Qualitative and quantitative evaluation of Simon™, a new CE-based automated Western blot system as applied to vaccine development

Many CE-based technologies such as imaged capillary IEF, CE-SDS, CZE, and MEKC are well established for analyzing proteins, viruses, or other biomolecules such as polysaccharides. For example, imaged capillary isoelectric focusing (charge-based protein separation) and CE-SDS (size-based protein separation) are standard replacement methods in biopharmaceutical industries for tedious and labor intensive IEF and SDS-PAGE methods, respectively. Another important analytical tool for protein characterization is a Western blot, where after size-based separation in SDS-PAGE the proteins are transferred to a membrane and blotted with specific monoclonal or polyclonal antibodies. Western blotting analysis is applied in many areas such as biomarker research, therapeutic target identification, and vaccine development. Currently, the procedure is very manual, laborious, and time consuming. Here, we evaluate a new technology called Simple Western? (or Simon?) for performing automated Western analysis. This new technology is based on CE-SDS where the separated proteins are attached to the wall of capillary by a proprietary photo activated chemical crosslink. Subsequent blotting is done automatically by incubating and washing the capillary with primary and secondary antibodies conjugated with horseradish peroxidase and detected with chemiluminescence. Typically, Western blots are not quantitative, hence we also evaluated the quantitative aspect of this new technology. We demonstrate that Simon? can quantitate specific components in one of our vaccine candidates and it provides good reproducibility and intermediate precision with CV <10%.     

Interlaboratory method validation of capillary electrophoresis sodium dodecyl sulfate (CE-SDS) methodology for analysis of mAbs

Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is an analytical method to assess the purity of proteins, commonly applied to monoclonal antibodies (mAbs) in the biopharmaceutical industry. To address the need to standardize the CE-SDS method in the pharmaceutical industry and to enhance the confidence in method transfer between laboratories operating different commercial capillary electrophoresis (CE) instrument platforms, an interlaboratory CE-SDS method validation was organized involving 13 laboratories in 13 companies on four different types of commercial capillary electrophoresis instruments. In the validation, a commercial mAb therapeutic was used as the sample. The validation process followed the analytical guidelines set by the ICH guidelines (International Conference for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use). The method's precision, accuracy, linearity and range, and limit of quantitation (LOQ) were validated in the study. Variations of all the parameters validated in the study passed the pre-set criteria defined at the beginning of the study. The definition was based on previously published works and the intended application purpose of the CE-SDS method for mAbs. The study proved that the CE-SDS method fits its intended application purpose as a size impurity assay and size heterogeneity characterization assay for mAb therapeutic products. This study is the first time a CE-SDS method is validated by multiple laboratories using different commercial CE instrument platforms and on a commercial mAb therapeutic. Its results will enhance the confidence of the biopharmaceutical industry to develop CE-SDS methods and transfer CE-SDS methods between different laboratories.     

A comparative study of CE-SDS,SDS-PAGE,and Simple Western: Influences of sample preparation on molecular weight determination of proteins

The development of capillary electrophoresis, especially CE-SDS devices, has led CE-SDS to become an established tool in a wide range of applications in the analysis of biopharmaceuticals and is increasingly replacing its method of origin, SDS-PAGE. The goal of this study was to evaluate the comparability of molecular weight (MW) determination especially by CE-SDS and SDS-PAGE. For ensuring comparability, model proteins that have little or no posttranslational modifications and an IgG antibody were used. Only a minor influence of sample preparation conditions, including sample buffer, temperature conditions, and different reducing agents on the MW determination were found. In contrast, the selection of the MW marker plays a decisive role in determining the accurate apparent MW of a protein. When using different MW markers, the deviation in MW determination can exceed 10%. Interestingly, CE-SDS and 10% SDS-PAGE hardly differ in their trueness of MW determination. The trueness in relation to the reference MW for each protein was calculated. Although the trueness values for the model proteins considered range between 1.00 and 1.11 using CE-SDS, they range between 0.93 and 1.03 on SDS-PAGE, depending on the experimental conditions chosen.     

Minimization of artifact protein aggregation using tetradecyl sulfate and hexadecyl sulfate in capillary gel electrophoresis under reducing conditions

In the biopharmaceutical industry, CE-SDS assesses the purity, heterogeneity, and stability of therapeutic proteins. However, for mAb-1 and mAb-2, typical CE-SDS under reducing conditions produced atypical protein peak profiles, which led to biased purity results, thus were not acceptable for biologics manufacturing. This bias was caused by the formation of method-induced higher molecular weight artifacts, the levels of which correlated with protein concentration. Here we show that adding sodium tetradecyl and hexadecyl sulfates to the sample and the sieving gel buffer solutions was required to prevent formation of aggregate artifacts and to maintain detergent:protein uniformity, suggesting their importance during the sample preparation steps of heat denaturation and subsequent cooling as well as during capillary migration. For these proteins, we show that this uniformity was likely due to the ability of these detergents to bind proteins with markedly higher affinities compared to SDS. “CE-SC_XS” methods (where CE-SC_XS is CGE using detergent composed of a sodium sulfate head group and a hydrocarbon tail, with “C_X” representing various tail lengths), were developed with a sodium tetradecyl sulfate sample buffer and a sodium hexadecyl sulfate containing sieving gel buffer that minimized artifacts and provided robust characterization and release results for mAb-1 and mAb-2.     

Optimization of CE-SDS Method for Antibody Separation Based on Multi-Users Experimental Practices

Capillary Electrophoresis-Sodium Dodecyl Sulfate (CE-SDS) method with UV detection was developed and satisfactorily used for determination of purity and manufacturing consistency of a monoclonal antibody (MAb) at Amgen Inc. (Seattle, WA). When this method was applied to some other MAbs, several problems with method robustness became apparent. These issues resulted in abnormal Electropherogram (e-gram) profiles potentially linked to various parameters specific molecules analyzed, sample formulation buffer composition, CE-SDS gel matrix type, and operators. A multi-users interest group (called CE Users Forum) was formed to systematically investigate and understand these issues. The CE Users Forum first identified the issues which needed resolution, defined group experiments to better understand the problem and to test potential solutions, and together defined a generic (platform) CE-SDS method for MAbs. Two CE instruments, Agilent HP^3DCE and Beckman PA 800, two CE-SDS gel matrices, BioRad and Beckman gels, as well as different types of MAbs in various buffers were used in this investigation. We present here a platform CE-SDS method for purity determination of MAbs. Method optimization and trouble-shooting procedures by the CE Users Forum played a key role in delivering a robust analytical method for characterization of antibodies by improving instrumental and experimental parameters such as instrument variability, instrument operating parameters, operator training, and reagent stability. The optimized CE-SDS method is used during process development and has been transferred to the quality control (QC) lab as a purity assay for lot release testing of therapeutic antibodies. Any trained analyst can successfully perform this method. A group such as the CE Users Forum is a good way to integrate best practices and solve technical issues in a cooperative environment.Presented at: CE in the Biotechnology & Pharmaceutical Industries: 7th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules, Montreal, Canada, August 12–16, 2005     

Meat species identification by linear discriminant analysis of capillary electrophoresis protein profiles

The objective of this study was to utilize linear discriminant analysis (LDA) in the interpretation of capillary electrophoresis-sodium dodecyl sulfate polymer-filled capillary gel electrophoresis (CE-SDS) meat protein profiles for the identification of meat species. The specific objectives were 1) to collect quantitative data on water-soluble and saline-soluble proteins of different meat species obtained by CE-SDS and 2) to apply LDA on collected CE-SDS protein data for the development of a pattern recognition statistical model useful in the differentiation of meat species. Samples were raw beef top and eye round, boneless fresh pork ham and loin, turkey leg and breast meat, and mechanically deboned turkey meat collected on six different occasions, making a total of 42 samples. Additionally, 14 samples were used as test samples to determine the classification ability of the procedure. Quantitative protein data obtained by CE-SDS was used to generate separate LDA models for either water- or saline-soluble protein extracts. Although a saline solution was a more efficient meat protein-extracting agent, as shown by a higher total protein concentration and a larger number of peaks, water-soluble CE-SDS protein profiles gave more distinctive discrimination among meat species. The correct classification given by LDA on water-soluble protein data was 100% for all meat species, except pork (94%). Conversely, the correct classification on saline-soluble protein data was 88% for beef and mechanically deboned turkey meat, and 94% and 100% for turkey and pork meat, respectively. LDA proved to be a useful pattern recognition procedure in the interpretation of CE-SDS protein profiles for the identification of meat species.     

A comparative study of CE-SDS,SDS-PAGE,and Simple Western—Precision,repeatability, and apparent molecular mass shifts by glycosylation

SDS gel electrophoresis is a commonly used approach for monitoring purity and apparent molecular mass (Mr) of proteins, especially in the field of quality control of biopharmaceutical proteins. The technological installation of CE-SDS as the replacement of the slab gel technique (SDS-PAGE) is still in progress, leading to a continuous improvement of CE-SDS instruments. Various CE-SDS instruments, namely Maurice (CE-SDS/CE-SDS PLUS) and Wes by ProteinSimple as well as the microchip gel electrophoresis system LabChip® GXII Touch™ HT by PerkinElmer were tested for precision and repeatability compared to SDS-PAGE (Bio-Rad). For assessing these quality control parameters, standard model proteins with minor post-translational modifications were used. Overall, it can be concluded that the CE-SDS-based methods are similar to SDS-PAGE with respect to these parameters. Quality characteristics of test systems gain more significance by testing proteins that do not behave like model proteins. Therefore, glycosylated proteins were analyzed to comparatively investigate the influence of glycosylation on Mr determination in the different instruments. In some cases, high deviations were found both among the methods and with regard to reference values. This article provides possible explanations for these findings.     

Sixteen capillary electrophoresis applications for viral vaccine analysis

A broad range of CE applications from our organization is reviewed to give a flavor of the use of CE within the field of vaccine analyses. Applicability of CE for viral vaccine characterization, and release and stability testing of seasonal influenza virosomal vaccines, universal subunit influenza vaccines, Sabin inactivated polio vaccines (sIPV), and adenovirus vector vaccines were demonstrated. Diverse CZE, CE-SDS, CGE, and cIEF methods were developed, validated, and applied for virus, protein, posttranslational modifications, DNA, and excipient concentration determinations, as well as for the integrity and composition verifications, and identity testing (e.g., CZE for intact virus particles, CE-SDS application for hemagglutinin quantification and influenza strain identification, chloride or bromide determination in process samples). Results were supported by other methods such as RP-HPLC, dynamic light scattering (DLS), and zeta potential measurements. Overall, 16 CE methods are presented that were developed and applied, comprising six adenovirus methods, five viral protein methods, and methods for antibodies determination of glycans, host cell-DNA, excipient chloride, and process impurity bromide. These methods were applied to support in-process control, release, stability, process- and product characterization and development, and critical reagent testing. Thirteen methods were validated. Intact virus particles were analyzed at concentrations as low as 0.8 pmol/L. Overall, CE took viral vaccine testing beyond what was previously possible, improved process and product understanding, and, in total, safety, efficacy, and quality.     

Quantitative impurity analysis of monoclonal antibody size heterogeneity by CE-LIF: example of development and validation through a quality-by-design framework

This paper describes the framework of quality by design applied to the development, optimization and validation of a sensitive capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) assay for monitoring impurities that potentially impact drug efficacy or patient safety produced in the manufacture of therapeutic MAb products. Drug substance or drug product samples are derivatized with fluorogenic 3-(2-furoyl)quinoline-2-carboxaldehyde and nucleophilic cyanide before separation by CE-SDS coupled to LIF detection. Three design-of-experiments enabled critical labeling parameters to meet method requirements for detecting minor impurities while building precision and robustness into the assay during development. The screening design predicted optimal conditions to control labeling artifacts while two full factorial designs demonstrated method robustness through control of temperature and cyanide parameters within the normal operating range. Subsequent validation according to the guidelines of the International Committee of Harmonization showed the CE-SDS/LIF assay was specific, accurate, and precise (RSD ≤ 0.8%) for relative peak distribution and linear (R > 0.997) between the range of 0.5-1.5 mg/mL with LOD and LOQ of 10 ng/mL and 35 ng/mL, respectively. Validation confirmed the system suitability criteria used as a level of control to ensure reliable method performance.     

Use of CE-SDS gel for characterization of monoclonal antibody hinge region clipping due to copper and high pH stress

CE-SDS gel technique has been used extensively in the field of monoclonal antibody (mAb) as a tool for product purity, stability, and characterization. It offers many advantages over the traditional labor-intensive SDS-PAGE slab gel technology with respect to speed and resolution. Monoclonal antibodies are known to cleave in the hinge region due to extreme pH, high temperature and in the presence of metals, especially copper. This cleavage will impact the shelf lifetime of mAb product hence its quality. CESDS gel method using Beckman PA800 with UV detection is used to characterize the effects of copper and other metals such as iron and zinc on mAb clipping. In addition, mAb integrity under high temperature and high pH stress conditions was also evaluated and the results clearly show that CE-SDS gel can distinguish clipping due to copper versus heat and/or high pH. The data presented illustrate the power of this simple CESDS gel technique in supporting the development of mAb from product quality and stability to the final product characterization.     

Capillary electrochromatographic analysis of barbiturates in serum

A capillary electrochromatographic method was developed for the separation of barbiturates. The separation was optimized in a 75 microm ID capillary, packed with 3-(1,8-naphthalimido)propyl-modified silyl silica gel (NAIP), studying the effect of buffer pH, buffer concentration, and mobile phase composition. Using an applied voltage of 20 kV and the short-end injection method (9 cm capillary effective length), the mobile phase of 1.0 mM citrate buffer (pH 5.0) containing 40% methanol provided the baseline separation of barbital, phenobarbital, secobarbital, and thiopental (internal standard) in less than 4.5 min. The method was successfully applied to the analysis of barbiturates in human serum. Under the optimal conditions, good repeatability and linearity were obtained in the range of 2.90-43.29 microg/mL for barbital, phenobarbital, and secobarbital.     

Protein mapping of peanut extract with capillary electrophoresis

Protein separation can be achieved with different modes of capillary electrophoresis, such as with capillary gel electroporesis (CGE) or with capillary zone electrophoresis (CZE). CZE protein mapping of peanut extract was approached in four different ways, combining neutral-coated or multilayer-coated capillaries with pHs well over or under the isoelectric point range of the proteins of interest. At acidic pHs, the mobility ranges of the major peanut allergens Ara h1, Ara h2, Ara h3, and Ara h6 were identified. Although the pH is a major factor in CZE separation, buffers with different compositions but with the same pH and ionic strength showed significantly different resolutions. Different components of the electrolyte were studied in a multifactorial design of experiment. CE-SDS and CZE proved to be suitable for protein mapping and we were able to distinguish different batches of peanut extract and burned peanut extract.     

Therapeutic protein purity and fragmented species characterization by capillary electrophoresis sodium dodecyl sulfate using systematic hybrid cleavage and forced degradation

Analytical and Bioanalytical Chemistry - Positive identification of capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) electropherogram peaks provides information to understand protein...     

Development of a chip-based capillary gel electrophoresis method for quantification of a half-antibody in immunoglobulin G4 samples

A method based on microfluidic technology was developed to support quantitative analysis of recombinant monoclonal immunoglobulin G4 (IgG4) antibody samples. The assay was performed on an Agilent 2100 Bioanalyzer in combination with the Protein 200 Plus LabChip Kit and the Protein 200 Plus assay software. Capillary electrophoresis principles have been transferred to a chip format that integrates all separation, staining, virtual destaining, and detection steps. The method is referred to in this paper as chip-based capillary gel electrophoresis (GelChip-CE method). The GelChip-CE method under nonreducing conditions proved to be a quantitative test for half-antibody determination in IgG4 samples. Similar to the traditional nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method, the GelChip-CE method includes a denaturing step prior to separation. We showed that denaturing the sample by heating resulted in an artificial increase in the amount of half-antibody detected, which could be prevented by addition of N-ethylmaleimide to the sample buffer. The GelChip-CE method allowed for analysis of IgG4 samples with more accuracy, higher precision, and a faster turnaround time than SDS-PAGE and reversed-phase high-performance liquid chromatography (RP-HPLC).     

Rapid separation of barbiturates and benzodiazepines by capillary electrochromatography with 3-(1,8-naphthalimido)propyl-modified silyl silica gel

A capillary electrochromatographic (CEC) method was applied to the simultaneous separation of barbiturates (barbital, phenobarbital, secobarbital and thiopental) and benzodiazepines (nitrazepam, diazepam and triazolam). The separation was performed in a 75 microm i.d. capillary, packed with 3-(1,8-naphthalimido)propyl-modified silyl silica gel (NAIP), studying the effects of buffer pH and mobile phase composition. Using an applied voltage of 20 kV and the short-end injection method (9 cm capillary effective length), the mobile phase of 1.0 mM citrate buffer (pH 5.0) containing 45% methanol provided the baseline separation of seven toxic drugs in less than 9 min. In CEC with NAIP, the benzodiazepines were separated by the combination of hydrophobic and pi-pi interactions, whereas the separation of the barbiturates was based on the hydrophobic interaction.     

Characterization of quantum dot bioconjugates by capillary electrophoresis with laser-induced fluorescent detection

In this paper, we present a universal, highly efficient and sensitive method for the characterization of quantum dot (QD) bioconjugates based on capillary electrophoresis with laser-induced fluorescent (LIF) detection. We first prepared CdTe QDs in aqueous phase by a chemical route with mercaptopropionic acid as a ligand, and then were coupled to certain proteins using bifunctional linkage reagent or electrostatic attraction. The QD bioconjugates were characterized by capillary electrophoresis with LIF detection. We found that QD bioconjugates were efficiently separated with free QDs by the optimization of buffer pH. Furthermore, we found that ultrafiltration was an effective and simple approach to purify QD conjugates with bovine serum albumin (BSA). Due to their broad absorption spectra and size dependent emission wavelength tunability, QDs can be excited to emit different colour fluorescence using a single wavelength laser source, and therefore, we believe that CE with LIF detection will become a universal and efficient tool for the characterization of QD bioconjugates.     

Sol‐gel technique for the preparation of β‐cyclodextrin gold nanoparticles as chiral stationary phase in open‐tubular capillary electrochromatography

A novel open‐tubular capillary electrochromatography column coated with β‐cyclodextrin was prepared using the sol‐gel technique. In the sol‐gel approach, owing to the three‐dimensional network of sol‐gel and the strong chemical bond between the stationary phase and the surface of capillary columns, good chromatographic characteristics and unique selectivity in separating enantiomers were shown. The influences of capillary inner diameter, coating time, organic modifier, buffer pH, and buffer concentration on separation were investigated. The sol‐gel‐coated β‐cyclodextrin column has shown improved enantioseparation efficiency of chlorphenamine, brompheniramine, pheniramine, zopiclone in comparison with the sol‐gel matrix capillary column. The migration time relative standard deviation of the separation of the enantiomers was less than 0.89% over five runs and 2.9% from column to column. This work confirmed that gold nanoparticles are promising electrochromatographic support to enhance the phase ratio of open‐tubular capillary electrochromatography column in capillary electrochromatography.     

Applications of Monte-Carlo simulation and chemometric techniques for development of bioanalytical liquid chromatography method for estimation of rosuvastatin calcium

In the present work, we investigated the development of a bioanalytical HPLC method of rosuvastatin (RSV) calcium as per the Quality by Design (QbD)-based systematic chemometric tools. At first, the method objectives were framed and critical analytical attributes (CAAs) were chosen. Risk assessment and factor screening was performed using Hybrid Risk Matrix and Plackett–Burman design for identifying vital factors influencing the critical method parameters (CMPs). Monte-Carlo simulation analysis was conducted which confirmed excellent process robustness (Ppk >1.33) for the studied ranges of CMPs. Furthermore, systematic method development was carried out using custom experimental design, where mobile phase ratio, pH, and injection volume were taken as CMPs at three levels. The obtained trials were evaluated for peak area, retention time, theoretical plates, and peak tailing as CAAs. Mathematical response surface modeling was carried out and optimal chromatographic solution was identified using response optimizer plots. Method transfer was made to bioanalytical scale for estimation of the analyte in rat plasma samples. Extensive method validation was performed as per the ICH Q2 guideline, which indicated validation parameters within the acceptable limits. Overall, the studies construed successful development of QbD compliant HPLC method of rosuvastatin with potential utility bioanalytical testing.     

Serum iohexol analysis by micellar electrokinetic capillary chromatography

A simple and rapid ( approximately 4 min) method for the measurement of iohexol in serum for assessing the glomerular filtration rate is described. It is based on direct serum injection on the capillary by MEKC. The method is linear between 8 and 260 mg/L, with an RSD of peak height of 2.9%. Several simple steps have contributed to an improved daily precision, such as choosing a high pH buffer, increasing the SDS concentration, frequent standardization, and eliminating any sample pretreatment.     

改良型单纯形法优化硝基喜树碱的毛细管电泳分离

使用改良型单纯形法优化硝基喜树碱(Nitrocamprothecinum,NC)一对同分异构体的毛细管电泳分离。考察了运行电压、缓冲液的pH、硼砂和十二烷基磺酸钠(SDS)的浓度等4个影响因子,与传统的单因子优化方法及其结果进行比较。改良型单纯形法(采用CRS函数为响应函数)在分析方法的建立过程中显著减少了实验步骤,获得了更好的电泳分离条件。