A comprehensive investigation of the interactions of human serum albumin with polymeric and hybrid nanoparticles.

Nanoparticles (NPs) engineered as drug delivery systems continue to make breakthroughs as they offer numerous advantages over free therapeutics. However, the poor understanding of the interplay between the NPs and biomolecules, especially blood proteins, obstructs NP translation to clinics. Nano-bio interactions determine the NPs' in vivo fate, efficacy and immunotoxicity, potentially altering protein function. To fulfill the growing need to investigate nano-bio interactions, this study provides a systematic understanding of two key aspects: (i) protein corona (PC) formation and (ii) NP-induced modifications on protein's structure and stability. A methodology was developed by combining orthogonal techniques to analyze both quantitative and qualitative aspects of nano-bio interactions, using human serum albumin (HSA) as a model protein. Protein quantification via liquid chromatography-mass spectrometry, and capillary zone electrophoresis (CZE) clarified adsorbed protein quantity and stability. CZE further unveiled qualitative insights into HSA forms (native, glycated HSA and cysteinylated), while synchrotron radiation circular dichroism enabled analyzing HSA's secondary structure and thermal stability. Comparative investigations of NP cores (organic vs. hybrid), and shells (with or without polyethylene glycol (PEG)) revealed pivotal factors influencing nano-bio interactions. Polymeric NPs based on poly(lactic-co-glycolic acid) (PLGA) and hybrid NPs based on metal-organic frameworks (nanoMOFs) presented distinct HSA adsorption profiles. PLGA NPs had protein-repelling properties while inducing structural modifications on HSA. In contrast, HSA exhibited a high affinity for nanoMOFs forming a PC altering thereby the protein structure. A shielding effect was gained through PEGylation for both types of NPs, avoiding the PC formation as well as the alteration of unbound HSA structure.

Infliximab aggregates produced in severe and mild elevated temperature stress conditions induce an extended specific CD4 T-cell response.

Aggregation has been widely described as a factor contributing to therapeutic antibody immunogenicity. Although production of high-affinity anti-drug antibodies depends on the activation of CD4 T lymphocytes, little is known about the T-cell response induced by antibody aggregates, especially for aggregates produced in mild conditions resulting from minor handling errors of vials. Large insoluble infliximab (IFX) aggregates produced in severe elevated temperature stress conditions have been previously shown to induce human monocyte-derived dendritic cell (moDC) maturation. We here showed that large IFX aggregates recruit in vitro a significantly higher number of CD4 T-cells compared to native IFX. Moreover, a larger array of T-cell epitopes encompassing the entire variable regions was evidenced compared to the native antibody. We then compared the responses of moDCs to different types of aggregates generated by submitting IFX to mild conditions of various times of incubation at an elevated temperature. Decreasing stress duration reduced aggregate size and quantity, and subsequently altered moDC activation. Of importance, IFX aggregates generated in mild conditions and not altering moDC phenotype generated an in vitro T-cell response with a higher frequency of CD4 T cells compared to native IFX. Moreover, cross-reactivity studies of aggregate-specific T cells showed that some T cells could recognize both native and aggregated IFX, while others responded only to IFX aggregates. Taken together, our results suggest that aggregation of antibodies in mild elevated temperature stress conditions is sufficient to alter moDC phenotype in a dose-dependent manner and to increase T-cell response.

Imaged capillary isoelectric focusing associated with multivariate analysis: A powerful tool for quality control of therapeutic monoclonal antibodies.

Monoclonal antibodies are increasingly used in cancer therapy. To guarantee the quality of these mAbs from compounding to patient administration, characterization methods are required (e.g. identity). In a clinical setting, these methods must be fast and straightforward. For this reason, we investigated the potential of image capillary isoelectric focusing (icIEF) combined with Principal Component Analysis (PCA) and Partial least squares-discriminant analysis (PLS-DA). icIEF profiles obtained from monoclonals antibodies (mAbs) analysis have been pre-processed and the data submitted to principal component analysis (PCA). This pre-processing method has been designed to avoid the impact of concentration and formulation. Analysis of four commercialized mAbs (Infliximab, Nivolumab, Pertuzumab, and Adalimumab) by icIEF-PCA led to the formation of four clusters corresponding to each mAb. Partial least squares-discriminant analysis (PLS-DA) applied to these data allowed us to build models to predict which monoclonal antibody is analyzed. The validation of this model was obtained from k-fold cross-validation and prediction tests. The selectivity and the specificity of the model performance parameters were assessed by the excellent classification obtained. In conclusion, we established that the combination of icIEF and chemometric approaches is a reliable approach for unambiguously identifying compounded therapeutic monoclonal antibodies (mAbs) before patient administration.

On-line dual-stage enrichment via magneto-extraction and electrokinetic preconcentration: A new concept and instrumentation for capillary electrophoresis.

This study reports on the development of a new concept of on-line dual preconcentration stages for capillary electrophoresis (CE), in which two completely different preconcentration approaches can be realized in the same capillary. In the first stage, a dynamic magneto-extraction of target analytes on circulating magnetic beads is implemented within the capillary. In the second one, electrokinetic preconcentration of eluted analytes via large volume sample stacking is carried out to focus them into a nano band, prior to CE separation of enriched analytes. To implement the dual-stage preconcentration operation, a purpose-made instrument was designed, combining electrophoretic and microfluidic modules to allow precise control of the movement of magnetic beads and analyte's flow. The potential of this new enrichment principle and its associated instrument was demonstrated for CE separation with light-emitting-diode-induced fluorescent (LEDIF) detection of target double-stranded DNA (ds-DNA). The workflow consists of purification and preconcentration of a target DNA fragment (300 bp) on negatively charged magnetic beads, followed by in-capillary elution and fluorescent labelling of the enriched DNA. Large volume sample stacking of the DNA eluent was then triggered to further preconcentrate the labelled DNA before its analysis by CE-LEDIF. An enrichment factor of 125 was achieved for the target DNA fragment. With our new approach, dual-stage sample pretreatment and CE separation can now be performed in-capillary without any mismatch of working volumes, nor any waste of pretreated samples.

Analytical methods of antibody surface coverage and orientation on bio-functionalized magnetic beads: application to immunocapture of TNF-α.

The use of magnetic beads bio-functionalized by antibodies (Ab) is constantly increasing with a wide range of biomedical applications. However, despite an urgent need for current methods to monitor Ab's grafting process and orientation, existing methods are still either cumbersome and/or limited. In this work, we propose a new simple and rapid analytical approach to evaluate antibody orientation and density on magnetic beads. This approach relies on the cleavage by IdeS, a highly specific protease for human immunoglobulin G (hIgG), of immobilized antibodies. The F(ab)2 and Fc fragments could be then accurately quantified by size exclusion chromatography (SEC)-coupled to fluorescent detection (FLD), and the ratio of these fragments was used to give insight on the IgG orientation at the bead surface. Four different commercially available magnetic beads, bearing carboxyl groups, tosyl groups, streptavidin, or protein G on their surface have been used in this study. Results obtained showed that this approach ensures reliable information on hIgG orientation and bead surface coverage. Protein G magnetic beads demonstrated an optimal orientation of antibodies for antigen capture (75% of accessible F(ab)2 fragment) compared to tosylactivated, carboxylated, and streptavidin ones. Capture efficiency of the different functionalized beads towards human TNF-α immunocapture, a biomarker of inflammation, has been also compared. Protein G beads provided a more efficient capture compared to other beads. In the future, this approach could be applied to any type of surface and beads to assess hIgG coverage and orientation after any type of immobilization. A rapid and simple approach to evaluate orientation and density of antibodies immobilized on magnetic beads.

Electroosmotic flow modulation for improved electrokinetic preconcentration: Application to capillary electrophoresis of fluorescent magnetic nanoparticles.

It is reported in this study a new approach for modulation and even suppression of the electroosmotic flow (EOF) to achieve better electrokinetic preconcentration in capillary electrophoresis. This is based on the augmentation of the buffer's concentrations to very high levels (more than a thousand of mM) without recourse to any dynamic/permanent coating nor viscous gel. The use of large weakly charged molecules as background electrolyte's constituents allows working at extreme concentration ranges without penalty of high electric currents and Joule heating. By this way, the electroosmotic mobility could be modulated over a wide range (2-60 × 10-5 cm2 V-1 s-1 under alkaline conditions), and suppressed to levels equivalent to those obtained with several neutral coatings. The highest buffer concentrations, and the lowest EOF magnitudes, accordingly, were achieved with diethanolamine/3-(Cyclohexylamino)-1-propanesulfonic acid (ionic strength (IS) of 250 mM, pH 9.5), Tris(hydroxymethyl)aminomethane (Tris)/2-(Cyclohexylamino)ethanesulfonic acid (CHES) (IS of 280 mM, pH 8.7) and triethanolamine/2-(Cyclohexylamino)ethanesulfonic acid (IS of 250 mM, pH 8.5). For demonstration, this new approach was applied for sensitive determination of core-shell magnetic nanoparticles (CSMNPs) having high potential for healthcare applications such as imaging agents for diagnostics and controllable cargos for nanomedicine. Different profiles were achieved for purpose-made and commercial magnetic nanoparticles using CE coupled with light-emitting-diode induced fluorescence (LEDIF) detection. The best performance for EOF-assisted preconcentration and CE-LEDIF of CSMNPs was achieved with these nanoparticles prepared in TRIS/CHES (IS 10 mM, pH 8.4) for preconcentration, and separation under BGE of TRIS/CHES (IS 100 mM, pH 8.4). Compared to the conventional capillary electrophoresis (CE-UV) method for characterization of magnetic nanoparticles, our proposed approach with fluorescent detection and EOF-assisted preconcentration offers almost 350-fold sensitivity improvement. Furthermore, our scheme can be used for monitoring the interaction between CSMNPs and target pharmaceutical molecules, serving for drug delivery development. A preliminary study with two antibiotics using this approach revealed that kanamycin interacts better with the target nanoparticles than amikacin.

Recent Electrokinetic and Microfluidic Strategies for Detection of Amyloid Beta Peptide Biomarkers: Towards Molecular Diagnosis of Alzheimer's Disease.

Among all neurodegenerative diseases, Alzheimer's Disease (AD) is the most prevalent worldwide, with a huge burden to the society and no efficient AD treatment so far. Continued efforts have been being made towards early and powerful diagnosis of AD, in the hope for a successful set of clinical trials and subsequently AD curative treatment. Towards this aim, detection and quantification of amyloid beta (Aß) peptides in cerebrospinal fluid (CSF) and other biofluids, which are established and validated biomarkers for AD, have drawn attention of the scientific community and industry over almost two decades. In this work, an overview on our major contributions over 15 years to develop different electrokinetic and microfluidic strategies for Aß peptides detection and quantification is reported. Accordingly, discussions and viewpoints on instrumental and methodological developments for microscale electrophoresis, microfluidic designs and immuno-enrichment / assays on magnetic beads in microchannels for tracing Aß peptides in CSF are given in this review.

Surface Biochemical Modification of Poly(dimethylsiloxane) for Specific Immune Cytokine Response.

Recent evidence suggests that proinflammatory cytokines, such as tumor necrosis factor α (TNF-α), play a pivotal role in the development of inflammatory-related pathologies (covid-19, depressive disorders, sepsis, cancer, etc.,). More importantly, the development of TNF-α biosensors applied to biological fluids (e.g. sweat) could offer non-invasive solutions for the continuous monitoring of these disorders, in particular, polydimethylsiloxane (PDMS)-based biosensors. We have therefore investigated the biofunctionalization of PDMS surfaces using a silanization reaction with 3-aminopropyltriethoxysilane, for the development of a human TNF-α biosensor. The silanization conditions for 50 µm PDMS surfaces were extensively studied by using water contact angle measurements, electron dispersive X-ray and Fourier transform infrared spectroscopies, and fluorescamine detection. Evaluation of the wettability of the silanized surfaces and the Si/C ratio pointed out to the optimal silanization conditions supporting the formation of a stable and reproducible aminosilane layer, necessary for further bioconjugation. An ELISA-type immunoassay was then successfully performed for the detection and quantification of human TNF-α through fluorescent microscopy, reaching a limit of detection of 0.55 µg/mL (31.6 nM). Finally, this study reports for the first time a promising method for the development of PDMS-based biosensors for the detection of TNF-α, using a quick, stable, and simple biofunctionalization process.

The FcγRIIa-Syk Axis Controls Human Dendritic Cell Activation and T Cell Response Induced by Infliximab Aggregates.

The development of anti-drug Abs in response to biological products (BP) is a major drawback in the treatment of patients. Factors related to the patient, the treatment, and the product can influence BP immunogenicity. Among these factors, BP aggregates have been suggested to promote immunogenicity by acting as danger signals recognized by dendritic cells (DC) facilitating the establishment of an anti-BP CD4 T cell-dependent adaptive immune response leading to anti-drug Abs production. To date, little is known on the mechanism supporting the effect of aggregates on DCs and consequently on the T cell response. The aim of this work was to identify key signaling pathways involved in BP aggregate DC activation and T cell response. We generated aggregates by submitting infliximab (IFX), an immunogenic anti-TNF-α chimeric Ab, to heat stress. Our results showed that IFX aggregates were able to induce human monocyte-derived DC (moDC) maturation in a concentration-dependent manner. Aggregate-treated moDCs enhanced allogeneic T cell proliferation and IL-5, IL-9, and IL-13 production compared with native Ab-treated moDCs. We then investigated the implication of FcγRIIa and spleen tyrosine kinase (Syk) in DC activation and showed that they were both strongly implicated in moDC maturation induced by IFX aggregates. Indeed, we found that neutralization of FcγRIIa inhibited DC activation, and consequently, Syk inhibition led to a decrease in T cell proliferation and cytokine production in response to IFX aggregates. Taken together, our results bring new insight, to our knowledge, on how protein aggregates could induce DC and T cell activation via the FcγRIIa-Syk signaling pathway.

Conformation assessment of therapeutic monoclonal antibodies by SEC-MS: Unravelling analytical biases for application to quality control.

Immunogenicity related to the degradation of therapeutic monoclonal antibodies (mAbs) remains a major concern for their therapeutic efficacy and safety. Therefore, an analytical method allowing characterization and detection of mAbs degradation is mandatory. In this study, a simultaneous coupling of size exclusion chromatography (SEC) to native mass spectrometry (MS) and fluorescence detection (FLD) is proposed to detect degraded therapeutic mAbs and biases of structural changes (e.g. dimerization, denaturation) that may occur during native MS. A comprehensive study on infliximab behaviors have been performed under different mobile phase conditions (e.g. composition, pH, organic solvent, etc.) and MS parameters (e.g. gas temperatures, CID energies, etc.). Experimental conditions avoiding artificial denaturation and/ or dimerization have been defined. We have also demonstrated that under the developed conditions infliximab affinity towards its biological target TNFα is preserved. In addition, using this method dimers, denatured monomers and fragments could be detected in trastuzmab samples stressed by a long-term storage. These results were confirmed by using SEC coupled to ion mobility mass spectrometry as an orthogonal method for the detection of denatured monomer.

Capillary zone electrophoresis-native mass spectrometry for the quality control of intact therapeutic monoclonal antibodies.

Therapeutic monoclonal antibodies (mAbs) are complex glycoproteins and ensuring their safety, efficacy and quality is still challenging. Indeed, during their manufacturing process, they are exposed to several stresses that can lead to their denaturation, misfolding or dimerization. We report here a new method based on capillary electrophoresis coupled to native mass spectrometry (MS) with a sheath liquid interface to analyze an intact therapeutic mAb, Infliximab, under non-denaturing conditions that preserve its conformational heterogeneity as well as self-association without inducing further unfolding / denaturation. For capillary zone electrophoresis (CZE) separation, a triple layer coating using polybrene-dextran sulfate-polybrene was employed. A sheath liquid composed of isopropanol - water - acetic acid with a flow rate of 10 µL min-1 and mild MS conditions allowed optimal signal intensities. A specific mass spectrum was obtained for each Infliximab conformation in a "stressed" formulated preparation. This is the first time that within a single analysis different conformational states, i.e. native and unfolded monomers as well as dimers are simultaneously detected. The results and the lack of analytical bias arising from the CZE-MS conditions were confirmed by using atomic force microscopy (AFM) as an orthogonal technique. A middle-up approach combined to CZE-MS analysis of the stressed samples suggested that the dimer formation involved mostly Fab-Fab interactions.

In-capillary immuno-preconcentration with circulating bio-functionalized magnetic beads for capillary electrophoresis.

This study reports on the conception of magneto-Capillary Electrophoresis (magneto-CE), an approach integrating immuno-capture on circulating bio-functionalized magnetic beads into a unique capillary for preconcentration and electrokinetic separation. This hybrid mode is an evolution of in-capillary magnetic bead-based operation from static cluster format to dynamic configuration where beads are allowed to controllably circulate inside a CE capillary for interaction improvement. To implement the magneto-CE operation, a purpose-made instrument was constructed, allowing visual observation of the movement of the magnetic beads. We applied a new methodological strategy for determination of the amyloid ß peptide (Aß 1-42), which is as an established biomarker for molecular diagnosis of Alzheimer's disease (AD). The methodology is based on magneto-immuno-capture of fluorescently labeled Aß 1-42 followed by a chemical elution with a basic solution prior to CE separation with laser induced fluorescent (LIF) detection. The superiority of this dynamic configuration of magneto-CE was demonstrated for this target analyte, with sample pretreatment and separation being performed in-capillary without any delay in between and without any waste of pretreated sample, which otherwise would not be the case with offline/batch-wise operation.

On-a-chip tryptic digestion of transthyretin: a step toward an integrated microfluidic system for the follow-up of familial transthyretin amyloidosis.

A microfluidic microreactor for trypsin mediated transthyretin (TTR) digestion has been developed as a step towards the elaboration of a fully integrated microdevice for the detection of a rare and disabling disease, the familial transthyretin amyloidosis (ATTR) which is related to specific TTR mutations. Therefore, an enzymatic microreactor coupled to an analytical step able to monitor the mutation of TTR on specific peptide fragments would allow an accurate monitoring of the treatment efficiency of ATTR. In this study, two types of immobilized trypsin microreactors have been investigated: a new miniaturized, microfluidic fluidized bed packed with trypsin functionalized magnetic particles (MPs), and a thiol-ene (TE) monolith-based chip. Their performances were first demonstrated with N-benzoyl-dl-arginine-4-nitroanilide hydrochloride BApNA, a low molecular weight substrate. High reaction yields (75.2%) have been reached within 0.6 min for the TE-based trypsin microreactor, while a lower yield (12.4%) was obtained for the micro-fluidized bed within a similar residence time. Transposition of the optimized conditions, developed with BApNA, to TTR digestion in the TE-based trypsin microreactor was successfully performed. We demonstrated that the TE-chip can achieve an efficient and reproducible digestion of TTR. This has been assessed by MS detection. In addition, TTR hydrolysis led to the production of a fragment of interest allowing the therapeutic follow-up of more than twenty possible ATTR mutations. High sequence coverage (90%), similar to those obtained with free trypsin, was achieved in a short time (2.4 min). Repeated experiments showed good reproducibility (RSD = 6.8%). These promising results open up the route for an innovative treatment follow-up dedicated to ATTR.

Hydrophilic interaction liquid chromatography for dalargin separation from its structural analogues and side products.

Retention behaviour of Dalargin and five peptide analogues of Leu-enkephalin, has been extensively studied by hydrophilic interaction liquid chromatography (HILIC) on a bare silica stationary phase (Atlantis® HILIC silica). The influence of buffer pH, ionic strength, and organic modifier content on peptide retentions was examined. Variation of organic modifier content (70-90% ACN) shows that, as expected, the most polar peptide, Dalargin, is the most retained. Moreover, at acidic pH, the retention mechanism for all the peptides studied seems to rely, mainly, on adsorption phenomenon. By varying the pswH buffer (between 4.4-7.5), we observed that the retention of all the peptides was mainly governed by their total number of charges, whatever the variation (increase or decrease) of their retention factor. At pswH 7.5, an increase of the cationic counter-ion concentration (NH4+) lead to a decrease of the retention factor of Dalargin, suggesting a weak cation exchange for this peptide. For the other peptides, the variation of the retention factors was negligible between 5-15mM. Above 15mM, the retention factors of all the peptides increased, probably due to an increase of the water layer thickness at the surface of the stationary phase. In the second part of the study, qualitative analysis of non-purified dalargin, resulting from solid-phase synthesis, was realized. Optimisation of the separation of the target peptide from its side products has been first performed with UV detection. Then, by coupling the HILIC column with ESI-MS, using the optimal separation conditions, it was possible to identify Dalargin and to propose the amino-acids sequence of its side-products.

Solid supports for extraction and preconcentration of proteins and peptides in microfluidic devices: A review.

Determination of proteins and peptides is among the main challenges of today's bioanalytical chemistry. The application of microchip technology in this field is an exhaustively developed concept that aims to create integrated and fully automated analytical devices able to quantify or detect one or several proteins from a complex matrix. Selective extraction and preconcentration of targeted proteins and peptides especially from biological fluids is of the highest importance for a successful realization of these microsystems. Incorporation of solid structures or supports is a convenient solution employed to face these demands. This review presents a critical view on the latest achievements in sample processing techniques for protein determination using solid supports in microfluidics. The study covers the period from 2006 to 2015 and focuses mainly on the strategies based on microbeads, monolithic materials and membranes. Less common approaches are also briefly discussed. The reviewed literature suggests future trends which are discussed in the concluding remarks.

Dyneon THV, a fluorinated thermoplastic as a novel material for microchip capillary electrophoresis.

In this work, we have investigated Dyneon THV, a fluorinated material, as a new material to afford electrokinetic separations in microfluidic devices. To overcome protein adsorption, two poly(ethylene oxide) (PEO)-based coatings have been investigated: Pluronic F127 and PEO stearate 40. The best results were obtained with the PEO stearate 40 coating which allowed decreasing the surface contact angle from 91 ± 3 to 76°± 3. With this surface treatment, a 66% reduction of the electroosmotic mobility at pH 8.0 and a marked suppression of protein adsorption were observed compared to a native Dyneon THV microchip. Finally, a separation of fluorescently labeled proteins (bovine serum albumin and trypsin inhibitor), well-known for their strong tendency to adsorb on hydrophobic surfaces, was successfully achieved in an HEPES buffer with a PEO stearate 40 treated microchip by capillary zone electrophoresis. Furthermore, we demonstrated the possibility to perform non-aqueous capillary electrophoresis analysis of hydrophobic dyes using various solvents in untreated microchips. The overall results demonstrated not only the suitability of the Dyneon THV microchip for electrokinetic separations, but also its versatility allowing different separation modes to be implemented with the same microchip material.

Quality Control of Therapeutic Monoclonal Antibodies at the Hospital After Their Compounding and Before Their Administration to Patients.

Monoclonal antibodies (mAbs) are widely used in cancer therapy and recently many new mAbs have gained EMA and FDA approvals for oncology indications. Here we describe a highly reproducible CZE method, relying on a cationic coating allowing separation and identification of a complex mixture of four compounded mAbs widely used in cancer therapy (cetuximab, rituximab, bevacizumab, and trastuzumab).

High-throughput identification of monoclonal antibodies after compounding by UV spectroscopy coupled to chemometrics analysis.

Monoclonal antibodies (mAbs) compounded into the hospital pharmacy are widely used nowadays. Their fast identification after compounding and just before administration to the patient is of paramount importance for quality control at the hospital. This remains challenging due to the high similarity of the structure between mAbs. Analysis of the ultraviolet spectral data of four monoclonal antibodies (cetuximab, rituximab, bevacizumab, and trastuzumab) using unsupervised principal component analysis led us to focus exclusively on the second-derivative spectra. Partial least squares-discriminant analysis (PLS-DA) applied to these data allowed us to build models for predicting which monoclonal antibody was present in a given infusion bag. The calibration of the models was obtained from a k-fold validation. A prediction set from another batch was used to demonstrate the ability of the models to predict well. PLS-DA models performed on the spectra of the region of aromatic amino acid residues presented high ability to predict mAb identity. The region corresponding to the tyrosine residue reached the highest score of good classification with 89 %. To improve the score, standard normal variate (SNV) preprocessing was applied to the spectral data. The quality of the optimized PLS-DA models was enhanced and the region from the tyrosine/tryptophan residues allowed us excellent classification (100 %) of the four mAbs according to the matrix of confusion. The sensitivity and specificity performance parameters assessed this excellent classification. The usefulness of the combination of UV second-derivative spectroscopy to multivariate analysis with SNV preprocessing demonstrated the unambiguous identification of commercially available monoclonal antibodies. Graphical abstract PLS-DA models on the spectra of the region of aromatic amino acid residues allows mAb identification with high prediction.

Magneto-immunocapture with on-bead fluorescent labeling of amyloid-ß peptides: towards a microfluidized-bed-based operation.

A new sample treatment approach for sensitive determination of three amyloid-ß peptides (Aß 1-42, Aß 1-40 and Aß 1-38) with capillary electrophoresis coupled with laser induced fluorescent detection is reported herein. These Aß peptides are considered an important family of biomarkers in the cerebrospinal fluid (CSF) for early diagnosis of Alzheimer's disease (AD). Due to their extremely low abundance in CSF (down to sub nM ranges), batch-wise preconcentration via magneto-immunocapture with enrichment factors up to 100 was implemented. The Aß peptides were first captured onto magnetic micro-beads. Then, on-beads fluorescent labeling of the captured Aß peptides were carried out to avoid the unwanted presence of extra fluorescent dye in the eluent as in the case of in-solution labeling. Finally thermal elution was performed and eluted labeled peptides were analyzed off line with CE-LIF. The Aß-capturing efficiencies of different commercially available antibodies grafted onto magnetic beads were tested. Aß peptides in CSF samples collected from AD's patients and healthy persons (used as controls) were measured and evaluated. As a proof of concept, the developed strategy was adapted into a miniaturized fluidized bed configuration that has the potential for coupling with a microchip separation system.

Capillary electrophoresis for rapid identification of monoclonal antibodies for routine application in hospital.

mAbs are widely used in cancer therapy. Their compounding, performed just before their administration to patients, is executed in a production unit of the hospital. Identification of these drugs, individually prepared in bags for infusion before patient administration, is of paramount importance to detect potential mistakes during compounding stage. A fast and reliable analytical method based on CZE combined to a cationic capillary coating (hexadimethrine bromide) was developed for identification of the most widely used compounded therapeutic for cancer therapy (bevacizumab, cetuximab, rituximab, and trastuzumab). Considering the high structural and physico-chemical similarities of these mAbs, an extensive optimization of the BGE composition has been performed. The addition of perchlorate ions and polysorbate in the BGE greatly increased the resolution. To validate the method, an internal standard was used and the relative migration times (RTm) were estimated. Very satisfactory RSDs of the RTm for rituximab (0.76%), cetuximab (0.46%), bevacizumab (0.31%), and trastuzumab (0.60%) were obtained. The intraday and interday RSD of the method were less than 0.32 and 1.3%, respectively for RTm. Significant differences between theses RTms have been demonstrated allowing mAbs identification. Finally, accurate mAbs identification has been demonstrated by a blind test.