Neutral Capillary Research Articles

Heterogeneity assessment of antibody-derived therapeutics at the intact and middle-up level by low-flow sheathless capillary electrophoresis-mass spectrometry

Antibody-based pharmaceuticals often encompass a complex structural heterogeneity requiring enhanced analytical methods for reliable characterization of variants and degradation products. We have explored the capabilities of low-flow sheathless capillary electrophoresis-mass spectrometry (CE-MS) for the high-resolution and sensitive profiling of antibody therapeutics. Near-zero electroosmotic flow was achieved by employing a novel neutral capillary coating that also prevents protein adsorption. CE-MS analysis of intact model proteins using an acidic background electrolyte demonstrated satisfactory performance, with overall migration-time RSDs below 2.2% from three different capillaries tested. For system evaluation, three nanobody preparations, including mono- and bivalent forms, and three monoclonal antibodies (mAbs) were analyzed. Intact nanobodies were resolved from their degradation products, which could be assigned to deamidated, cleaved, and truncated forms at the C-terminal tag. Excellent resolution of isomeric deamidated products was obtained. The mAbs were analyzed intact and after digestion by the endoproteinase IdeS (middle-up approach). CE-MS of intact mAbs provided resolution of clipped species (e.g. light chain and light chain-heavy chain fragments) from the native protein. Moreover, glycoforms containing sialic acids were resolved from their non-sialylated counterparts. For IdeS-digested, F (ab)2 and Fc/2 portions where efficiently resolved for the three mAbs. Whereas the migration time of the Fc/2 fragments was fairly similar, the migration time of the F (ab)2 part was strongly varied among the mAbs. For all mAbs, separation of Fc/2 charge variants - including sialylated glycoforms and other post-translational modifications, such as loss of C-terminal lysine or asparagine deamidation - was achieved. This allowed a detailed and reliable assessment of the Fc/2 heterogeneity (18–33 proteoforms) of the three analyzed mAbs.

Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary.

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The biological activity of these proteins depends on their physicochemical properties. The use of high-performance techniques like chromatography and capillary electrophoresis has been described for the analysis of physicochemical heterogeneity of mAbs. Nowadays, capillary zone electrophoresis (CZE) technique constitutes one of the most resolutive and sensitive assays for the analysis of biomolecules. Besides, the electro-driven separation in CZE is governed by extensive properties of matter and offers the advantage of analyzing proteins close to their native state. However, the successful implementation of this technique for routine analysis depends on the skills of the analyst at the critical steps during sample and system preparation. The purpose of this tutorial is to detail the steps to succeed in the CZE analysis of mAbs. Further, this protocol can be used for the development and improvement of skills of the personnel involved in protein analytical chemistry laboratories.

Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

A comparative study of various physicochemically modified capillaries used in CE technique for the three distinct analytical purposes.

The five different commercially offered capillaries, bare silica, one dynamically and three permanently coated, have been tested with respect to the three distinct bioanalytical issues: (i) achiral and chiral separation of small mass molecules, warfarin and its six isomeric hydroxy-derivatives; (ii) ultraselective separation of transferrin and albumin including differentiation between the diferric, two monoferric and iron-free forms of transferrin; and (iii) pioneering identification of albumin-induced shifts of acid dissociation constant by using CE instrumentation, revealed for warfarin and six hydroxywarfarins. As a result all of these pharmacologically and biochemically-relevant purposes have been reached, but using different capillaries. The dynamically coated silica capillary has allowed for the first time to separate warfarin from its six main hydroxy-metabolites by CE with high resolution. The bare silica capillary has performed well in cyclodextrin-assisted enantioseparation of these compounds. The neutral capillary has provided excellent resolution and performance in separation of proteins. The amine capillary, in turn, has occurred to be the best choice for identification of albumin-induced pKa shifts occurring in vivo, omitting capillary clogging and providing better sensitivity than the neutral capillary. This work reveals specific predispositions and shows that there is no universal capillary which may be applied to all issues.

Capillary isoelectric focusing method development and validation for investigation of recombinant therapeutic monoclonal antibody

Capillary isoelectric focusing (cIEF) is a basic and highly accurate routine analytical tool to prove identity of protein drugs in quality control (QC) and release tests in biopharmaceutical industries. However there are some “out-of-the-box” applications commercially available which provide easy and rapid isoelectric focusing solutions for investigating monoclonal antibody drug proteins. However use of these kits in routine testings requires high costs.A capillary isoelectric focusing method was developed and validated for identification testing of monoclonal antibody drug products with isoelectric point between 7.0 and 9.0. A method was developed providing good pH gradient for internal calibration (R2>0.99) and good resolution between all of the isoform peaks (R=2), minimizing the time and complexity of sample preparation (no urea or salt used). The method is highly reproducible and it is suitable for validation and method transfer to any QC laboratories. Another advantage of the method is that it operates with commercially available chemicals which can be purchased from any suppliers. The interaction with capillary walls (avoid precipitation and adsorption as far as possible) was minimized and synthetic isoelectric small molecular markers were used instead of peptide or protein based markers.The developed method was validated according to the recent ICH guideline (Q2(R1)). Relative standard deviation results were below 0.2% for isoelectric points and below 4% according to the normalized migration times. The method is robust to buffer components with different lot numbers and neutral capillaries with different type of inner coatings. The fluoro-carbon coated column was chosen because of costs-effectivity aspects.

A Comprehensive Study of Silanization and Co-Condensation for Straightforward Single-Step Covalent Neutral Capillary Coating

We performed a comprehensive theoretical and experimental study on concurrent silanization and co-condensation of tetraethyl orthosilicate (TEOS) and 2-Methoxy(polyethyleneoxypropyl)trimethoxysilane (PEOS) under basic hydro-organic medium for straightforward production of a covalent neutral coating for capillary electrophoresis. It is proposed that the steric hindrance induced by the large structure of PEOS renders it difficult to obtain a facile direct reaction with the silanols on the capillary’s internal surface. Rather, the addition of TEOS for silanization and cross-linkage is efficient to retain PEOS anchored on the silanol surface, thus producing a neutral layer. Performance of the produced neutral capillary in terms of suppression and stability of the electroosmotic flow, as well as application of this new neutral coating for the separation of low molecular weight carboxylic anions and proteins of both positive and negative charges were evaluated.

Separation of iron-free and iron-saturated forms of transferrin and lactoferrin via capillary electrophoresis performed in fused-silica and neutral capillaries

A capillary electrophoresis-based method for the cost-effective and high efficient separation of iron-free and iron-saturated forms of two members of transferrin family: transferrin and lactoferrin has been developed. The proposed qualitative method relying on the SDS application allowed us to separate iron-free and iron-saturated forms of these proteins, as well as human serum albumin, used as an internal standard. Owing to the distinct migration times under established conditions, the combination of transferrin and lactoferrin assays within a single analytical procedure was feasible. The performance of the method using a fused-silica capillary has been compared with the results obtained using the same method but performed with the use of a neutral capillary of the same dimensions. Neutral capillary has been used as an alternative, since the comparable resolution has been achieved with a concomitant reduction of the electroosmotic flow. Despite of this fact, the migration of analytes occurred with similar velocity but in opposite order, due to the reverse polarity application. A quantitative method employing fused-silica capillary for iron saturation study has been also developed, to evaluate the iron saturation in commercial preparations of lactoferrin.

Use of neutral capillaries for the enantioseparation ofN-benzoylated amino acids by capillary electrophoresis with bromobalhimycin as chiral selector

In this study, the partial filling technique on both polycationic polymer hexadimethrine bromide (HDB) modified capillary and eCAP neutral capillary were systematically compared in order to enhance the enantioseparation ability of bromobalhimycin as CE additive. The separation conditions, such as pH, the plug length, and the concentration of bromobalhimycin, etc., were optimized in order to obtain satisfactory separations. As expected, for all tested 28 N-benzoylated amino acids, up to five times higher enantioresolutions were obtained on the eCAP neutral capillary compared to that on the polycationic polymer hexadimethrine bromide modified capillary. Moreover, 26 of 28 tested racemic compounds were almost baseline- resolved without observing any interference from the front of the plug of bromobalhimycin. Although the limitation of longer running time on the neutral capillary, it allows the use of higher content of bromobalhimycin in the running buffer without any interference on the detection of analytes when enantioseparations are more difficult to obtain.

Determination of biapenem in a medicinal product by micellar electrokinetic chromatography with sweeping in an enhanced electric field

The elaborated method of micellar electrokinetic chromatography (MEKC) used to separate biapenem from its related substances was successfully implemented using sweeping under an enhanced electric field, followed by UV absorption detection at 200nm. The best results were obtained with formic buffer (22.5mM) pH 4.3 and sodium dodecyl sulfate (150mM) added to the electrolyte as the sweeping agent. Neutral capillary (60/50cm; 50μm ID) with reverse polarity and voltage values of 22kV, were used throughout the investigation.The optimized method of biapenem determination, validated in terms of linearity, accuracy and precision, provides a detection limit of 0.5μg/mL at S/N=3 for biapenem. The repeatability of the CE system, expressed by relative standard deviations (RSD) in the migration times, for biapenem and its degradation products varied from 0.14 to 1.48%, whereas for the corrected peak areas RSD were about 0.68–8.43%. Satisfactory separation was achieved within 20min of electrophoresis; moreover all carbapenems (imipenem, meropenem, ertapenem, doripenem and biapenem) were separated from each other during analysis. The evaluated MEKC method was applied to the analysis of a medicinal product containing biapenem – Omegacin® 0.3g for intravenous drip infusion.

Coupling porous sheathless interface MS with transient‐ITP in neutral capillaries for improved sensitivity in glycopeptide analysis

IgG antibodies are modulated in their function by the specific structure of the N-glycans attached to their Fc (fragment crystallizable) portions. However, the glycosylation analysis of antigen-specific IgGs is a challenging task as antibody levels to a given antigen only represent a fraction of the total IgG levels. Here, we investigated the use of a transient-ITP (t-ITP)--MS method for highly sensitive IgG1 glycosylation profiling as a complementary method to a high-throughput nano-RPLC-MS method. It was found that t-ITP-CZE using neutrally coated separation capillaries with a large volume injection (37% of capillary volume) and interfaced to MS with a sheathless porous sprayer yielded a 40-fold increase in sensitivity for IgG1 Fc glycopeptide analysis when compared to the conventional strategy. Furthermore, the glycoform profiles found with the t-ITP-CZE strategy were comparable to those from nano-RPLC-MS. In conclusion, the use of the highly sensitive t-ITP-CZE-MS method will provide information on IgG Fc glycosylation for those samples with IgG1 concentrations below the LODs of the conventional method.

Prediction of the Ripening Times of Ewe's Milk Cheese by Multivariate Regression Analysis of Capillary Electrophoresis Casein Fractions

The effect of the ripening time on the proteolytic process in cheeses made from ewe's milk during a 139-day ripening period was monitored by the use of capillary electrophoresis of pH 4.6 insoluble fraction. Totals of 18 and 21 peaks were recognized and matched in the electropherograms obtained with a fused-silica capillary and a neutral capillary (hydrophilically coated), respectively. These peaks correspond to intact ovine caseins and their hydrolysis products (alpha(s1)-casein I, alpha(s1)-casein II, alpha(s1)-casein III, alpha(s2)-casein, beta(1)-casein, beta(2)-casein, p-kappa-casein, alpha(s1)-I-casein, gamma(1)-casein, gamma(2)-casein, and gamma(3)-casein). The alpha(s)-caseins (alpha(s1)- and alpha(s2)-casein) displayed similar degradation pattern to one another, but different from those of beta-caseins (beta(1)- and beta(2)-casein). beta-Caseins were very much undergoing lesser degradation during the ripening time than alpha(s)-casein. Finally, partial least-squares regression and principal components regression were used to predict the ripening time in cheeses. The models obtained yielded good results since the root-mean-square error in prediction by cross validation was <8.6 days in all cases.

Chemometrical Analysis of Capillary Electrophoresis Casein Fractions for Predicting Ripening Times of Milk Mixture Cheese

The effect of the ripening time on the proteolytic process in cheeses manufactured from mixtures of cow's and ewe's milk during a 167-day ripening period was monitored by capillary electrophoresis of the pH 4.6-insoluble fraction. Totals of 21 and 16 peaks were recognized and matched in the electropherograms obtained with a fused-silica capillary and a neutral capillary (hydrophilically coated), respectively. These peaks corresponded to intact bovine and ovine caseins and their hydrolysis products (e.g., alpha(s1)-casein, gamma-caseins). In 167-day-old cheeses, bovine alpha(s0)-casein (alpha(s1)-casein 9P) had been completely degraded and 6% of the residual bovine alpha(s1)-casein remained intact. Breakdown of the beta-casein fraction was lower than that of the alpha(s)-casein fraction. Finally, partial least-squares regression and principal component regression were used to predict the ripening time in cheeses. The root-mean-square errors in prediction by cross-validation were <7.8 days in all cases.

The affinity of different MBD proteins for a specific methylated locus depends on their intrinsic binding properties.

The methyl-CpG binding domain (MBD) family of proteins was defined based on sequence similarity in their DNA binding domains. In light of their high degree of conservation, it is of inherent interest to determine the genomic distribution of these proteins, and their associated co-repressor complexes. One potential determinant of specificity resides in differences in the intrinsic DNA binding properties of the various MBD proteins. In this report, we use a capillary electrophoretic mobility shift assay (CEMSA) with laser-induced fluorescence (LIF) and neutral capillaries to calculate MBD-DNA binding affinities. MBD proteins were assayed on pairs of methylated and unmethylated duplex oligos corresponding to the promoter regions of the BRCA1, MLH1, GSTP1 and p16(INK4a) genes, and binding affinities for each case were calculated by Scatchard analyses. With the exception of mammalian MBD3 and Xenopus MBD3 LF, all the MBD proteins showed higher affinity for methylated DNA (in the nanomolar range) than for unmethylated DNA (in the micromolar range). Significant differences between MBD proteins in the affinity for methylated DNA were observed, ranging within two orders of magnitude. By mutational analysis of MBD3 and using CEMSA, we demonstrate the critical role of specific residues within the MBD in conferring selectivity for methylated DNA. Interestingly, the binding affinity of specific MBD proteins for methylated DNA fragments from naturally occurring sequences are affected by local methyl-CpG spacing.

Development and validation of a capillary electrophoresis method for direct measurement of isocitric, citric, tartaric and malic acids as adulteration markers in orange juice

Fruit juices each have very distinct organic acids profiles that can be used as fingerprints for establishing authenticity. A method has been developed, optimised and validated for measuring by capillary electrophoresis citric, isocitric, malic and tartaric acids as authenticity markers in orange juices, without any sample treatment other than dilution and filtration. Final conditions were phosphate buffer 200 m M, pH 7.50, −14 kV as applied potential, and 57 cm length neutral capillary. Detection was direct UV at 200 nm. Different kinds and marks of orange juice, chosen from the great variety existent in the market, were analysed and clear differences could be found between them and just pressed orange juice.

Phosphopeptide isomer separation using capillary zone electrophoresis for the study of protein kinases and phosphatases.

Methods for the rapid separation of phosphopeptide isomers (peptides with the same sequence but with phosphates on different residues) were developed using capillary zone electrophoresis with ultraviolet (CZE-UV) detection. Uncoated, cationic and neutral capillaries were used with both acidic and basic peptides. These methods enabled the assay of several protein kinases (mitogen activated protein kinase, protein kinase A, GST-tyrosine kinase) and phosphatases (acid, alkaline, and protein tyrosine phosphatase) and the determination of the sites of phosphorylation and dephosphorylation. Incubations of nonphosphorylated or phosphorylated peptide with kinases or phosphatases took place directly in the instrument's autosampler and were monitored over several hours using CZE-UV.

Development of a urinary free cortisol assay using solid-phase extraction–capillary electrophoresis

In clinical practice, the measurement of urinary free cortisol (UFC) provides the most sensitive and specific diagnostic information for excess adrenal production of cortisol. The existing methodologies (RIA and HPLC) are time consuming, costly, involve tedious extractions, derivatizations and problems with non-specific interactions with cortisol metabolites in urine. In the present study, we describe the development of an SPE–CE method for the rapid analysis of UFC. UFC was concentrated using SPE C 18 cartridges (3M Empore) under a vacuum and eluted with acetonitrile–SDS. The use of 10% acetone to wash cartridges before final elution with acetonitrile–SDS showed significant improvements in the free cortisol recovery. The complete extraction was accomplished in 10–15 min with a recovery of 89–94%. CE analysis was done on a Beckman P/ACE 5010 with detection at 254 nm using a neutral capillary. Detection limits of free cortisol in urine was improved to 10 μg/l with SPE compared to 500 μg/l without SPE. No interferences either from BSA or other urinary cortisol metabolites affected the free cortisol determinations. The results showed the feasibility of a rapid UFC detection with improved sample handling capacity.

Specific Determination of Urinary Free Cortisol by Solid-Phase Microparticle Extraction Capillary Electrophoresis with Fused Silica Capillaries

Measurement of 24-h urinary free cortisol (UFC) provides the most sensitive and specific diagnostic information for adrenal malfunctions, especially for Cushing syndrome (1)(2). The existing methodologies include immunoassays (3)(4)(5) and HPLC, with and without solid-phase extraction and derivatization (6)(7). Immunoassays for UFC are precise but overestimate the concentration because of antibodies’ cross-reactivity with various metabolites and with synthetic corticoids having a chemical configuration similar to that of cortisol (8). HPLCs, on the other hand, although very specific, require large volumes of mobile phase, as well as sample pretreatment. Capillary electrophoresis (CE) overcomes many of these problems through the use of an open-tubular format, thus avoiding interaction of analytes with the solid-phase resin. Earlier, we reported the feasibility of rapid UFC detection in solid-phase extraction CE with a neutral capillary (9). Using a neutral capillary, we could demonstrate a detection limit of 552 nmol/L for UFC in human urine. However, neutral capillaries have several disadvantages: Not only are they expensive, they also are unstable and lack a uniform capillary coating. This makes the neutral capillary unattractive for the development of a routine clinical laboratory assay. In the present study, we developed a solid-phase microparticle extraction (SPME) coupled micellar electrokinetic capillary chromatography (MEKC) with use of a fused-silica capillary as a …

Capillary electrophoresis of r-hirudin and a polyethylene glycol derivative of r-hirudin (PEG-hirudin).

Recombinant (r-) hirudins and PEG-hirudin are currently tested for anticoagulant therapy. For their concentration measurement, radioimmunoassay and HPLC methods are available. The separation of r- and PEG-hirudin is currently performed by HPLC. However, the sensitivity of the method is low. Capillary electrophoresis is a rapid, selective technique that requires low sample amounts. Our aim was the development of a capillary electrophoresis method to measure r- and PEG-hirudin. The results are as follows: In a borate solution (0.3% boric acid and 0.4% sodium tetraborate, pH 9.5) r-hirudin was separated from PEG-hirudin in a purified system using a fused silica capillary (50 cm long and 75-micron i.d. and reversed polarity). A neutral capillary with a 20 mM tricine buffer (pH 8.0, field strength 500 V/cm) was also effective in resolving r- from PEG-hirudin. A linear correlation was found between the peak area and the concentration between 20 micrograms/mL and 10 mg/mL for hirudin (r2 = 0.99) and between 1.25 and 10 mg/mL for PEG-hirudin (r2 = 0.99). In human plasma mixtures, r- and PEG-hirudin were completely separated. The linear correlation between the peak area and the concentration was r2 = 0.99. In a fused silica capillary, r- and PEG-hirudin are separated in a purified system. Capillary electrophoresis which is performed in a neutral capillary, resolves r- from PEG-hirudin in a purified system, in plasma and in urine. The sensitivities of the methods are comparable. Capillary electrophoresis separates r- from PEG-hirudin and may be applied to biologic systems to measure the concentration and purity of r- and PEG-hirudin.