Element of a validation method for MU-B3 monoclonal antibody using an imaging capillary isoelectric focusing system

This paper presents an imaging capillary isoelectric focusing (CIEF) assay for the determination of the identity, stability, and isoform distribution of a murine monoclonal antibody (MU-B3). The experiments were conducted using a Convergent Bioscience iCE280 instrument. The optimum carrier ampholyte composition that gave the best peak separation was found to be 25% Pharmalyte pH 3-10 and 75% Pharmalyte pH 5-8. The antibody gave a highly reproducible CIEF profile with three major peaks having average isoelectric point (pI) values of 6.83, 6.99, and 7.11. Intraday and interday reproducibility of pI values was found to be within RSD of 0.5%. The CIEF profile was also the same, with an alternate column cartridge and alternate batches of methyl cellulose. A plot of peak areas versus MU-B3 concentration was linear (R2 = 0.995) up to a concentration of 0.5 mg/mL in the sample solution. Peak area measurements were reproducible to within 7% RSD. The CIEF profiles of two other antibodies were distinctly different from the profile of MU-B3, showing that the assay is specific. After a sample of MU-B3 was subjected to heat stress by exposure to heat at 55 degrees C for 4 h, its CIEF profile was altered with extra peaks appearing at lower pI values, indicating that the assay could be used to monitor stability. The result of the heat stress experiment was also confirmed with a parallel slab-gel IEF analysis of the antibody sample before and after application of the heat stress. The results of this work suggest that imaging CIEF can be used for product testing under a quality control environment. The assay can be used for pI profiling of proteins and for monitoring structural changes (deamidation, glycosylation, etc.) during the manufacturing process and upon storage.     

Rapid separation of protein isoforms by capillary zone electrophoresis with new dynamic coatings

Many cellular functions are regulated through protein isoforms. Changes in the expression level or regulatory dysfunctions of isoforms often lead to developmental or pathological disorders. Isoforms are traditionally analyzed using techniques such as gel- or capillary-based isoelectric focusing. However, with proper electro-osmotic flow (EOF) control, isoforms with small pI differences can also be analyzed using capillary zone electrophoresis (CZE). Here we demonstrate the ability to quickly resolve isoforms of three model proteins (bovine serum albumin, transferrin, alpha1-antitrypsin) in capillaries coated with novel dynamic coatings. The coatings allow reproducible EOF modulation in the cathodal direction to a level of 10(-9) m2V(-1)s(-1). They also appear to inhibit protein adsorption to the capillary wall, making the isoform separations highly reproducible both in peak areas and apparent mobility. Isoforms of transferrin and alpha1-antitrypsin have been implicated in several human diseases. By coupling the CZE isoform separation with standard affinity capture assays, it may be possible to develop a cost-effective analytical platform for clinical diagnostics.     

Interlaboratory reproducibility of mobility parameters in capillary electrophoresis for substance identification in systematic toxicological analysis

An interlaboratory pilot study was performed to determine the reproducibility of mobility parameters in capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). The study was performed by an intended small number of laboratories (three) that used different brands of instruments (two). The effective mobility was corrected using standards by a method that was recently introduced to obtain a more reproducible migration parameter. A test set of 20 acidic test compounds and 5 reference compounds were analyzed during five days in each laboratory using CZE and MEKC. Buffers used consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate, 50 mM sodium dodecyl sulfate set at pH 7.5 (MEKC). Analyses were carried out using fused-silica capillaries at an electric field strength of either 52.6 kV/m or 37.5 kV/m. The interlaboratory reproducibility (mean RSD) of the effective mobility was 3.0% for CZE and 6.7% for MEKC. After applying the correction method, these values became 3.0% for CZE and 3.3% for MEKC, which is adequate for systematic toxicological analysis (STA) applications. A significant improvement of reproducibility for the calculated corrected effective mobility mu(eff)c was observed when variations are high. Therefore, it is recommended to use the correction method in interlaboratory situations, especially when instruments and capillaries from different manufacturers are used.     

Intercompany Study to Evaluate the Robustness of Capillary Isoelectric Focusing Technology for the Analysis of Monoclonal Antibodies

Interlaboratory comparisons are essential to bringing emerging technologies into biopharmaceutical industry practice and regulatory acceptance. As a result, an international team including 12 laboratories from 10 independent biopharmaceutical companies in the United States and Switzerland was formed to evaluate the precision and robustness of capillary isoelectric focusing (CIEF) to assess the charge heterogeneity of monoclonal antibodies. The different laboratories determined the apparent pI and the relative distribution of the charge isoforms of a representative monoclonal antibody (rMAb) sample using the same CIEF method. Statistical evaluation of the data was performed to determine within and between-laboratory consistencies and outlying information. The apparent pI data generated for each charge variant peak showed very good precision between laboratories with percentage of RSD values of ??0.5%. Similarly, the percentage of RSD for the rMAb charge variants percent peak area values are ??4.4% across different laboratories with different analysts using different lots of ampholytes and multiple instruments. Taken together, these results validate the appropriate use of CIEF in the biopharmaceutical industry in support of regulatory submissions.     

Capillary isoelectric focusing of proteins utilizing poly(vinylpyrrolidone)- and plexiglas-coated columns

Fused-silica capillaries chemically derivatized with silane/poly(vinylpyrrolidone) (PVP) or dynamically modified with plexiglas [poly(methyl methacrylate)] were prepared and evaluated with regard to column stability and separation performance for capillary isoelectric focusing of standard proteins. The PVP coating showed the better stability and was good for at least 100 runs while the plexiglas coating started to deteriorate after about 30 runs. The time spent for the plexiglas coating is about 40 minutes while the PVP coating requires two days. The migration time reproducibility was better with the PVP capillary (RSD 0.7-1.6%, n = 5) compared to the plexiglas-coated column (RSD 1.2-2.9%, n = 5) while peak area and height varied over a similar interval (RSD 2-28.1% area; 0.9-22.7% height, n = 5). The two most consistent proteins in this evaluation, viz. myoglobin A and carbonic anhydrase II, showed linear dynamic ranges between 5-150 and 5-50 microg/mL, and limits of detections at 2 and 1 microg/mL, respectively, employing UV detection at 280 nm.     

新型共聚物涂层毛细管电泳柱及其分离蛋白质的研究

研究新型共聚物——ＺＢ系列表面键合剂在毛细管电泳中的应用。采用物理吸附的方法制备了ＺＢ－００４,ＺＢ－０１４,ＺＢ－０１６等３种涂层毛细管柱,在ｐＨ３～５范围内,３种涂层均能有效地降低管壁对蛋白质的吸附作用和电渗流,其中亲水性较弱的ＺＢ－００４涂层的分离性能最好。在ｐＨ＜５时,涂层具有较高的稳定性和良好的分析重复性,但在更高的ｐＨ值条件下,仍然存在着峰形畸变和电渗流迅速增加的现象。     

CE coupled to MALDI with novel covalently coated capillaries

CE offers the advantage of flexibility and method development options. It excels in the area of separation of ions, chiral, polar and biological compounds (especially proteins and peptides). Masking the active sites on the inner surface of a bare fused silica capillary wall is often necessary for CE separations of basic compounds, proteins and peptides. The use of capillary surface coating is one of the approaches to prevent the adsorption phenomena and improve the repeatability of migration times and peak areas of these analytes. In this study, new capillary coatings consisting of (i) derivatized polystyrene nanoparticles and (ii) derivatized fullerenes were investigated for the analysis of peptides and protein digest by CE. The coated capillaries showed excellent run‐to‐run and batch‐to‐batch reproducibility (RSD of migration time ≤0.5% for run‐to‐run and ≤9.5% for batch‐to‐batch experiments). Furthermore, the capillaries offer high stability from pH 2.0 to 10.0. The actual potential of the coated capillaries was tested by combining CE with MALDI‐MS for analysing complex samples, such as peptides, whereas the overall performance of the CE‐MALDI‐MS system was investigated by analysing a five‐protein digest mixture. Subsequently, the peak list (peptide mass fingerprint) generated from the mass spectra of each fraction was entered into the Swiss‐Prot database in order to search for matching tryptic fragments using the MASCOT software. The sequence coverage of analysed proteins was between 36 and 68%. The established technology benefits from the synergism of high separation efficiency and the structure selective identification via MS.     

毛细管等电聚焦-毛细管区带电泳二维蛋白质分离平台的构建及其性能

通常采用二维聚丙烯酰胺凝胶电泳 ( 2 D- PAGE)分析组织或细胞的全蛋白质 [1] ,但难与质谱 ( MS)直接联用 .用高效液相色谱 ( HPLC)和毛细管电泳 ( CE)分离分析蛋白质和多肽的一维分离模式的分辨率和峰容量有限 .多维柱联用技术比一维分离有更高的分辨率和峰容量 [2 ] ,便于和 MS直接联用 [3,4 ] ,易于实现自动化 .目前 ,有关 2 D- CE的报道相对较少 [5～ 7] ,我们初步实现了将 2 D- PAGE由平板转移到毛细管中[6 ,7] ,但凝胶柱的制作烦琐 ,存在交叉污染 ,不能与 MS直接联用 .本文用微透析中空纤维膜为接口构建了毛细管等电聚焦 ( …     

Characterization of antithrombin III from human plasma by two-dimensional gel electrophoresis and capillary electrophoretic methods

The isoforms distribution of the glycoprotein antithrombin III (ATIII) derived from human plasma was investigated by means of isoelectric focusing (IEF) in polyacrylamide gels with immobilized pH gradients (IPG) and two-dimensional gel electrophoresis (2-DE) as well as capillary electrophoretic methods. It turned out that the presence of high concentrations of chaotropics (urea, thiourea) and zwitterionic detergents (3-[(3-cholamidepropyl)dimethylammonio]-1-propanesulfonate (CHAPS)) was decisive for attaining good resolution of the protein isoforms. Resolution by IPG-IEF was obtained with excellent reproducibility and pI differences down to 0.01 pH units could be distinguished. ATIII-alpha and ATIII-beta-fractions preseparated by heparin affinity chromatography showed an analogous but shifted spot pattern consisting each of one major and three minor isoforms. The main isoforms of ATIII-alpha and ATIII-beta exhibit pI values of 5.18 and 5.32, respectively, both values determined in the presence of high concentrations of urea. The pI difference of 0.14 pH units correspond to the effect of two sialic acids absent in ATIII-beta. The formation and occurrence of ATIII dimers and trimers turned out to be dependent on the sample preparation. The results obtained by 2-DE were compared with those of capillary zone electrophoresis (CZE) and capillary IEF (CIEF). Quantitative analysis regarding the CZE separated isoforms of plasma derived ATIII yielded a content of about 70% ATIII-alpha main isoform and about 6.6% of ATIII-beta. The pI values of ATIII determined by CIEF with internal calibration were in fair agreement with the pI values of the main isoforms achieved with 2-DE.     

毛细管电泳中获得稳定电渗流的毛细管预处理方法

报道了一种毛细管电泳分析中获得重复性分析结果的毛细管柱预处理方法。通过采用有机溶剂的碱性溶液对毛细管柱进行预冲洗,可得到内壁均一的能产生稳定电渗流的毪 细管术,实现了强极性有机化合物如硝基酚的毛细管区带电泳分析。     

Identification and quantification of natural isoxazolinone compounds by capillary zone electrophoresis.

A capillary zone electrophoresis (CZE) method that is specific, simple, rapid and also cheap was developed to analyse some natural UV-absorbing isoxazolinone compounds with toxic potential present in legume seedlings. The six most common natural isoxazolinone compounds were separated within 10 min with 25 mM potassium phosphate (pH 7.5) containing 8% 1-propanol as running buffer. A 60 cm coated fused-silica capillary (52.6 cm effective length x 75 microm I.D.), with an electric field of 375 V/cm at 30 degrees C was used. The limit of detection ranged from 0.01 mM (3.0 microg/ml) to 0.03 mM (7.7 microg/ml). Linearity between peak areas and concentrations ranging from 0.05 mM to 1.75 mM were determined for each isoxazolinone. The correlation coefficient was 0.9954 or greater. Both relative migration time and peak area were reproducible. The RSD of relative migration time is between 0.44 and 1.94% and RSD of peak area is between 1.26 and 6.86%. The concentrations of isoxazolinones in Lathyrus odoratus and L. sativus seedlings obtained by CZE were in agreement with the previous results from HPLC.     

毛细管柱表面鸡卵清蛋白快速改性并用于等电聚焦电泳分离蛋白质

开发了一种利用鸡卵清蛋白改性毛细管柱内表面的快速方法。改性后的毛细管柱可避免蛋白质样品的吸附作用，在较温和的条件下，柱性能有很好的稳定性，可适用于等电聚焦电泳分离生物样品的需要。     

Decreased protein peak asymmetry and width due to static capillary coating with hydrophilic derivatives of polydimethylacrylamide

We have recently described [1] a fast and simple method for the "adsorbed static" coating of capillaries in capillary zone electrophoresis (CZE) with epoxy-poly(dimethylacrylamide) (EPDMA). Protein CZE peaks in the EPDMA-coated capillaries exhibited a peak asymmetry similar to that obtained in capillaries with "covalent static" coating of polyacrylamide, suggesting a similar degree of adsorption of the protein onto the coating [2]. Instability of such coating at very low ionic strength and its stripping from the capillary in the presence of sodium dodecyl sulfate (SDS) also indicated a hydrophilic bonding of EPDMA to the silanol surface of the capillary, while its stripping in the CZE of "carboxylate-modified" polystyrene suggested a competition between carboxylate and EPDMA for the hydrophilic bonds to silanol. To test those propositions, a number of EPDMA-derived coating agents with increased hydrophilicity were synthesized. Of a number of the hydrophilic coating agents tested (Table 1) only two, 2% hydrolyzed EPDMA (HPDMA) hydrolyzed in sulfuric acid to effect the conversion of the epoxy groups to diols (Table 1, No. 38), and 20% EPDMA (Table 1, No. 44) exhibited for representative proteins a decreased peak asymmetry and width while the stability of the suppression of electroosmotic flow (EOF), and the stability of mobility in consecutive CZE runs was reduced relative to EPDMA. Coating agents which were more highly hydrophilic than those two (Table 1, No. 49) or less hydrophilic than 2% EPDMA (Table 1, Nos. 57, 53, 46) provided no stable static coating.     

Use of a native affinity ligand for the detection of G proteins by capillary isoelectric focusing with laser-induced fluorescence detection

Affinity probe capillary isoelectric focusing (CIEF) with laser-induced fluorescence was explored for detection of Ras-like G proteins. In the assay, a fluorescent BODIPY FL GTP analogue (BGTPgammaS) and G protein were incubated resulting in formation of BGTPgammaS-G protein complex. Excess BGTPgammaS was separated from BGTPgammaS-G protein complex by CIEF using a 3-10 pH gradient and detected in whole-column imaging mode. In other cases, a single point detector was used to detect zones during the focusing step of CIEF using a 2.5-5 pH gradient. In this case, analyte peaks passed the detector in approximately 5 min at an electric field of 350 V/cm. Detection during focusing allowed for more reproducible assays at shorter times but with a sacrifice in sensitivity compared to detection during mobilization. Resolution was adequate to separate BGTPgammaS-Ras and BGTPgammaS-Rab3A complexes. Formation of specific complexes was confirmed by adding GTPgammaS to samples containing BGTPgammaS-G protein. GTPgammaS competed with BGTPgammaS for G protein binding sites resulting in decreased BGTPgammaS-G protein peak heights. The concentrating effect of CIEF enabled detection limits of 30 pM.     

New physically adsorbed polymer coating for reproducible separations of basic and acidic proteins by capillary electrophoresis

In this work, a new physically adsorbed coating for capillary electrophoresis (CE) is presented. The coating is based on a N,N-dimethylacrylamide-ethylpyrrolidine methacrylate (DMA-EPyM) copolymer synthesized in our laboratory. The capillary coating is simple and easy to obtain as only requires flushing the capillary with a polymer aqueous solution for 2 min. It is shown that by using these coated capillaries the electrostatic adsorption of a group of basic proteins onto the capillary wall is significantly reduced allowing their analysis by CE. Moreover, the DMA-EPyM coating provides reproducible separations of the basic proteins with RSD values for migration times lower than 0.75% for the same day (n = 5) and lower than 3.90% for three different days (n = 15). Interestingly, the electrical charge of the coated capillary wall can be modulated by varying the pH of the running buffer which makes possible the analysis of basic and acidic proteins in the same capillary. The usefulness of this coating is further demonstrated via the reproducible separation of whey (i.e. acidic) proteins from raw milk. The coating protocol should be compatible with both CE in microchips and CE-MS of different types of proteins.     

Recent developments in capillary electrophoresis-mass spectrometry of proteins and peptides.

Many researchers have invested considerable efforts toward improving capillary electrophoresis (CE)-mass spectrometry (MS) systems so they can be applied better to standard analyses. This review highlights the developments in CE-MS of proteins and peptides over the last five years. It includes the developments in interfaces, sample-enrichment techniques, microfabricated devices, and some applications, largely in capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF) and capillary isotachophoresis formats.     

Analysis of orotic acid in human urine by on-line combination of capillary isotachophoresis and zone electrophoresis.

The techniques of the on-line combination of capillary isotachophoresis with zone electrophoresis in two coupled capillaries (ITP-CZE) and a single capillary zone electrophoresis (CZE) were used for the sensitive determination of orotic acid (OA) in human urine. The simple CZE system was successfully applied for fast and reliable analyses of urine of healthy adult volunteers (the detection limit 1.7.10(-6) M OA, the total time of analysis 6 min). However, this method failed in analyses of OA in urine of ill children due to more complex matrix of the samples. Here, the ITP preconcentration and preseparation step coupled on-line with CZE proved to serve well with an electrolyte system developed and optimized for this purpose. The maximum selectivity and resolution of OA from other sample constituents in ITP-CZE was achieved by use of an electrolyte system of very low pH 2.15 both for ITP and CZE stage. The sensitivity of detection and simplicity of OA identification were enhanced by use of an external UV scanning detector. High sensitivity of ITP-CZE combination (limit of detection 3.10(-7) M OA), low sample consumption (1 microliter), good reproducibility of migration times (inter-day RSD < 1.86%) and acceptable reproducibility of the determination of OA in urine samples (average RSD = 7.27%) make this technique suitable for routine determination of trace concentration of OA especially in urine of ill children under various pathological conditions and medication.     

Evaluation of capillary isoelectric focusing in glycerol-water media with a view to hydrophobic protein applications

Capillary isoelectric focusing (CIEF) separations are usually performed with neutral coated fused-silica capillaries in aqueous anticonvective media. Glycerol, a very viscous solvent (eta = 945 mPa x s at 25 degrees C), known to help stabilize any kind of proteins and solubilize hydrophobic ones, was tested as an alternative to using commercial gels. Viscosity and electroosmotic mobility were measured as a function of gel or glycerol content in water, and a 30:70 v/v glycerol-water medium appeared as a good compromise for performing CIEF in a bare fused-silica capillary without imposing too high a viscosity. To demonstrate the feasibility of this new CIEF system, a standard mixture of nine model proteins was separated according to their pI with a good agreement between experimental and literature aqueous pIs. Moreover, better resolution was achieved with this system than with the conventional aqueous CIEF system, as two of the model proteins could not be separated in the latter system. Glycerol-water CIEF in bare silica capillary was next applied to the separation of horse radish peroxidase, a complex mixture of protein isoforms. The good concordance with the separation obtained by the conventional CIEF system indicated the adequacy of this new system. Finally, as anticipated from the results obtained for the separation of bacteriorhodopsin, a membrane protein, glycerol-water CIEF performed in bare silica capillary appears to be a promising alternative to conventional aqueous CIEF for hydrophobic protein characterization, under their native form.     

新型毛细管等电聚焦驱动方法的建立及其应用

毛细管等电聚焦(CIEF)在生物分离分析领域中的发展迅速．CIEF可分为两步法聚焦和一步法聚焦．电渗流(EOF)可作为一种新的驱动力．Rassi等根据串连体系中电渗流速率将被平均化的原理,将未涂层毛细管与聚乙二醇涂层的毛细管通过聚四氟乙烯管耦合起来,成功地利用毛细管区带电泳快速分离蛋白．此外,利用电渗流输液原理设计的电渗泵还可用作流动注射和微柱液相色谱的驱动系统。     

Evaluation of capillary electrophoretic techniques towards systematic toxicological analysis.

Two capillary electrophoresis (CE) methods were evaluated for their suitability in systematic toxicological analysis (STA). A test set of 25 barbiturates was analysed using capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Buffers used consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate, 50 mM sodium dodecyl sulphate set at pH 7.5 (MEKC). All analyses were carried out using fused silica capillaries using an electric field strength of 52.6 kV/m. The use of a reproducible identification parameter is very important in STA as it influences the identification power (IP). To deal with the poor reproducibility of the migration time, we introduced the corrected effective mobility. Inter-day reproducibilities of the latter parameter were < 0.6% for CZE and < 0.5% for MEKC, using daily prepared buffers. The IP of the methods was expressed by calculation of the discriminating power and the mean list length. Data obtained were compared to gas chromatographic and high-performance liquid chromatographic data, and correlations between all methods were calculated. It was shown that little correlation exists between chromatographic and electrophoretic techniques. The results indicated that CE has a good identification power for the application in STA, especially when a combination of methods having a low correlation is used.