照组（PPCES溶液）（P组）和EPO+SP600125（SP组）,通过阻断左肺门制作动物模型并予相应处理。原位末端标记法（TUNEL）检测肺细胞凋亡情况并计算凋亡指数（AI）;RT-PCR法、免疫组化法测定肺组织Bcl-2、Bax基因和蛋白的表达;光镜下观察肺组织的病理变化及测定肺泡损伤数（IQA）。结果与C组比较,IR组肺组织IQA显著升高,AI显著升高,Bcl-2基因和蛋白表达明显下降,Bax基因和蛋白表达明显上调,Bcl-2/Bax的比值降低（均P＜0.05）,肺组织形态学发生异常改变;与IR组比较,EPO组、P组、SP组的IQA显著降低,AI显著降低,Bcl-2基因和蛋白表达上调,Bax基因和蛋白和表达下降,Bcl-2/Bax的比值增高（均P＜0.05）,肺组织形态学结构异常改变有所减轻;与EPO组比较,SP组的IQA降低,AI显著降低,Bcl-2基因和蛋白表达上调,Bax基因和蛋白表达下降,Bcl-2/Bax的比值增高（均P＜0.05）,SP组的肺组织形态学结构损伤较EPO组减轻。结论EPO后处理能减轻LIRI,其机制可能通过抑制JNK信号转导通路的激活,上调凋亡抑制因子Bcl-2的表达,下调促凋亡基因Bax的表达,提高Bcl-2/Bax比值,使肺组织细胞凋亡减少,改善其结构。     

非糖基化促红细胞生成素的聚乙二醇定点修饰及修饰产物性质

临床用重组人促红细胞生成素(rhEpo)是中国仓鼠卵巢细胞(Chinese hamster ovary cell, CHO)表达的糖蛋白, 糖基对稳定蛋白的结构和生物活性非常重要, 但CHO表达体系生产成本高、产量低. 以大肠杆菌表达的促红细胞生成素为非糖基化蛋白(rh-ngEpo), 对其进行聚乙二醇(PEG)修饰可以提高蛋白稳定性和体内循环半衰期. 本文采用分子量为20000的N-末端专一性的单甲氧基聚乙二醇-丙醛(mPEG-ALD)修饰rh-ngEpo, 对影响修饰反应的因素进行了考察. 结果表明, 在最佳反应条件下, 单修饰率可达55%. 修饰混合物经离子交换层析分离, 获得了纯度大于95%的单修饰产物, 其二、三级结构证明与原蛋白相似. 肽图分析结果表明, PEG绝大部分修饰在蛋白N-末端的氨基酸残基上. ELISA分析表明, 单修饰产物的体外活性虽然比修饰前减少30%, 但热稳定性得到显著增强, 在SD大鼠体内的药代动力学性质得到显著提高. 研究结果表明, PEG可以在一定程度上替代糖基的作用, PEG修饰的非糖基化Epo有望成为一种新型的促红细胞生成蛋白药物.