Methodological Challenges of Protein Analysis in Blood Serum and Cerebrospinal Fluid by Capillary Electrophoresis

Summary High-performance capillary electrophoresis (HPCE or CE) is an ultrasensitive analytical technique with high resolving power and a wide area of applications including peptide/protein analysis. Its applicability is greatly enhanced by the short separation times, the ease of method development and the minimum sample and organic solvent requirements. Various HPCE modes have been developed for peptide/protein analysis, including capillary zone electrophoresis, micellar elektrokinetic capillary chromatography, capillary isoelectric focusing, isotachophoresis, capillary gel electrophoresis and microemulsion elektrokinetic chromatography. HPCE can easily be applied to quality control of manufacturing processes or to clinical routine for diagnostic purposes due to its potential to provide information on the identity, the purity of the samples and the quantities of the constituents. Furthermore, interactions of a peptide or a protein with other molecules can be studied by HPCE. The separation principles of the various operation modes applied to peptide/protein analysis are presented in this article. Furthermore, in order to exemplify the application of the separation principles in the area of serum protein analysis, which is of importance in clinical practice, the capillary electrophoretic methods developed for analysis of serum and cerebrospinal fluid proteins are also reviewed.Presented at: International Symposium on Separation and Characterization of Natural and Synthetic Macromolecules, Amsterdam, The Netherlands, February 5–7, 2003     

Assessment of aptamer-steroid binding using stacking-enhanced capillary electrophoresis

The binding affinity of 17β-estradiol with an immobilized DNA aptamer was measured using capillary electrophoresis. Estradiol captured by the immobilized DNA was injected into the separation capillary using pH-mediated sample stacking. Stacked 17β-estradiol was then separated using micellar electrokinetic capillary chromatography and detected with UV-visible absorbance. Standard addition was used to quantify the concentration of estradiol bound to the aptamer. Following incubation with immobilized DNA, analysis of free and bound estradiol yielded a dissociation constant of 70 ± 10 μM. The method was also used to screen binding affinity of the aptamer for estrone and testosterone. This study demonstrates the effectiveness of capillary electrophoresis to assess the binding affinity of DNA aptamers.     

In‐depth insight into the methods of plasma protein‐drug interaction studies: Comparison of capillary electrophoresis‐frontal analysis,isothermal titration calorimetry,circular dichroism and equilibrium dialysis

Plasma protein‐drug binding assays are routinely performed during the early stages of drug discovery and development, which creates demand for an automated high‐throughput screening assay to increase laboratory efficiency. A comprehensive comparison of the four methods typically used for determining the binding parameters is presented in this study with respect to the above demand. Capillary electrophoresis‐frontal analysis, isothermal titration calorimetry, circular dichroism and equilibrium dialysis were used to study the affinity of human serum albumin for diclofenac and lidocaine. These model drugs were chosen due to their different physico‐chemical properties and different binding sites on the albumin molecule, also resulting in different binding strength. The binding parameters estimated under the conditions as similar as possible were comparable among all these approaches as well as to the literature values. Besides this, the comparison of the results and especially other considerations demonstrated the benefits and drawbacks of the selected methods, with capillary electrophoresis‐frontal analysis being the best candidate for such studies.     

On-column ligand synthesis coupled to partial-filling affinity capillary electrophoresis to estimate binding constants of ligands to a receptor

This paper describes a two-step procedure whereby on-column ligand synthesis and partial-filling affinity capillary electrophoresis (PFACE) are sequentially coupled to each other to determine the binding constants of 9-fluorenylmethoxy carbonyl (Fmoc)-amino acid-D-Ala-D-Ala species to vancomycin (Van) from Streptomyces orientalis. In this technique four separate plugs of sample are injected onto the capillary column and electrophoresed. The initial sample plug contains a D-Ala-D-Ala terminus peptide and two non-interacting standards. Plugs two and three contain solutions of Fmoc-amino acid-N-hydroxysuccinimide (NHS) ester and running buffer, respectively. The fourth sample plug contains an increasing concentration of Van partially-filled onto the capillary column. Upon electrophoresis the initial D-Ala-D-Ala peptide reacts with the Fmoc-amino acid NHS ester yielding the Fmoc-amino acid D-Ala-D-Ala peptide. Continued electrophoresis results in the overlap of the plugs of Van and Fmoc-amino acid-D-Ala-D-Ala peptide and non-interacting markers. Analysis of the change in the relative migration time ratio of the Fmoc-amino acid-D-Ala-D-Ala peptide relative to the non-interacting standards, as a function of the concentration of Van, yields a value for the binding constant. These values agree well with those estimated using other binding and ACE techniques.     

Comparison of various capillary electrophoretic approaches for the study of drug–protein interaction with emphasis on minimal consumption of protein sample and possibility of automation†

The binding ability of a drug to plasma proteins influences the pharmacokinetics of a drug. As a result, it is a very important issue in new drug development. In this study, affinity capillary electrophoresis, capillary electrophoresis with frontal analysis, and Hummel Dreyer methods with internal and external calibration were used to study the affinity between bovine serum albumin and salicylic acid. The binding constant was measured by all these approaches including the equilibrium dialysis, which is considered to be a reference method. The comparison of results and other considerations showed the best electrophoretic approach to be capillary electrophoresis‐frontal analysis, which is characterized by the high sample throughput with the possibility of automation, very small quantities of biomacromolecules, simplicity, and a short analysis time. The mechanism of complex formation was then examined by capillary electrophoresis with frontal analysis. The binding parameters were determined and the corresponding thermodynamic parameters such as Gibbs free energy ΔG⁰, enthalpy ΔH⁰, and entropy changes ΔS⁰ at various temperatures were calculated. The results showed that the binding of bovine serum albumin and salicylic acid was spontaneous, and that hydrogen bonding and van der Waals forces played a major role in the formation of the complex.     

色谱技术在药物-血浆蛋白相互作用研究中的应用进展

小分子药物进入人体血液循环系统后与人血清白蛋白(HSA)、α₁ -酸性糖蛋白(AGP)等血浆蛋白存在广泛的相互作用,这些相互作用深刻影响药物在体内的分布及其与靶标蛋白的结合,进而影响药物效应的发挥。深入探究药物与血浆蛋白间的相互作用对于候选药物的成药性优化、新药研发、联合用药的风险评控等意义重大。而发展高效、灵敏、准确的分析检测方法是开展药物-血浆蛋白相互作用研究的关键。近年来,色谱技术由于其高通量、高分离性能、高灵敏度等特点在该领域得到了广泛的应用,包括测定血浆蛋白翻译后修饰对药物结合的影响,多种药物的竞争性结合等。其中,高效亲和色谱(HPAC)和毛细管电泳(CE)应用最为广泛,能够通过多种分析方法获取结合常数、结合位点数、解离速率常数等相互作用信息。该文着重综述了HPAC和CE在药物-血浆蛋白相互作用研究中的常用策略及最新研究进展,包括HPAC中常用的前沿色谱法、竞争洗脱法、超快亲和提取法、峰值分析法和峰衰减分析法,以及CE中常用的亲和毛细管电泳法(ACE)和毛细管电泳前沿分析法(CE-FA)等。最后,该文还对当前色谱方法存在的不足进行了总结,并对色谱技术在药物-血浆蛋白相互作用研究领域的应用前景和发展方向进行了展望。     

Evalution of capillary electrophoresis-frontal analysis for the study of low molecular weight drug-human serum albumin interactions

Capillary electrophoresis frontal analysis was applied to 12 low molecular weight compounds including 8 drug substances displaying a range of different properties with respect to binding affinity, binding location, structure, lipophilicity, charge at physiological pH, and electrophoretic mobility. It was found that capillary electrophoresis frontal analysis can be used as a general method to study and quantify drug-human serum albumin interactions. The binding parameters obtained were consistent with literature values. Dextran was in some cases added to the run buffer to improve separation of the drug and human serum albumin plateau peaks. Results indicate that mobility differences between free and complexed human serum albumin give rise to only minor errors. Capillary electrophoresis frontal analysis was also found applicable to the study of human serum albumin drug displacement reactions. Low sensitivity of the UV-detection system was found to be the major limitation of capillary electrophoresis frontal analysis. The method is simple, and minimal effort has to be put into method development, which makes it well suited for screening in early drug development.     

Affinity capillary electrophoresis method for investigation of bile salts complexation with sulfobutyl ether‐β‐cyclodextrin

Sulfobutyl ether‐β‐cyclodextrin (SBEβCD) is utilized in preformulation and drug formulation as an excipient for solubilization of drugs with poor aqueous solubility. Approximately seven negative charges of SBEβCD play a role with respect to solubilization and complexation, but also have an influence on the ionic strength of the background electrolyte when the cyclodextrin is used in capillary electrophoresis. Mobility‐shift affinity capillary methods for investigation of the complexation of taurocholate and taurochenodeoxycholate with the negatively charged cyclodextrin derivative applying constant power and ionic strength conditions as well as constant voltage and varying ionic strength were investigated. A new approach for the correction of background electrolyte ionic strength was developed. Mobility‐shift affinity capillary electrophoresis experiments obtained at constant voltage and constant power settings were compared and found to provide binding parameters that were in good agreement upon correction. The complexation of taurochenodeoxycholate with SBEβCD was significantly stronger than the corresponding interaction involving taurocholate. The obtained stability constants for the bile salts were in the same range as those previously reported for the interaction with neutral β‐cyclodextrins derivatives, i.e. the positions of the negative charges on SBEβCD and the bile salts within the complex did not lead to significant electrostatic repulsion.     

Determination of therapeutic oligonucleotides using capillary gel electrophoresis

Oligonucleotides have developed into highly versatile and selective therapeutics over the past 20 years. More than five discrete mechanisms of action have been reported and more than 10 different chemical modifications have been used to extend their in vivo half-life and reduce their toxicity. Capillary gel electrophoresis (CGE) has been used extensively for the quantitative analysis of oligonucleotide therapeutics in both preclinical and clinical studies since the 1990s. The success of CGE is based on its extraordinary resolving power, which allows for the simultaneous determination of the parent drug and its metabolites. More recently, capillary gel electrophoresis has seen renewed interest with the emergence of replaceable gels with single-base resolving power and new capillary electrophoresis-mass spectrometry interfaces. This review discusses the bioanalysis of therapeutic oligonucleotides showing the evolution of the field over the past two decades leading to the current new approaches. Included in this review are topics such as different gel types, sample introduction modes, sample extraction procedures, separation conditions and detection methods used in CGE, along with discussions of the successes and limitations associated with each.     

Capillary electrophoretic methods applied to the investigation of peptide complexes

This article gives an overview of the applications of capillary electrophoretic methods to investigate the non‐covalent interactions of peptides (peptide complexes) with variable middle‐ and high‐molecular‐mass receptors (ligands) as well as with small ions and molecules in the period 2007–2014. Different modes of capillary electrophoretic methods, such as mobility shift (vacancy) affinity capillary electrophoresis, multiple injection affinity capillary electrophoresis, partial filling affinity capillary electrophoresis, Hummel–Dryer method, vacancy peak method and (continuous) frontal analysis capillary electrophoresis, are briefly described and their applicability to determination of binding constants of peptide complexes is discussed. In addition, the detailed experimental conditions of individual applications and the values of binding constants of the particular peptide complexes are presented.     

Through-a-chip partial filling affinity capillary electrophoresis for estimating binding constants of ligands to receptors

In this paper, we describe the development of a microfluidic/capillary electrophoresis (CE) technique employing partial filling affinity capillary electrophoresis (PFACE) to estimate binding constants of ligands to receptors using as model systems carbonic anhydrase B (CAB, EC 4.2.1.1) and vancomycin from Streptomyces orientalis. Using multilayer soft lithography (MSL), a microfluidic device (MD) consisting of fluid and control channels is fabricated and fitted with an external capillary column. Multiple flow channels allows for manipulation of a zone of ligand and sample containing receptor and non-interacting standards into the MD and subsequently into the capillary column. Upon electrophoresis the sample components migrate into the zone of ligand where equilibrium is established. Changes in migration time of the receptor are used in the analysis to obtain a value for the binding interaction. The manipulation of small volumes of solution on the MD minimizes the need of time-consuming pipetting steps.     

Protonated diamines as anion-binding agents and their utility in capillary electrophoresis separations

Capillary zone electrophoresis is a proven method for separating small ions because of the inherent charge and differences in mobility of these analytes. Despite its resolving power, CZE can be insufficient for separating ions with similar mobilities. One remedy is to modify mobilities via the addition of background electrolyte complexation agents. However, this approach is not straightforward for inorganic anions, which lack complexation options. To address this shortfall, the diprotonated diamine moiety was investigated for complexation of dianions. Dicationic diamines significantly complexed dianions, and this interaction was not purely electrostatic in nature because affinities varied with dianion identity. Aqueous association constants were measured with affinity capillary electrophoresis (ACE) and found to be similar in magnitude but different in selectivity to those of dianions with magnesium ion. Binding was also investigated for zwitterionic buffers containing the protonated diamine moiety. Zwitterions exhibited binding constants as high as 18 M^?1 (30‐mM ionic strength). This work discusses the observed binding constants and their potential usefulness in CZE separations of inorganic anions. Also covered are improvements to ACE methodology and an evaluation of some of the assumptions employed.     

十二烷基硫酸钠毛细管电泳法分析非还原单克隆抗体药物纯度的前处理条件优化

十二烷基硫酸钠毛细管电泳法因具有快速、分辨率高的优势，已成为单克隆抗体纯度分析的主流方法。在非还原单克隆抗体纯度检测中，其样品前处理过程对于结果有显著影响。为优化样品前处理，考察了以碘乙酰胺和N-乙基马来酰亚胺为巯基封闭剂，在pH 6.0~9.0的样品缓冲液条件下，不同种类和批次的单抗的纯度。发现在两种巯基封闭剂中，高pH的样品缓冲液会影响巯基封闭的效果，产生较多的抗体片段；而在低pH条件下，抗体片段较少，单抗的纯度更高。因此在进行非还原单抗的纯度检测中，pH 6.0的样品缓冲液为最佳的前处理条件。     

Capillary electrophoresis frontal analysis: principles and applications for the study of drug-plasma protein binding

Capillary electrophoresis is a well-established technique for the study of noncovalent interactions. Various approaches exist and capillary electrophoresis-frontal analysis provides an interesting alternative to the migration shift affinity capillary electrophoresis methods and conventional methods. The present work reviews the principles on which the frontal analysis method is founded. Advantages and limitations of capillary electrophoresis frontal analysis in comparison with both conventional and other capillary electrophoresis based methods for quantification of binding interactions are discussed. Investigations utilizing capillary electrophoresis-frontal analysis have focused on the interaction of drugs with plasma proteins. These studies, primarily addressing the binding of drugs to human serum albumin, alpha1-acid glycoprotein, and lipoproteins are reviewed together with some recent developments in capillary electrophoresis-frontal analysis methodology.     

Development and validation of a capillary electrophoresis method for the enantiomeric purity determination of SLV307, a basic potential antipsychotic compound

A capillary electrophoresis method for the enantiomeric purity determination of SLV307, a basic potential antipsychotic compound, has been developed and validated. It is shown that the presence of the eutomer in the sample solution may have a significant effect on the peak shape of the distomer, due to electrodispersion. The method is shown to give good performance with respect to the validation parameters specificity, linearity, accuracy, precision, determination limits, stability of solutions, and robustness of extraction.     

Microscale characterization of the structure-activity relationship of a heparin-binding glycopeptide using affinity capillary electrophoresis and immobilized enzymes.

A heparin-binding glycopeptide (T3) from human serum amyloid P component was characterized by taking advantage of two important features of capillary electrophoresis: the low sample consumption and the possibility of doing on-line binding studies. Incubations with neuraminidase and proteolytic enzymes were carried out with enzymes immobilized on paramagnetic microbeads. Affinity capillary electrophoresis subsequently was used to characterize T3 and its fragments with respect to heparin binding. We find that an intact glycan moiety makes the C-terminal part of T3 relatively resistant to chymotryptic clevage. This protection is lost upon desialylation. Also, the C-terminus of T3 is involved in heparin binding while the N-terminal part of the molecule has no appreciable binding activity. The micromethods presented here make it feasible to perform structure-function studies even on the small amounts of analytes that are typically available when working with glycopeptides from natural sources.     

Chiral separation of gemifloxacin in sodium-containing media using chiral crown ether as a chiral selector by capillary and microchip electrophoresis

Chiral crown ether, (+)-(18-crown-6)-tetracarboxylic acid (18C6H(4)), is an effective chiral selector for resolving enantiomeric primary amines owing to the difference in affinities between 18C6H(4) and each of the amine enantiomers. In addition to the destacking effect of sodium ion in the sample solution, the strong affinity of sodium ion to the polyether ring of crown ether is unfavorable to chiral capillary electrophoresis using 18C6H(4) as a chiral selector. In this report, the chiral separation of gemifloxacin dissolved in a saline sample matrix using 18C6H(4) was investigated. Adding a chelating agent, ethylenediaminetetraacetic acid (EDTA), to the run buffer greatly improved the separation efficiencies and peak shapes. The successful chiral separation of gemifloxacin in a urinary solution was demonstrated for both capillary and microchip electrophoresis.     

Use of a partial-filling technique in affinity capillary electrophoresis for determining binding constants of ligands to receptors

This work evaluates the concept of a partial-filling technique in affinity capillary electrophoresis (ACE) using two model systems: vancomycin from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). In this technique the capillary is first partially-filled with ligand followed by a sample of receptor and non-interacting standard and electrophoresed. Analysis of the change in the mobility ratio, M, of the receptor, relative to the non-interacting standard, as a function of the concentration of the ligand, yields a value for the binding constant. These values agree well with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method is presented.     

The immunoassay of methotrexate by capillary electrophoresis with laser-induced fluorescence detection.

Immunoassay is widely employed as a highly sensitive, specific analytical method for hormones and drugs in biological samples. A technique utilizing capillary electrophoresis with laser-induced fluorescence detection was examined based on the reaction process of these immunoassays in order to develop a protocol characterized by high sensitivity and high speed. The conditions of the antigen-antibody reaction and capillary electrophoresis were variously examined using fluorescein-labeled methotrexate and the antibody of methotrexate. As a result, the immunoassay could be completed within a few minutes. Moreover, detection in the pg range could be accomplished. The sensitivity corresponded to that of radioimmunoassay. A simultaneous multi-component analysis of the immunoassay is also possible due to the high resolving power of capillary electrophoresis. In this study, the possibility of a simultaneous analysis of methotrexate and vancomycin was also investigated.