Analyzing intact proteins by capillary electrophoresis (CE) is challenging but highly valuable, as it holds potential for future clinical applications via the separation and quantification of protein biomarkers. To achieve this, the development of CE methods requires improvements in separation efficiency, reproducibility, and reduction of sample adsorption on the capillary wall. Double-chained surfactant coatings were described as potential candidate leading to high separation efficiency. In this work, a capillary coating based on dioctadecyldimethylammonium bromide (DODAB), a double-chained cationic surfactant, was investigated by assessing the critical parameters affecting DODAB-vesicle preparation and coating performances. These parameters include temperature & sonication of the coating solution, nature of the coating buffer, and capillary inner diameter. The plate height (H) of proteins as a function of their linear migration velocity (u) was systematically plotted to evaluate the separation performances under different kinetic conditions by applying different separation voltages. DODAB coating showed high surface homogeneity leading to low slope (p) of the H vs u curve (2.23 ± 0.99 ms in average on 3 different capillaries) on 5 model basic proteins (lysozyme, carbonic anhydrase, ribonuclease A, β-lactoglobulin A and myoglobin) using 2 M acetic acid, pH 2.2, as background electrolyte in a 40 cm total length and 25 μm inner diameter capillary. Excellent separation performances were achieved reaching up to 606,000 ± 27,000 plates/m at -12 kV for lysozyme, with excellent repeatability (RSD on migration times = 0.21 %).

Enzymatic degradation of biopolymers underpins critical processes in biotechnological applications and natural processes, such as food digestion and cancer development, yet the interplay between enzyme propagation in a dense substrate and substrate degradation remains unresolved. It remains especially unclear whether degradation of a dense matrix facilitates or impedes enzyme propagation. We simultaneously track with spatiotemporal resolution unidirectional enzyme diffusion and biopolymer degradation in a model system. We demonstrate that enzyme diffusion is decoupled from catalytic activity, while the degradation front progression is dictated by enzyme diffusion, reaction kinetics and slow enzyme deactivation. These findings establish a quantitative framework to optimize enzymatic processes for applications in biomedicine, biomass valorization, and nanotechnology.

This publication constitutes a comparison of two apparently different communities, namely, synthetic polymer self-assemblies and amyloid aggregates, to cross-fertilize each other. The starting point is shared recognized issues in the reproducibility of the fabrication of the aggregates for each community. Based on this, this publication compares the underlying mechanistic principles of self-assembly, the methods for the preparation of assemblies, and the techniques used for their characterization. This highlights some common practices, while also revealing notable differences. Interestingly, some techniques may be used by both communities but with different perspectives and aims, whereas others are preferably employed by one community. The article finally suggests possible cross-advice for each one.

For intact protein separation by capillary electrophoresis, the application of a proper capillary coating is crucial to achieve high separation efficiencies. There are a large number of different capillary coatings and coating protocols, making it crucial to compare the coating performances in identical experimental conditions for intact protein separations. Here, the plate height vs. migration velocity was systematically plotted by doing electrophoretic experiments at different electric voltages, on five model proteins. For the first time, this study allows comparing the performances of seven different neutral and cationic coatings in a 2 M acetic acid background electrolyte. For all tested capillary coatings, i.e. polyvinyl alcohol (PVA), covalent and physically adsorbed polyethylene oxide (PEO), hydroxypropyl cellulose (HPC), linear polyacrylamide (LPA), 5-layers cationic polyelectrolyte coating formed from polydiallyldimethyl ammonium chloride and sodium polystyrene sulfonate (PDADMAC-PSS)2.5, and dioctadecyldimethylammonium bromide (DODAB), the plate height of all proteins increases when higher voltages are applied, leading to better separation at lower voltages. For neutral coatings, a non-linear increase of H was observed at higher voltages, leading to poor separation efficiency for certain proteins at high voltages. This effect was not observed on cationic coatings and was explained by the stretching of the protein chain, promoting the interaction with the capillary coating. Regarding the average performances of the coatings in terms of separation efficiency, the ranking of coatings followed (best to worst): DODAB > HPC > μSIL-WAX > LPA > PEO > SMIL > PVA. It is worth noting that the resolution depends not only on the separation efficiency, but also on the apparent selectivity, which is strongly dependent on the EOF magnitude relative to the effective mobility of the protein. Therefore, the choice of the coating should also consider the range of desired EOF relative to the targeted proteins.

BACKGROUND:Liposomes and lipid nanoparticles (LNPs) are widely used as biomimetic systems and drug delivery vehicles, yet their isoelectric point (pI) remains underutilized as a characterization parameter. Capillary isoelectric focusing (cIEF) enables direct pI determination, but carrier ampholytes used in cIEF separations may compromise nanoparticle stability. Their influence on nanoparticle integrity remains poorly understood, hindering cIEF analysis development. Taylor dispersion analysis (TDA) is a particle-sizing technique that determines the hydrodynamic radius (Rh) of analytes, including nanoparticles and can be used for sensitive monitoring of nanoparticle stability. RESULTS:TDA was used to characterize synthetic liposomes, lipid extract vesicles, and mRNA vaccine-derived LNPs in the presence of two carrier ampholyte (CA) systems, Pharmalyte 3-10 and AESlyte SH 3-10. Liver-derived vesicles exhibited pronounced ampholyte sensitivity, with rapid population loss and aggregation at relatively low CA concentrations, consistent with their failure in subsequent cIEF analysis. In contrast, POPC:DOPE:Chol liposomes and vaccine LNPs maintained nanoparticle populations across a broad CA concentration range, although concentration-dependent shifts in size distributions were observed. Both ampholyte systems enabled successful cIEF analysis of POPC:DOPE:Chol liposomes and vaccine LNPs. However, differences in nanoparticle stability were observed with different CA types at elevated concentrations. SIGNIFICANCE AND NOVELTY:This work demonstrates that TDA can serve as a predictive screening tool for evaluating ampholyte-induced lipid nanoparticle destabilization. By linking ampholyte concentration and type, vesicle composition, and electrophoretic focusing performance, the study identifies stability windows that support reliable pI determination of lipid-based nanoparticles.

BACKGROUND:Vaccines based on Outer Membrane Vesicles (OMVs) against Neisseria meningitidis require the quantification and characterization of residual capsular polysaccharides (CapsPS) that are not completely removed during the production process. This task is challenging due to the complexity of the CapsPS composition and the presence of OMVs in the samples. RESULTS:Micellar Electrokinetic Chromatography (MEKC) using a negatively charged polyelectrolyte multilayer coating (SMIL) enabled complete separation of CapsPS and OMV residues in approximately 40 min. The use of a 4-layer SMIL coating was crucial for achieving good repeatability of migration times (tm). The method allowed the quantification of CapsPS at concentrations between 80 and 600 mg L-1. The average number and weight degrees of polymerization were also determined for each batch of OMVs, with polydispersity indexes between 1.2 and 1.5 and degrees of polymerization (DPn) between 30 and 70. SIGNIFICANCE:This method can be readily implemented in the pharmaceutical industry for the monitoring and documentation of process development.

We understand your surprise regarding our data showing a GBCA retention on Amicon® filters [...]

Mass spectrometry-based top-down protein analysis requires efficient separation. In the context of proteoform analysis, capillary zone electrophoresis (CZE) is very valuable. The resolution of two peaks in CZE can be increased when the absolute mobility of the counter-directed electroosmotic flow (EOF) is close to the effective mobility of the analytes, resulting in a low apparent mobility of the analytes. The mobility of the EOF of highly efficient sulfobetaine-modified poly(α-L-lysine) (α-PLL) coatings changes depending on the number of modified side chains. Here, such coatings are used to selectively increase the peak resolution of proteoforms of model proteins and analytes in a complex protein sample (intact yeast protein extract). Whereas a high EOF system allows for the separation of proteins of a wide mobility range (complete proteome), lower EOF systems allow for a much better separation of proteins and proteoforms of low mobility, including those containing acidic post-translation modifications (PTMs). This leads to the identification of 2.5 times more proteoforms by MS/MS experiments in the lower mobility range of the yeast proteome. The sulfobetaine-modified α-PLL coatings presented here exhibit a toolbox for highly resolved separation of proteins and proteoforms in targeted or untargeted top-down protein analysis. SUMMARY: Sample complexity is one of the main challenges when analyzing a proteome on the proteoform level. In the course of this, capillary electrophoresis-mass spectrometry turned out to be an excellent tool because of its high-performing separation, particularly for large molecules. Here, we present a method enabling the best possible separation due to efficient and EOF-tunable coatings, allowing for flexible and dedicated selection of a range of proteins and proteoforms to be analyzed under ideal separation conditions. The high performance is demonstrated by the separation of proteoforms of common PTM-rich model proteins as well as complex proteome samples.

BACKGROUND:Capillary electrophoresis (CE) is a highly efficient and versatile analytical method for the separation of biomacromolecules such as proteins and peptides. One major concern to reach high separation efficiency is the adsorption of analytes on the capillary wall and the heterogeneity of the capillary surface charge which generates hydrodynamic dispersion due to local electroosmotic (EOF) fluctuations. RESULTS:Double chain surfactants have been described as potential interesting candidates for capillary coatings in CE. They are notably offering a very homogenous surface charge leading to very high separation efficiency with reported values up to 1 million plates per meter. SIGNIFICANCE:This review provides an overview of double chain surfactant coatings used in CE with an emphasis on the coating protocol, the nature of the surfactant, the preparation of the coating solution (concentration, temperature, sonication or extrusion), the physicochemical parameters affecting their properties (pH, ionic strength, nature of the anion in the coating solution, coating additives, capillary internal diameter), and the coating stability/durability.

BACKGROUND:Gadolinium-based contrast agents (GBCA) are widely used in magnetic resonance imaging (MRI) to enhance image contrast. However, their interactions with biological macromolecules remain an important area of investigation due to potential safety concerns. Developing robust analytical methods to study these interactions is essential for improving safety assessments and guiding the design of next-generation contrast agents. RESULTS:In this study, we employed Frontal Analysis Continuous Capillary Electrophoresis (FACCE) to investigate the binding interactions between lysozyme, a model protein, and several GBCA, including Gd-DTPA (Gadopentetate, Magnevist®), Gd-BOPTA (Gadobenate, Multihance®), Gd-DOTA (Gadoterate, Dotarem®), Gd-PCTA D2 (Gadopiclenol, Elucirem™) and Gd-HP-DO3A (Gadoteridol, Prohance®). Our results demonstrate that GBCA exhibit varying degrees of cooperative binding to lysozyme, with stoichiometry (n) of 4 for Gd-DTPA, Gd-BOPTA, and Gd-DOTA. The strength of interactions followed the trend: Gd-BOPTA (Multihance®) > Gd-DTPA (Magnevist®) > Gd-DOTA (Dotarem®), while Gd-PCTA D2 (Elucirem™) showed no significant binding. However, FACCE was unable to resolve interactions for Gd-HP-DO3A (Prohance®) due to interference with lysozyme. SIGNIFICANCE:FACCE is a well-adapted method to investigate GBCA-protein interaction. It requires low sample volumes and allows determining the stoichiometry, binding constant, and cooperativity of the interaction. This work emphasizes the cooperative nature of the GBCA-lysozyme interactions and indicates that the Hill model is suitable for analysing the interactions within the tested concentration range of GBCAs.

Capillary electrophoresis (CE) is a robust, selective and highly efficient technique for the analysis of peptides and (intact) proteins. The anionic and/or hydrophilic character of the fused silica surface generally leads to protein adsorption by electrostatic interaction or H-bonding. Successive multiple ionic-polymer layer (SMIL) coatings are often used to limit adsorption and to improve the repeatability of migration times. Besides these adsorption phenomena, the electroosmotic flow (EOF) also has a strong influence on the resolution. Here, the frequently used and efficient cationic SMIL coating agent poly(alpha-l-lysine) (alpha-PLL) is modified for the systematic modulation of the EOF. In particular, by converting stepwise the epsilon-amino functions of this polycation into carboxamides, the total number of positive charges decreases, leading to reduced EOF. To simultaneously keep the analyte-surface interactions as small as possible, carboxylic acids bearing a zwitterionic functionality based structurally on a sulfobetaine motif were developed. Using the water-soluble and highly hydrolysis-resistant condensation agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), the degree of functionalization could be adjusted between 8 and 99%, depending on the applied stoichiometry. The obtained set of mixed polycationic/polyzwitterionic polymers based on the alpha-PLL scaffold were investigated as the outermost layers of the SMIL coatings, clearly showing that the EOF decreases depending on the degree of functionalization while maintaining high efficiency.

Coatings with zwitterionic compounds have been developed as antifouling surface to reduce or eliminate nonspecific adsorption to the solid/liquid interface. In this work, zwitterionic silica (SBSi) coating was modified on fused silica capillary for the first time by silanization (covalent bonding). The self-assembled SBSi coating was characterized using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and contact angle. A comparative study of electroosmotic flow (EOF) with bare fused silica capillary showed that the SBSi coating could provide effective suppression of EOF at pH 5.5. CE separations of three intact proteins (lysozyme, ribonuclease A, and myoglobin) were performed at pH 3.5 using different voltages ranging between 10 kV and 30 kV. Accordingly, the coating performances were assessed by plotting the plate height H versus the analyte velocity u, leading to similar performances as compared with polyelectrolyte multilayers (SMIL) coatings in similar conditions.

Capillary electrophoresis (CE) has emerged as a relevant technique for protein and biopharmaceutical analysis, as it combines high separation efficiency, sensitivity, and versatility. The use of capillary coatings, including successive multiple ionic-polymer layers (SMILs), reduces interactions between analytes and the capillary, further improving the CE performance. Nevertheless, separations done on SMIL coatings rarely surpass 500 × 103 plates/m. To obtain the best out of the CE, it is interesting to have a detailed look at the sources of peak dispersion. Separations of a mix of model proteins were performed on (poly(diallyldimethylammonium chloride)/poly(styrenesulfonate))2.5-coated capillaries at different electrical field strengths, leading to plate height H against migration velocity u plots that enabled a quantitative analysis of each contribution. Using this model, capillary lengths and injected volumes were systematically varied. For the first time, the contribution of sample electrophoretic heterogeneity to the total peak dispersion was deciphered for model proteins and a monoclonal antibody. Dispersion due to electromigration was seen to have an impact on plate heights in the case of triangular peaks of small molecules but not for proteins under the present conditions. UV and mass spectrometry detections were compared on the same capillary, providing valuable information on the impact of the detection type on separation efficiency. Close to 1 million plates/m were reached in the best conditions.

In this work, we describe an optical setup to determine the internal diameter of narrow bore fused silica capillary used in capillary electrophoresis and Taylor dispersion analysis (TDA). Indeed, fluctuations up to about ±3-4 µm on the capillary I.D. can generate important inaccuracy on the hydrodynamic radius determination by TDA. Calibration of the optical set-up, impact of the operator and of the placement of the capillary in the focal plane, and the influence of the way to cut the capillary were investigated and discussed. This optical set-up was next used to determine capillary I.D. on a 60 m long capillary spool. Relatively small variations were observed along a 60 m capillary spool (0.3 µm maximum variation), while important I.D. fluctuations can be observed from capillary batch to batch. Taking three capillaries of three different nominal I.D. values, Rh values of sodium benzoate obtained by TDA were not significantly different if the capillary I.D. were optically measured, while significant variations were observed with the nominal I.D. values. A protocol based on TDA of sodium benzoate was proposed for calibrating narrow bore fused silica capillary I.D. without the use of optical measurements for researchers that would not have access to such optical equipment.

Background: Gadolinium-based contrast agents (GBCA) are widely used in magnetic resonance imaging (MRI) to enhance image contrast by interacting with water molecules, thus improving diagnostic capabilities. However, understanding the residual accumulation of GBCA in tissues after administration remains an area of active research. This highlights the need for advanced analytical techniques capable of investigating interactions between GBCAs and biopolymers, such as type I collagen, which are abundant in the body. Objective: This study explores the interactions of neutral and charged GBCAs with type I collagen under physiological pH conditions (pH 7.4) using Taylor dispersion analysis (TDA) and frontal analysis continuous capillary electrophoresis (FACCE). Methods: Collagen from bovine achilles tendon was ground using a vibratory ball mill to achieve a more uniform particle size and increased surface area. Laser granulometry was employed to characterize the size distributions of both raw and ground collagen suspensions in water. TDA was used to assess the hydrodynamic radius (Rh) of the soluble collagen fraction present in the supernatant. Results: From the TDA and FACCE results, it was shown that there were no significant interactions between the tested GBCAs and either the ground collagen or its soluble fraction at pH 7.4. Interestingly, we also observed that collagen interacts with filtration membranes, indicating that careful selection of membrane material, or the absence of filtration in the experimental protocol, is essential in interaction studies involving collagen. Conclusion: These findings bring valuable insights into the behavior of GBCAs in biological systems with potential implications for clinical applications.

Successive multiple ionic-polymer layers (SMILs) have long since proved their worth in capillary electrophoresis as they ensure stable electroosmotic flow (EOF) and relatively high separation efficiency. Recently, we demonstrated that plotting the plate height (H) against the solute migration velocity (u) enabled a reliable quantitative evaluation of the coating performances in terms of separation efficiency. In this work, various physicochemical and chemical parameters of the SMIL coating were studied and optimized in order to decrease the slope of the ascending part of the H vs u curve, which is known to be controlled by the homogeneity in charge of the coating surface and by the possible residual solute adsorption onto the coating surface. SMILs based on poly(diallyldimethylammonium chloride) (PDADMAC) and poly(sodium styrene sulfonate) (PSS) were formed and the effect of each polyelectrolyte molar mass and of the number of polyelectrolyte layers (up to 21 layers) was studied. The use of polyethylene imine as an anchoring first layer was considered. More polyelectrolyte couples based on PDADMAC, polybrene, PSS, poly(vinyl sulfate), and poly(acrylic acid) were tested. Finally, zwitterionic polymers based on the poly(α-L-lysine) scaffold were synthesized and used as the last layer of SMILs, illustrating their ability to finetune the EOF, while maintaining good separation efficiency.

Improving separation efficiency in capillary electrophoresis (CE) requires systematic study of the influence of the electric field (or solute linear velocity) on plate height for a better understanding of the critical parameters controlling peak broadening. Even for poly(diallyldimethylammonium chloride) (PDADMAC)/poly(sodium styrenesulfonate) (PSS) successive multiple ionic-polymer layer (SMIL) coatings, which lead to efficient and reproducible separations of proteins, plate height increases with migration velocity, limiting the use of high electric fields in CE. Solute adsorption onto the capillary wall was generally considered as the main source of peak dispersion, explaining this plate height increase. However, experiments done with Taylor dispersion analysis and CE in the same conditions indicate that other phenomena may come into play. Protein adsorption with slow kinetics and few adsorption sites was established as a source of peak broadening for specific proteins. Surface charge inhomogeneity was also identified as a contribution to plate height due to local electroosmotic fluctuations. A model was proposed and applied to partial PDADMAC/poly(ethylene oxide) capillary coatings as well as PDADMAC/PSS SMIL coatings. Atomic force microscopy with topography and recognition imaging enabled the determination of roughness and charge distribution of the PDADMAC/PSS SMIL surface.

The characterization of the impurities of pharmaceutical monoclonal antibodies (mAbs) is crucial for their function and safety. Capillary zone electrophoresis (CZE) is one of the most efficient tools to separate charge variants of mAbs; however, peak characterization remains difficult, since the hereby used background electrolytes (BGEs) are not compatible with electrospray ionization-mass spectrometry (ESI-MS). Here, a method that allows the separation of intact mAb charge variants is presented using CZE-ESI-MS, combining a cationic capillary coating and an acidic BGE. Therefore, a successive multiple ionic-polymer layer coating was developed based on diethylaminoethyl-dextran-poly(sodium styrene sulfonate). This coating leads to a relatively low reversed electroosmotic flow (EOF) with an absolute mobility slightly higher than that of antibodies, enabling the separation of variants with slightly different mobilities. The potential of the coating is demonstrated using USP mAb003, where it was possible to separate C-terminal lysine variants from the main form, as well as several acidic variants and monoglycosylated mAb forms. The presented CZE-MS method can be applied to separate charge variants of a range of other antibodies such as infliximab, NISTmAB (Reference Material from the National Institute of Standards and Technology), adalimumab, and trastuzumab, demonstrating the general applicability for the separation of proteoforms of mAbs.

Taylor Dispersion Analysis (TDA) allows diffusion coefficient (D) or hydrodynamic radius (Rh) determination on a wide range of size between angstroms and about 300 nm. However, solute adsorption phenomena can affect the repeatability and reproducibility of TDA. Several numerical studies addressed the theoretical impact of solute adsorption in TDA, but very few experimental studies focus on this topic and no experimental methodologies were proposed so far to reduce the impact of adsorption in TDA. In this work, an experimental protocol, called plug-in-front TDA, consisting of adding the solute in the eluent at a lower concentration compared to the injected sample, was proposed to strongly limit the impact of adsorption on the Rh determination. This protocol was suggested based on the evidence that adsorption / desorption phenomena impacting narrow bore fused silica TDA in aqueous conditions are typically slow processes that can be counteracted by saturating the interaction sites during the experiments. Successful applications to proteins and mRNA lipid nanoparticles (LNP) in vaccine against Covid 19 and protein analysis were reported. TDA of proteins in conditions of strong interactions with the capillary surface was possible using the plug-in-front methodology. We anticipate that such experimental methodology will greatly help the experimentalist for implementing TDA in various applications.

Taylor dispersion analysis (TDA) is a simple and absolute method to determine the hydrodynamic radius of solutes that respond to UV or fluorescence detections. To broaden the application range of TDA, it is necessary to develop new detection modes. This study aims to study capacitively coupled contactless conductivity detector (C4D) for the analysis of charged macromolecules. The detection sensitivities and hydrodynamic radii were compared for a C4D detector and a UV detector on positively or negatively charged polymers responding both to UV and C4D (poly-L-lysine and poly(acrylamide-co-2-acrylamido-1-methyl-propanesulfonate). The influence of the composition of the background electrolyte on the detection sensitivity has been studied and optimized for C4D detection. The influence of the molar mass and of the polymer chemical charge density on the C4D and UV sensitivities of detection have been investigated based on well-characterized copolymers samples of different molar masses and charge densities. The advantages and disadvantages compared to UV detection, as well as the range of applicability of C4D detection in TDA were identified. C4D detection can be an alternative method for sizing charged polymers of reasonable molar mass (typically below 105 g mol-1) that do not absorb in UV. A decline in the sensitivity of detection in C4D was observed for higher molar masses.