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.     

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.     

Immunoaffinity chromatographic isolation of prostate-specific antigen from seminal plasma for capillary electrophoresis analysis of its isoforms

Prostate-specific antigen (PSA) concentration in serum has been the biomarker employed for prostate cancer diagnosis in the last two decades. However, new more specific biomarkers allowing a better differentiation of cancer from non-malignant prostate diseases are necessary. Glycosylation of PSA gives rise to different forms of the protein which can be separated into several isoforms by analytical techniques, such as CE. Because PSA glycosylation is influenced by pathological conditions, the CE pattern of PSA isoforms could be different in prostate cancer than in non-malignant prostate diseases. To study this CE pattern of PSA, prior purification of the protein from the biological fluid is mandatory. In this study an immunoaffinity chromatography method which allows PSA purification without altering the CE pattern is developed. An in-house prepared column produced with commercial anti-PSA antibodies is employed. The use of 1 M propionic acid as elution agent provides higher than 40% recovery of high purity PSA. CE analysis of PSA immunopurified from seminal plasma of a healthy individual shows the same 8 peaks as the commercially available PSA standard. Sample preparation only requires dilution with phosphate buffered saline prior to immunoaffinity purification. High repeatability for the sample preparation step was achieved (RSD% for percentage of corrected peak area in the range 0.6–5.3 for CE analysis of three independently purified seminal plasma aliquots compared to range 0.8–4.9 for a given aliquot analyzed three times by CE). IAC of five microliters seminal plasma provided enough PSA to achieve signal/noise ratio larger than 5 for the smallest CE isoforms.     

High resolution separation methods for the determination of intact human erythropoiesis stimulating agents. A review

Human erythropoietin (hEPO), a hormone involved in the formation of red blood cells, is a 30 kDa glycoprotein with a high carbohydrate content. The production of recombinant hEPO has made possible its widespread therapeutic use and its banned use in competition sports. Methods to analyze EPO and other erythropoiesis stimulating agents (ESAs) are necessary for the characterization and quality control of these biopharmaceuticals and also for doping control. In this paper, high resolution separation methods, namely high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), with special attention to CE-coupled mass spectrometry, are reviewed. The usefulness of these techniques when applied in different modes to separate the glycoprotein isoforms, aggregates or excipients are detailed. In addition, sample preparation methods that have been applied to ESA samples for subsequent determination by HPLC or CE, as well as the potential compatibility of other preparation methods, are discussed. Applications of the HPLC and CE methods regarding regulatory considerations for biopharmaceuticals analysis, with emphasis on biosimilars, and doping control are also included. Finally, limitations of the present methods and their possible solutions are considered.     

Simultaneous determination of nandrolone, testosterone, and methyltestosterone by multi-immunoaffinity column and capillary electrophoresis

A multitarget antibody immunoaffinity column was proposed for the purification and enrichment of nandrolone, testosterone, and methyltestosterone from urine. Nandrolone-3-site substituted antigen was designed and synthesized and the polyclonal antibody was prepared with immunizing rabbits. The stationary phase of the immunoaffinity column was synthesized by covalently bonding the antibodies specific to nandrolone, testosterone, and methyltestosterone onto CNBr-actived Sepharose 4B. The analytes of interest were extracted with a methanol/water mixture in one step. The immunoaffinity column showed high affinity and high selectivity to a class of structurally related compounds. The elution was then transferred to a micellar electrokinetic CE system with a running buffer of sodium borate and sodium cholate for separation and determination. Recoveries of the three steroids from complex matrix were 88-94% with RSD values less than 5.2%. Optimization of the immunoaffinity column purification was achieved and the feasibility of the technique for the analysis of steroid hormone was discussed. The results indicated that the combination of multi-immunoaffinity column and CE was an effective technique, which was rapid, simple, and sensitive for the assay of steroids.     

Fluorescence-labelled antigen-binding fragments (Fab) from monoclonal antibody 5F12 detect human erythropoietin in immunoaffinity capillary electrophoresis

A faster and more convenient method is required for the detection of recombinant erythropoietin (Epo) in human body fluids. In the present study we wanted to elucidate the principal suitability of immunoaffinity capillary electrophoresis (CE) in this respect. CE offers itself as a high-speed, high-throughput technique provided a suitable affinity reagent is available. We chose monoclonal antibody 5F12 from Amgen which binds to a conformation-independent epitope in the N-terminal region of the human Epo protein. For CE with laser-induced fluorescence detection it was necessary to produce fluorescently labelled antibody with one single antigen binding site. Monomeric antigen-binding fragments (Fab) were obtained by site-selective cleavage of the pure antibody and labelled with the fluorescent dye, Alexa Fluor 488. The mixture of labelled isomers was partially resolved by ion exchange HPLC and isoelectric focusing. The fluorescent Fab could be used to detect erythropoietin by immunoaffinity capillary isoelectric focusing and zone capillary electrophoresis via its antigen complex.Abbreviations BGE background electrolyte - CE capillary electrophoresis - Epo Erythropoietin - Fab antigen-binding fragment - FITC fluorescein isothiocyanate - IEF isoelectric focusing - mAb monoclonal antibody - PBS phosphate-buffered saline - rHuEpo recombinant human erythropoietin - scFv (recombinant) single chain variable fragment - SDS-PAGE denaturing polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - ECL enzyme-coupled chemoluminescence - v_H variable domain - c_H1–3 constant domains of an antibody's heavy chain     

On-line coupling of sol-gel-generated immunoaffinity columns with high-performance liquid chromatography.

The paper demonstrates the possibility to use sol-gel-generated immunoaffinity columns as selective sample preparation step in on-line combination with HPLC. In the past sol-gel-generated immunoaffinity columns have only been included in off-line sample preparation schemes. Compared with conventional RP-materials on-line coupling of sol-gel-generated silica matrices with a pore structure designed to retain antibodies poses additional problems caused by their lower pressure tolerance and by the necessity to match the mobile phases not only to take into account the chromatographic properties but also the conformational stability of the antibodies. These problems have been overcome by an on-line system which can be regarded as a prototype for similar systems which exploit the selectivity of sol-gel immunoaffinity columns. The system consists of a sol-gel-generated immunoaffinity column coupled to an RP enrichment column and an analytical column. The practicality of such systems is demonstrated using the example of anti-pyrene immunoaffinity columns applied for the determination of pyrene in aqueous solutions.     

Immunoaffinity CE in clinical analysis of body fluids and tissues

This paper reviews immunoaffinity CE procedures developed since 1998 for drug, hormone, and disease marker analyses of body fluids and tissues. Immunoaffinity CE and related techniques are described. Examples of clinical applications are included.     

Recent advances in pharmacokinetic applications of capillary electrophoresis

This article reviews recent capillary electrophoresis (CE)-based assays which were published for pharmacokinetic studies. Both the advantages and disadvantages of these CE-based assays are discussed based on their feasibility and the significance towards the better understanding of pharmacokinetics. In addition, as a future outlook, novel assays such as immunoaffinity CE and chip-based CE for analyzing drugs in biological fluids are summarized in view of their potential for pharmacokinetic applications.     

Combined use of supported liquid membrane and solid-phase extraction to enhance selectivity and sensitivity in capillary electrophoresis for the determination of ochratoxin A in wine

This paper proposes a novel strategy to enhance selectivity and sensitivity in CE, using supported liquid membrane (SLM) and off-line SPE simultaneously. The determination of ochratoxin A (OA) in wine has been used to demonstrate the potential of this methodology. In the SLM step, the donor phase (either a 20 mL volume of a standard solution at pH 1 or a wine sample at pH 8) was placed in a vial, where a micromembrane extraction unit accommodating the acceptor phase (1 mL water, pH 11) in its lumen was immersed. The SLM was constructed by impregnating a porous Fluoropore Teflon (PTFE) membrane with a water-immiscible organic solvent (octanol). In the off-line SPE step, the nonpolar sorbent (C-18, 4 mg) selectively retained the target ochratoxin, enabling small volumes of acceptor phase (1 mL) to be introduced. The captured analytes were eluted in a small volume of methanol (0.1 mL). This procedure resulted in sample cleanup and concentration enhancement. The method was evaluated for accuracy and precision, and its RSD found to be 5%. The LODs for OA in the standard solutions and wine samples were 0.5 and 30 microg/L, respectively. The results obtained demonstrate that SLM combined with off-line is a good alternative to the use of immunoaffinity columns prior to CE analysis.     

Capillary electrophoresis and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry for analysis of the novel erythropoiesis-stimulating protein (NESP)

NESP (novel erythropoiesis-stimulating protein) is a recently approved hyperglycosylated analogue of human erythropoietin (EPO) with a long-lasting effect. In this work, the capillary electrophoresis (CE) methodology proposed by the European Pharmacopoeia for the separation of EPO glycoforms has been modified for the separation of NESP glycoforms. Optimization of pH of the separation electrolyte has been fundamental in order to achieve baseline resolution of seven peaks corresponding to NESP glycoforms. Intact NESP has also been characterized by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). An accurate approximation to an average molecular mass of the NESP molecule has been obtained, taking into account the strong influence of laser intensity upon the MALDI-TOF mass spectra found.     

Recent advances of capillary electrophoresis in pharmaceutical analysis

This review covers recent advances of capillary electrophoresis (CE) in pharmaceutical analysis. The principle, instrumentation, and conventional modes of CE are briefly discussed. Advances in the different CE techniques (non-aqueous CE, microemulsion electrokinetic chromatography, capillary isotachophoresis, capillary electrochromatography, and immunoaffinity CE), detection techniques (mass spectrometry, light-emitting diode, fluorescence, chemiluminescence, and contactless conductivity), on-line sample pretreatment (flow injection) and chiral separation are described. Applications of CE to assay of active pharmaceutical ingredients (APIs), drug impurity testing, chiral drug separation, and determination of APIs in biological fluids published from 2008 to 2009 are tabulated.     

On-line immunoaffinity capillary electrophoresis based on magnetic beads for the determination of alpha-1 acid glycoprotein isoforms profile to facilitate its use as biomarker

An immunoaffinity purification method coupled on-line to capillary electrophoresis (IACE) which allows the determination of several isoforms of intact alpha-1 acid glycoprotein (AGP) in serum samples using UV detection is developed. The immunoaffinity step is based on anti-AGP antibodies (Abs) covalently bound to magnetic beads (MBs) which are captured at the inlet end of the capillary using permanent magnets placed inside the cartridge of the CE instrument. The on-line method includes injection of the MBs with the Ab bound (MBs–Ab) and their trapping by the magnets at the entrance of the separation column, injection of serum sample and capture of AGP by the Abs, release of captured AGP, focus of desorbed protein, separation of AGP isoforms, and removal of MBs–Ab. The optimization of the different factors involved in each step allowed purification, separation and detection of AGP isoforms in a single electrophoretic analysis in about 1 h. Automation, sample and reagents consumption as well as analysis time was improved compared to off-line alternatives which use purification of AGP in an immunochromatographic column and CE separation of AGP isoforms in two independent operations. The analytical methodology developed allows the separation of 10 AGP isoforms in serum samples from a healthy donor. For a serum sample, precision (expressed as relative standard deviation) in terms of corrected area percentage was better than 0.5% for each peak accounting for more than 10% of total AGP and it was better than 4.0% in terms of relative migration time of each AGP isoform considering the whole process.     

Analysis of recombinant human erythropoietin and novel erythropoiesis stimulating protein digests by immunoaffinity capillary electrophoresis–mass spectrometry

In this work, we demonstrate that detection of a specific peptide marker by immunoaffinity capillary electrophoresis–mass spectrometry (IA-CE–MS) could be used to confirm the presence of recombinant human erythropoietin (rhEPO) in solution. Besides the carbohydrate content, the amino acid sequence of novel erythropoiesis stimulating protein (NESP) differs from human erythropoietin (hEPO) at five positions (Ala30Asn, His32Thr, Pro87Val, Trp88Asn, and Pro90Thr). After digesting both glycoproteins in solution by trypsin and PNGase F, two specific proteotypic peptides, EPO (77–97) and NESP (77–97) which differ in three amino acids, were selected as rhEPO and NESP markers, respectively. Both digests and their mixtures were analyzed by IA-CE–MS. The IA stationary phase was prepared from a custom made polyclonal anti-EPO (81–95) antibody immobilized on a solid support of CNBr-Sepharose 4B and was packed in a microcartridge near the inlet of the separation capillary. As the antibody was directed to a synthetic peptide EPO (81–95), only the proteotypic peptide EPO (77–97) was retained. The retained peptide was eluted, separated by electrophoresis and detected by MS. The method was specific to confirm the presence of rhEPO in solution. Although the limits of detection for the peptide marker were similar to those obtained with CE–MS (a few mg/L), these results show the potential of this novel approach to detect in the future rhEPO and its analogues selectively and unambiguously at the levels expected in biological fluids.     

Extending the application potential of capillary electrophoresis/frontal analysis for drug-plasma protein studies by combining it with mass spectrometry detection

CE/frontal analysis (CE/FA) is probably one of the most frequently used modes of CE for studying affinity interactions. It is typically performed with classic UV-Vis detection that suffers from low concentration sensitivity. To overcome this limitation, the applicability of CE/FA in combination with ESI-MS detection for the investigation of drug–HSA interactions was demonstrated. The developed new method combines the advantages of CE/FA, such as low sample consumption and no labeling or immobilization of interacting partners, with the benefits of MS detection, such as higher selectivity and sensitivity; moreover, it can be used for molecules lacking a fluorophore or chromophore. The binding parameters of tolbutamide (TL) and glimepiride (GLP), first- and second-generation antidiabetics that differ strongly in their solubility in aqueous solutions, were investigated by this CE/FA-MS method. This method, in contrast to the CE/FA method with the most commonly used UV-Vis detection, is more sensitive; an almost three times lower LOD was reached. The binding parameters of TL and GLP were investigated by this CE/FA-MS method and compared with the literature data. The binding constant value of TL obtained by UV-Vis detection was lower than the value obtained by the method hyphenated with MS detection, which is probably given by the influence of the ESI parameters on the stability of drug–HSA complex. In addition, the ratio of TL and HSA concentrations was divergent in both of the experimental approaches. Finally, it can be concluded that both detection methods have their strengths and weaknesses.     

Nanomaterials and chip-based nanostructures for capillary electrophoretic separations of DNA

Capillary electrophoresis (CE) and microchip capillary electrophoresis (MCE) using polymer solutions are two of the most powerful techniques for the analysis of DNA. Problems, such as the difficulty of filling polymer solution to small separation channels, recovering DNA, and narrow separation size ranges, have put a pressure on developing new techniques for DNA analysis. In this review, we deal with DNA separation using chip-based nanostructures and nanomaterials in CE and MCE. On the basis of the dependence of the mobility of DNA molecules on the size and shape of nanostructures, several unique chip-based devices have been developed for the separation of DNA, particularly for long DNA molecules. Unlike conventional CE and MCE methods, sieving matrices are not required when using nanostructures. Filling extremely low-viscosity nanomaterials in the presence and absence of polymer solutions to small separation channels is an alternative for the separations of DNA from several base pairs (bp) to tens kbp. The advantages and shortages of the use of nanostructured devices and nanomaterials for DNA separation are carefully addressed with respect to speed, resolution, reproducibility, costs, and operation.     

Synergism of crown ethers in the thenoyl trifluoroacetone extraction of americium(III) in benzene medium

The synergism of the crown ethers (CE) dicyclohexano-18-crown-6 (DC18C6), dibenzo-18-crown-6 (DB18C6) and 18-crown-6 (18C6) has been investigated in the thenoyl trifluoroacetone (HTTA) extraction of americium(III) in benzene medium from an aqueous phase of ionic strength 0.5 and pH 3.50 at room temperature (23°C). The extracted synergistic species have the general formula Am(TTA)₃ · CE except for DC18C6 in which case the species Am(TTA)₃·2CE was also observed at high CE concentrations. The order of synergism was found to be DC18C6>DB18C6>18C6, which is the order of the basicity of CE as indicated by their ability to extract hydrogen ions from nitric acid solutions.     

Evidences of cyclodextrin-mediated enantioselective photodegradation of rac-nicardipine by capillary electrophoresis

Capillary electrophoresis (CE) was applied to photostability studies on rac-nicardipine, a dihydropyridine chiral drug. CE methods were developed able to provide the enantioresolution of drug and its separation from the photodegradation products. Enantioresolution was achieved using 5% sulfated-beta-cyclodextrin (S-beta-CD) as chiral selector in 20 mM triethanolammonium phosphate solution (pH 3). The photostability studies were carried out on inclusion complexes of rac-nicardipine with beta-cyclodextrin (beta-CD) and (2-hydroxypropyl)-beta-cyclodextrin (HP-beta-CD) in aqueous solutions (pH 7.4 and 5). The CE analysis of the solutions exposed to UV-A and UV-B radiations showed a photoprotective effect by beta-CD; conversely, HP-beta-CD proved to favor the drug photodegradation. Moreover, evidences for CDs-mediated stereoselective photodegradation of rac-nicardipine were obtained. In fact, two distinct photodegradation profiles were observed for the nicardipine enantiomers in the presence of the CDs. The photodegradation was found to follow an apparent first-order kinetics and two different kinetic constants (k) were obtained for the two enantiomers. After exposure to UV-A and UV-B radiations, the solutions contained residual nicardipine with a significant change in the enantiomeric ratio; this effect was depending on the CD used for the inclusion complexation.     

Combination of Aqueous and Non-Aqueous Capillary Electrophoresis with Electrospray Mass Spectrometry for the Determination of Drug Residues in Water

 The work presented in this paper deals with the combination of capillary electrophoresis (CE) with electrospray mass spectrometry (MS) for the determination of drug residues in water. CE/MS methods have been developed based on either aqueous or non-aqueous ammonium acetate solutions as the carrier electrolyte for the separation of selected drugs. The different separation conditions were compared in terms of selectivity and detection limits; both aqueous and non-aqueous CE proved to be suitable for the present analytical task, exhibiting detection limits between 3 and 93 μg/dm³ (injected standard concentration) corresponding to concentrations between 5 and 19 ng/dm³ in the sample. A combination of liquid-liquid extraction and solid-phase extraction was investigated for sample pretreatment, yielding enrichment factors of 10000. The applicability of CE/MS was demonstrated for the analysis of several river water samples.     

Improved solid-phase microextraction device for use in on-line immunoaffinity capillary electrophoresis

A simple solid-phase microextraction device was fabricated for use in on-line immunoaffinity capillary electrophoresis (CE). The device, designed in the form of a four-part cross-shaped or cruciform configuration, includes a large-bore tube to transport samples and washing buffers and a small-bore fused-silica capillary for separation of analytes. At the intersection of the transport and separation tubes, a small cavity was fabricated, termed the analyte concentrator-microreactor, which contains four porous walls or semipermeable membranes (one for each inlet and outlet of the tubes) permitting the confinement of beads or suitable microstructures. The surface of the beads in the analyte concentrator carried a molecular recognition adsorbing chemical or affinity ligand material. The improved cruciform configuration of the analyte concentrator-microreactor device, designed for use in on-line immunoaffinity CE, enables it to specifically trap, enrich, and elute an analyte from any biological fluid or tissue sample extract without any sample pretreatment except filtration, centrifugation, and/or dilution allowing the separation and characterization of target analyte(s) with improved speed, sensitivity, and lower cost than existing techniques. As a model system, Fab' fragments derived from a purified immunoglobulin G (IgG) antibody were covalently bound to controlled-porosity glass and used as constituents of the analyte-microreactor device. The high-specificity polyclonal antibodies employed in these experiments were individually raised against the acidic nonsteroidal anti-inflammatory drugs ibuprofen and naproxen, and the neuropeptides angiotensin II, and neurotensin. These compounds, which were present in simple and complex matrices were captured by and eluted from the analyte concentrator-microreactor using a 50 mM sodium tetraborate buffer solution, pH 9.0, followed by a 100 nL plug of 300 mM glycine buffer, pH 3.4. Two analyte concentrators were tested independently: one containing Fab' fragments derived from antibodies raised against ibuprofen and naproxen; the other containing Fab' fragments derived from antibodies raised against angiotensin II and neurotensin. Each resulting electropherogram demonstrated the presence of two eluted materials in less than 20 min. Immunoaffinity CE performed in a cruciform structure was simpler and faster than previously reported in the literature using on-line microextraction devices designed in a linear format. The new concentration-separation system operated consistently for many runs, maintaining reproducible migration times and peak areas for every analyte studied.