A bioinspired approach for the modulation of electroosmotic flow and protein-surface interactions in capillary electrophoresis using silylated amino-amides blocks and covalent grafting.

We explore a bioinspired approach to design tailored functionalized capillary electrophoresis (CE) surfaces based on covalent grafting for biomolecules analysis. First, the approach aims to overcome well-known common obstacles in CE protein analysis affecting considerably the CE performance (asymmetry, resolution, and repeatability) such as the unspecific adsorption on fused silica surface and the lack of control of electroosmotic flow (EOF). Then, our approach, which relies on new amino-amide mimic hybrid precursors synthesized by silylation of amino-amides (Si-AA) derivatives with 3-isocyanatopropyltriethoxysilane, aims to recapitulate the diversity of protein-protein interactions (π-π stacking, ionic, Van der Waals…) found in physiological condition (bioinspired approach) to improve the performance of CE protein analysis (electrochromatography). As a proof of concept, these silylated Si-AA (tyrosinamide silylation, serinamide silylation, argininamide silylation, leucinamide silylation, and isoglutamine silylation acid) have been covalently grafted in physiological conditions in different amount on bare fused silica capillary giving rise to a biomimetic coating and allowing both the modulation of EOF and protein-surface interactions. The analytical performances of amino-amide functionalized capillaries were assessed using lysozyme, cytochrome C and ribonuclease A and compared to traditional capillary coatings poly(ethylene oxide), poly(diallyldimethylammonium chloride), and sodium poly(styrenesulfonate). EOF, protein adsorption rate, protein retention factor k, and selectivity were determined for each coating. All results obtained showed this approach allowed to modulate the EOF, reduce unspecific adsorption, and generate specific interactions with proteins by varying the nature and the amount of Si-AA in the functionalization mixture.

Fast in-line bottom-up analysis of monoclonal antibodies: Toward an electrophoretic fingerprinting approach.

For their characterization and quality control, monoclonal antibodies are frequently analyzed at the bottom-up level to generate specific fingerprints that can be used to tackle post-translational modifications or ensure production consistency between lots. To circumvent time-consuming and labor-intensive off-line sample preparation steps, the implementation of integrated methodologies from sample preparation to separation and detection is highly valuable. In this perspective, capillary zone electrophoresis appears as a choice technique since the capillary can subsequently be used as a vessel for sample preparation and electrophoretic discrimination/detection of the reaction products. Here, a fast in-line methodology for the routine quality control of mAbs at the bottom-up level is reported. Simultaneous denaturation and reduction (pretreatment step) were conducted with RapiGest® surfactant and dithiothreitol before in-line tryptic digestion. Reactant mixing was realized by transverse diffusion of laminar flow profile under controlled temperature. In-line digestion was carried out with a resistant trypsin to autolysis. The main parameters affecting the digestion efficiency (trypsin concentration and incubation conditions) were optimized to generate mAb electrophoretic profiles free from trypsin interferences. An acidic MS-compatible BGE was used to obtain high resolution separation of released peptides and in-line surfactant cleavage. The whole methodology was performed in less than two hours with good repeatability of migration times (RSD = 0.91%, n = 5) and corrected peak areas (RSD = 9.6%, n = 5). CE-fingerprints were successfully established for different mAbs and an antibody-drug conjugate.

Development of Amino Acids Functionalized SBA-15 for the Improvement of Protein Adsorption.

Ordered mesoporous materials and their modification with multiple functional groups are of wide scientific interest for many applications involving interaction with biological systems and biomolecules (e.g., catalysis, separation, sensor design, nano-science or drug delivery). In particular, the immobilization of enzymes onto solid supports is highly attractive for industry and synthetic chemistry, as it allows the development of stable and cheap biocatalysts. In this context, we developed novel silylated amino acid derivatives (Si-AA-NH2) that have been immobilized onto SBA-15 materials in biocompatible conditions avoiding the use of toxic catalyst, solvents or reagents. The resulting amino acid-functionalized materials (SBA-15@AA) were characterized by XRD, TGA, EA, Zeta potential, nitrogen sorption and FT-IR. Differences of the physical properties (e.g., charges) were observed while the structural ones remained unchanged. The adsorption of the enzyme lysozyme (Lyz) onto the resulting functionalized SBA-15@AA materials was evaluated at different pHs. The presence of different functional groups compared with bare SBA-15 showed better adsorption results, for example, 79.6 nmol of Lyz adsorbed per m2 of SBA-15@Tyr compared with the 44.9 nmol/m2 of the bare SBA-15.

Capillary Electrophoresis-Mass Spectrometry at Trial by Metabo-Ring: Effective Electrophoretic Mobility for Reproducible and Robust Compound Annotation.

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (µeff) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The µeff was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the µeff for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.

An in-line enzymatic microreactor for the middle-up analysis of monoclonal antibodies by capillary electrophoresis.

Monoclonal antibodies (mAbs) are undergoing rapid growth in the pharmaceutical industry due to their clinical efficiency. Concomitantly, robust, cost-effective, and high throughput analytical methods are needed for their quality control. Among all analytical techniques, capillary electrophoresis (CE) presents alternative and attractive features because the capillary can be used both as a microreactor and as a support for separation. Transverse diffusion of laminar flow profiles was applied for the middle-up analysis of mAbs for the first time. Infliximab was selected as the model mAb. All middle-up analysis steps (enzymatic digestion, electrophoretic separation and UV detection) were integrated into the same capillary. The conditions for the separation of infliximab subunits (pH, ionic strength, and type of background electrolyte) and in-line digestion parameters (reactant injection conditions, time, temperature and enzyme/mAb ratio) were optimized. The in-line methodology was compared to the off-line methodology and evaluated in terms of proteolysis efficiency, repeatability, and applicability to different mAbs. Finally, the methodology was transferred to capillary electrophoresis coupled to mass spectrometry (sheathless interface) to identify infliximab subunits. The in-line methodology was successfully implemented with a simplified injection scheme, temperature control, fast enzymatic reaction and high resolution of separation of infliximab subunits under pseudo-native MS compatible conditions. In comparison with the off-line methodology, reactant consumption was reduced by a factor of 1000, and the numbers of theoretical plates were increased by a factor of 2.

Influence of salt and acetonitrile on the capillary zone electrophoresis analysis of imatinib in plasma samples.

A simple, sensitive, specific, and cost-effective analytical methodology was developed for the analysis of human plasma samples spiked with imatinib by CZE with on-line UV detection in the context of Therapeutic Drug Monitoring. Several analytical conditions such as the ionic strength (I) and the pH of the BGE composed of citric acid and ε-amino caproic acid were studied in regards of the presence of sodium chloride (NaCl) in plasma samples (1% m/v). Computer simulations (Simul software) were used to confirm the experimental results and to understand imatinib electrophoretic behavior in the presence of NaCl. Furthermore, the advantages of adding ACN to the sample containing NaCl to combine efficient protein precipitation and on-line CZE stacking of imatinib were demonstrated. LOD and LOQ values of 48 and 191 ng/mL were obtained from plasma sample supernatant after protein precipitation with ACN, which is much lower than mean imatinib plasma level observed for patients treated by imatinib mesylate (about 1000 ng/mL). Good linearity was obtained in the concentration range 191-5000 ng/mL (R2  > 0.997). RSD of less than 1.68% and 2.60% (n = 6) for migration times and corrected peak areas, respectively, were observed at the LOQ.

Evaluation of capillary zone electrophoresis for the quality control of complex biologic samples: Application to snake venoms.

Snake venoms constitute a very promising resource for the development of new medicines. They are mainly composed of very complex peptide and protein mixtures, which composition may vary significantly from batch to batch. This latter consideration is a challenge for routine quality control (QC) in the pharmaceutical industry. In this paper, we report the use of capillary zone electrophoresis for the development of an analytical fingerprint methodology to assess the quality of snake venoms. The analytical fingerprint concept is being widely used for the QC of herbal drugs but rarely for venoms QC so far. CZE was chosen for its intrinsic efficiency in the separation of protein and peptide mixtures. The analytical fingerprint methodology was first developed and evaluated for a particular snake venom, Lachesis muta. Optimal analysis conditions required the use of PDADMAC capillary coating to avoid protein and peptide adsorption. Same analytical conditions were then applied to other snake venom species. Different electrophoretic profiles were obtained for each venom. Excellent repeatability and intermediate precision was observed for each batch. Analysis of different batches of the same species revealed inherent qualitative and quantitative composition variations of the venoms between individuals.

Optimization of a nano-enzymatic reactor for on-line tryptic digestion of polypeptide conjugates by capillary electrophoresis.

This work aims at studying the optimization of an on-line capillary electrophoresis (CE)-based tryptic digestion methodology for the analysis of therapeutic polypeptides (PP). With this methodology, a mixture of surrogate peptide fragments and amino acid were produced on-line by trypsin cleavage (enzymatic digestion) and subsequently analyzed using the same capillary. The resulting automation of all steps such as injection, mixing, incubation, separation and detection minimizes the possible errors and saves experimental time. In this paper, we first study the differents parameters influencing PP cleavage inside the capillary (plug length, reactant concentration, incubation time, diffusion and electrophoretic plugs mixing). In a second part, the optimization of the electrophoretic separation conditions of generated hydrolysis products (nature, pH and ionic strength (I) of the background electrolyte (BGE)) is described. Using the optimized conditions, excellent repeatability was obtained in terms of separation (migration times) and proteolysis (number of products from enzymatic hydrolysis and corresponding amounts) demonstrating the robustness of the proposed methodology.

Modeling the electrophoresis of highly charged peptides: application to oligolysines.

The "coarse-grained" bead modeling methodology, BMM, is generalized to treat electrostatics at the level of the nonlinear Poisson-Boltzmann equation. This improvement makes it more applicable to the important class of highly charged macroions and highly charged peptides in particular. In the present study, the new nonlinear Poisson-Boltzmann, NLPB-BMM procedure is applied to the free solution electrophoretic mobility of low molecular mass oligolysines (degree of polymerization 1-8) in lithium phosphate buffer at pH 2.5. The ionic strength is varied from 0.01 to 0.10 M) and the temperature is varied from 25 to 50°C. In order to obtain quantitative agreement between modeling and experiment, a small amount of specific phosphate binding must be included in modeling. This binding is predicted to increase with increasing temperature and ionic strength.

Modeling the electrophoresis of oligolysines.

CE is used to measure the electrophoretic mobility of low molecular mass oligo-L-lysines (n=1-8) in aqueous LiH2PO4 buffer, BGE, at pH 2.5 over a range of temperatures (25-50 °C) and ionic strengths (10-100 mM). Mobilities are corrected for Joule heating and under the conditions of the experiment, interaction of the peptides with the capillary walls can be ignored. A "coarse grained" bead modeling methodology (BMM) (H. Pei et al., J. Chromatogr. A 2009, 1216, 1908-1916) is used to model the mobilities. This model partially accounts for peptide conformation as well as the assumed form of its secondary structure. For highly charged oligolysines, it is necessary to properly account for the relaxation effect. In the present study, the BMM approach tends to overestimate oligolysine mobility and that effect tends to increase with increasing ionic strength and peptide length. It is proposed that association between the oligolysines and buffer components (H2PO4⁻ in this case) that go beyond classical electrostatic interactions are responsible for this discrepancy. A simple binding model is introduced that illustrates how this association can reconcile model and experiment.

A rapid and reversible colorimetric assay for the characterization of aminated solid surfaces.

The covalent immobilization of synthetic or natural macromolecular compounds containing amino groups onto polystyrene (PS) solid surfaces is of great interest in diagnostic applications. A sensitive assay allowing the determination of reactive end groups is therefore a powerful tool for predicting the performance of the active surface. Recently, we reported the use of the Coomassie brilliant blue (CBB) colorimetric reagent to quantify protonated groups (N(+)) in linear and dendritic structures in solution (Coussot et al., Polym Int 58(5):511-518, 2009). In this work, a simple method using CBB dye for the characterization of PS aminated solid surfaces is developed. The proposed amino density estimation by colorimetric assay (ADECA) method is based on the reversible complexation of the dye with the N(+) groups on solid surfaces. The assay measures the released dye thanks to the use of a unique sodium carbonate-methanol buffer. Thereby, for the first time, the same surface can be used for characterization and for further coupling applications. A surface density of four N(+) groups per square nanometer can be measured in PS microwell format, the whole characterization being done within 30 min. Performances of this new colorimetric-based method are detailed. The ADECA method is further demonstrated to be useful for the characterization of aminated polypropylene and glass materials with various sizes and shapes.

In-capillary (electrophoretic) digestion-reduction-separation: A smart tool for middle-up analysis of mAb.

Comprehensive characterization of physicochemical properties of monoclonal antibodies (mAbs) is a critical process to ensure their quality, efficacy, and safety. For this purpose, mAb analysis at different levels (bottom-up, middle-up) is a common approach that includes rather complex multistep sample preparation (reduction, digestion). To ensure high analysis performance, the development of fully integrated methodologies is highly valuable. Capillary zone electrophoresis is a particularly well-adapted technique for the multistep implementation of analytical strategies from sample preparation to detection. This feature was employed to develop novel integrated methodologies for the analysis of mAb at the middle-up level. Multiple in-line reactions (simultaneous reduction and digestion) were performed for the first time in the separation capillary. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was used as an effective reducing agent under a broad pH range and IdeS (Immunoglobulin degrading enzyme from Streptococcus) as a highly specific enzyme for mAb digestion. Transverse diffusion of laminar flow profile (TDLFP) was applied for reactants mixing. Both in-line sample preparation and separation parameters were optimized under non-denaturing and denaturing conditions. The developed in-line methodologies provided good reproducibility and higher peak efficiencies comparing with off-line assays. They were successfully applied to different mAbs.

A fully automated on-line salting-out assisted liquid-liquid extraction capillary electrophoresis methodology: Application to tyrosine kinase inhibitors in human plasma.

A fully automated analytical methodology combining salting-out assisted liquid-liquid extraction (SALLE) and capillary electrophoresis (CE) for the analysis of three Tyrosine Kinase Inhibitors (TKIs) in plasma samples is proposed. The automated methodology, called A-SALLE-CE-UV, makes full use of the advantages of both techniques by combining desalting, protein precipitation, automated liquid-liquid extraction, in-line CE stacking and electrophoretic separation of analytes in plasma samples in a fully integrated way. At first, the capillary is used to deliver appropriate micro-volumes of extraction agent solutions (acetonitrile, salt) in the plasma sample. ACN and salting-out agent (NaCl) solutions are added by pressure from outlet vials into the sample vial (inlet) containing human plasma sample spiked with the three tested TKIs. After addition of both ACN and NaCl solutions, mixing is achieved by generating air bubbles leading to a two phases separation and extraction of TKIs in the upper mostly organic phase (ACN). The upper phase containing the TKIs is then injected and analysed by CE-UV. Due to the presence of ACN, the analytes are stacked in-line and successfully separated in the same capillary. The results obtained in terms of limit of detection (LOD), limit of quantification (LOQ), sensitivity enhancement factor (SEF), repeatability and linearity demonstrate the applicability of the proposed method for possible therapeutic drug monitoring (TDM) of TKIs.

Liquid chromatography-tandem mass spectrometric assay for the quantification of CDK4/6 inhibitors in human plasma in a clinical context of drug-drug interaction.

The CDK4/6 inhibitors palbociclib and ribociclib are kinase inhibitors used in association with hormonal therapy for the management of patients with metastatic breast cancer. Like most kinase inhibitors, therapeutic drug monitoring may be used for personalize their dosage. To this aim, we developed and validated a sensitive and specific HPLC-MS/MS method for palbociclib and ribociclib quantification in blood samples. We then quantified exposure to palbociclib (plasma trough concentration; Ctrough) in a real-life cohort of patients with locally invasive or metastatic breast cancer (n = 18) at day 15 of the first cycle of palbociclib treatment to characterize palbociclib concentration at steady state (Clinicaltrials.gov identifier NCT04025541, IdRCB n° 2018-A00064-51, 03/07/2018). The geometric mean (± standard deviation [min-max]) of palbociclib plasma Ctrough was 88.58 ng/mL (± 26.4 [46.5 ng/mL - 133 ng/mL]) at day 15. Some covariates, such as drug-drug interactions, could explain the concentration variations observed in our Caucasian cohort. These first results in real-life settings obtained with our HPLC-MS/MS method give important information on palbociclib monitoring and pharmacokinetic variability.

Influence of polyelectrolyte capillary coating conditions on protein analysis in CE.

CE of biomolecules is limited by analyte adsorption on the capillary wall. To prevent this, monolayer or successive multiple ionic-polymer layers (SMILs) of highly charged polyelectrolytes can be physically adsorbed on the inner capillary surface. Although these coatings have become commonly used in CE, no systematic investigation of their performance under different coating conditions has been carried out so far. In a previous study (Nehmé, R., Perrin, C., Cottet, H., Blanchin, M. D., Fabre, H., Electrophoresis 2008, 29, 3013-3023), we investigated the influence of different experimental parameters on coating stability, repeatability and peptide peak efficiency. Optimal coating conditions for monolayer and multilayer (SMILs) poly(diallyldimethylammonium) chloride/ poly(sodium 4-styrenesulfonate) coated capillaries were determined. In this study, the influence of polyelectrolyte concentration and ionic strength of the coating solutions, and the number of coating layers on coating stability and performance in limiting protein adsorption was carried out. EOF magnitude and repeatability were used to monitor coating stability. Coating ability to limit protein adsorption was investigated by monitoring variations of migration times, time-corrected peak areas and separation efficiency of test proteins. The separation performance of polyelectrolyte coatings were compared with those obtained with bare silica capillaries.

Use of coated capillaries for the electrophoretic separation of stereoisomers of a growth hormone secretagogue.

The diastereoisomeric separation of peptidomimetics of hexarelin, a strong growth hormone secretagogue, in CE has been studied. Highly sulfated-gamma-CD was found to be an appropriate selector for the separation of the stereoisomers. However, non-repeatable analyses were obtained on bare fused silica capillary due to the progressive adsorption of the analytes on the capillary wall. Two types of polyelectrolyte coating agents were tested to prevent this phenomenon. Coating with neutral polyethylene oxide was found to be efficient but resulted in a very long analysis time (about 40 min). Coating with cationic poly(diallyldimethylammonium) chloride was found both to prevent analyte adsorption, reduce analysis time and alter separation selectivity. EOF measurement revealed that the highly sulfated-gamma-CDs were strongly adsorbed on the poly(diallyldimethylammonium) chloride coating surface yielding a stable strong cathodic EOF, which considerably reduced analysis time (about 12 min). Very good repeatability of analysis was obtained (RSD(migration time)<1%).

Effect of the consumption of a fermented dairy product containing Bifidobacterium lactis DN-173 010 on constipation in childhood: a multicentre randomised controlled trial (NTRTC: 1571).

BACKGROUND: Constipation is a frustrating symptom affecting 3% of children worldwide. Randomised controlled trials show that both polyethylene glycol and lactulose are effective in increasing defecation frequency in children with constipation. However, in 30-50%, these children reported abdominal pain, bloating, flatulence, diarrhoea, nausea and bad taste of the medication. Two recent studies have shown that the fermented dairy product containing Bifidobacterium lactis strain DN-173 010 is effective in increasing stool frequency in constipation-predominant irritable bowel syndrome patients with a defecation frequency < 3/week and in constipated women with a defecation frequency < 3/week. Goal of this study is to determine whether this fermented dairy product is effective in the treatment of constipated children with a defecation frequency < 3/week. METHODS/DESIGN: It is a two nation (The Netherlands and Poland) double-blind, placebo-controlled randomised multicentre trial in which 160 constipated children (age 3-16 years) with a defecation frequency <3/week will be randomly allocated to consume a fermented dairy product containing Bifidobacterium lactis DN-173 010 or a control product, twice a day, for 3 weeks. During the study all children are instructed to try to defecate on the toilet for 5-10 minutes after each meal (3 times a day) and daily complete a standardized bowel diary. Primary endpoint is stool frequency. Secondary endpoints are stool consistency, faecal incontinence frequency, pain during defecation, digestive symptoms (abdominal pain, flatulence), adverse effects (nausea, diarrhoea, bad taste) and intake of rescue medication (Bisacodyl). Rate of success and rate of responders are also evaluated, with success defined as > or = 3 bowel movements per week and < or =1 faecal incontinence episode over the last 2 weeks of product consumption and responder defined as a subject reporting a stool frequency > or = 3 on the last week of product consumption. To demonstrate that the success percentage in the intervention group will be 35% and the success percentage in the control group (acidified milk without ferments, toilet training, bowel diary) will be 15%, with alpha 0.05 and power 80%, a total sample size of 160 patients was calculated. CONCLUSION: This study is aimed to show that the fermented dairy product containing Bifidobacterium lactis strain DN-173 010 is effective in increasing stool frequency after 3 weeks of product consumption in children with functional constipation and a defecation frequency < 3/week.

In-line tryptic digestion of therapeutic molecules by capillary electrophoresis with temperature control.

The main purpose of the present work is to provide a fully integrated temperature control of in-line tryptic digestion in order to facilitate the quality control of polypeptidic therapeutic compounds. The in-line enzymatic reaction was performed in 100 mM bicarbonate ammonium whereas a mixture of citric acid/ε-amino caproic acid (pH 5.0 and I 75 mM) was used as a background electrolyte (BGE). After the injection of all reactants (substrate, enzyme, proteolysis buffer), a BGE plug was injected to push all reactants until a position where the capillary is thermostated. Then, the enzymatic reaction was initiated during 15 min of incubation time and finally, a voltage was applied to separate the generated proteolysis products. The methodology was developped regarding the effects of the BGE plug length and pressure on the reactants plug mixing and on the advanced of the tryptic digestion. Successful temperature control of in-line proteolysis with excellent repeatability was obtained in optimal cleavage and separation conditions.

Coupling of salting-out assisted liquid-liquid extraction with on-line stacking for the analysis of tyrosine kinase inhibitors in human plasma by capillary zone electrophoresis.

Developing an easy to use, cheap and fast analytical methodology is highly demanded for clinical practices, such as therapeutic drug monitoring (TDM). The present work deals with the development of an analytical methodology for the analysis of four basic anticancer drugs, namely tyrosine kinase inhibitors (TKIs), in human plasma by combining salting-out assisted liquid-liquid extraction (SALLE) with capillary electrophoresis (CE). This SALLE-CE methodology makes a full use of the advantages of both techniques by combining extraction, on-line concentration and separation in a simple way. First, plasma samples containing TKIs are mixed with acetonitrile (ACN) in appropriate volumes to precipitate proteins. After vortexing and centrifugation, sodium chloride (NaCl) is added to the plasma-ACN mixture to induce a two phases separation. TKIs are efficiently extracted (60-100% extraction efficiency) in the upper (mostly organic) phase which is directly analyzed by capillary electrophoresis (CE) coupled to UV detection. The high content of ACN in the upper phase allows the stacking of the analytes in the capillary (on-line stacking) during analysis. For the first time thanks to this electrophoretic process, the injected sample volume can be as large as 80% of the capillary volume (till the detector window). Good linearity was obtained for each TKI in the concentration range 60-2000 ng/ml with correlation coefficient (r²) between 0.997 and 0.999. LOD and LOQ in human plasma with such large injected volume were determined from 16 to 280 ng/ml and from 62 to 900 ng/ml respectively depending on the TKI. Recoveries for the four TKIs ranged from 60 to 100%. The repeatability of the SALLE-CE methodology for the analysis of TKIs in human plasma was evaluated with injected sample volume equal to 80% of the capillary volume till detector window. Relative standard deviations (RSDs) of less than 1.24 and 2.84% on migration times and corrected peak areas respectively were obtained at the LOQ. The sensitivity was enhanced by 61 to 265 folds confirming the applicability of the proposed methodology for the assay of TKIs in patients' plasma.

Integrated microreactor for enzymatic reaction automation: An easy step toward the quality control of monoclonal antibodies.

The main purpose of the present work is to provide a fully integrated miniaturized electrophoretic methodology in order to facilitate the quality control of monoclonal antibodies (mAbs). This methodology called D-PES, which stands for Diffusion-mediated Proteolysis combined with an Electrophoretic Separation, permits to perform subsequently mAb tryptic digestion and electrophoresis separation of proteolysis products in an automated manner. Tryptic digestion conditions were optimized regarding the influence of enzyme concentration and incubation time in order to achieve similar enzymatic digestion efficiency to that obtained with the classical methodology (off-line). Then, the optimization of electrophoretic separation conditions concerning the nature of background electrolyte (BGE), ionic strength and pH was realized. Successful and repeatable electrophoretic profiles of three mAbs digests (Trastuzumab, Infliximab and Tocilizumab), comparable to the off-line digestion profiles, were obtained demonstrating the feasibility and robustness of the proposed methodology. In summary, the use of the proposed and optimized in-line approach opens a new, fast and easy way for the quality control of mAbs.