Getting the Best out of Capillary Electrophoresis and Capillary Electrophoresis-Mass Spectrometry by Quantifying Sources of Peak Broadening for Proteins Using Polyelectrolyte Multilayer Coated Fused Silica Capillaries.

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.

Charge variant analysis of monoclonal antibodies by CZE-MS using a successive multiple ionic-polymer layer coating based on diethylaminoethyl-dextran.

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.

Preventing the impact of solute adsorption in Taylor dispersion analysis: Application to protein and lipid nanoparticle analysis.

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.

Dual Contributions of Analyte Adsorption and Electroosmotic Inhomogeneity to Separation Efficiency in Capillary Electrophoresis of Proteins.

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.

Taylor dispersion analysis for measurement of diffusivity and size of gadolinium-based contrast agents.

Gadolinium-based contrast agents (GBCA) are complexes of a Gadolinium metal center and a linear or macrocyclic polyamino-carboxylic acid chelating agent. These agents are employed to enhance the visibility of deep abnormalities through MRI techniques. Knowing the precise dimensions of various GBCA is key parameter for understanding their in-vivo and pharmaco-kinetic behaviors, their diffusivity, as well as their relaxivity. However, conventional size characterization techniques fall short when dealing with these tiny molecules (≤1 nm). In this work, we propose to determine the size and diffusivity of gadolinium-based contrast agents using Taylor dispersion analysis (TDA). TDA provided a reliable measurement of the hydrodynamic diameter and the diffusion coefficient. The obtained results were compared to DOSY NMR (Diffusion-ordered Nuclear Magnetic Resonance Spectroscopy) and DFT (Density Functional Theory).

Investigating cationic and zwitterionic successive multiple ionic-polymer layer coatings for protein separation by capillary electrophoresis.

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.

Dual Cross-Linked Stimuli-Responsive Alginate-Based Hydrogels.

Sodium alginate with different molecular weights (55, 170, and 320 kg mol-1) were chemically modified by grafting methacrylic moieties onto the hydroxyl groups of the alginate backbone. The methacrylation was optimized to obtain different degrees of modification. Chemically cross-linked hydrogels were obtained following UV-light irradiation in the presence of a photoinitiator. The swelling behavior and the mechanical properties were observed to depend on both the degree of methacrylation and the alginate molecular weight. Due to the chain entanglement present in high-viscosity sodium alginate, lower degrees of modification were required to tune the hydrogel properties. Moreover, in the presence of Ca2+, secondary cross-linking was introduced by the coordination of the alginate guluronate moieties with the Ca2+ ions. The addition of this secondary cross-linking caused fast volume shrinkage and a reinforcement of the mechanical properties. The secondary cross-linking was reversible, and the hydrogels regained their original shape for at least three cycles. Additionally, the dual cross-linked network can be used to induce adhesion between hydrogels and serve as a building block for self-folding actuators.

Size-based characterization of dendrigraft poly(L-lysine) by free solution capillary electrophoresis using polyelectrolyte multilayer coatings.

Dendrigraft poly(L-lysine) (DGL) constitutes a promising dendritic-like drug vehicle with high biocompatibility and straightforward access via ring-opening polymerization of N-carboxyanhydride in water. The characterization of the different generations of DGL is however challenging due to their heterogeneity in molar mass and branching ratio. In this work, free solution capillary electrophoresis was used to perform selective separation of the three first generations of DGL, and optimized conditions were developed to maximize inter-generation resolution. To reduce solute adsorption on the capillary wall, successive multiple ionic polymer layer coatings terminated with a polycation were deposited onto the inner wall surface. PEGylated polycation was also used as the last layer for the control of the electroosmotic flow (EOF), depending on the PEGylation degree and the methyl-polyethylene glycol (mPEG) chain length. 1 kDa mPEG chains and low grafting densities were found to be the best experimental conditions for a fine tuning of the EOF leading to high peak resolution. Molar mass polydispersity and polydispersity in effective electrophoretic mobility were successfully determined for the three first generations of DGL.

Critical parameters for highly efficient and reproducible polyelectrolyte multilayer coatings for protein separation by capillary electrophoresis.

Since the introduction of polyelectrolyte multilayers to protein separation in capillary electrophoresis (CE), some progress has been made to improve separation efficiency by varying different parameters, such as buffer ionic strength and pH, polyelectrolyte nature and number of deposited layers. However, CE is often overlooked as it lacks robustness compared to other separation techniques. In this work, critical parameters for the construction of efficient and reproducible Successive multiple ionic-polymer layers (SMIL) coatings were investigated, focusing on experimental conditions, such as vial preparation and sample conservation which were shown to have a significant impact on separation performances. In addition to repeatability, intra- and inter-capillary precision were assessed, demonstrating the improved capability of poly(diallyldimethylammonium chloride) / poly(sodium styrene sulfonate) (PDADMAC / PSS) coated capillaries to separate model proteins in a 2 M acetic acid background electrolyte when all the correct precautions are put in place (with run to run%RSD(tm) < 1.8%, day to day%RSD(tm) < 3.2% and cap to cap%RSD(tm) < 4.6%). The approach recently introduced to calculate retention factors was used to quantify residual protein adsorption onto the capillary wall and to assess capillary coating performances. 5-layer PDADAMAC / PSS coatings led to average retention factors for the five model proteins of ∼4×10-2. These values suggest a relatively low residual protein adsorption leading to reasonably flat plate height vs linear velocity curves, obtained by performing electrophoretic separations at different electrical voltages (-10 to -25 kV).

Metabolism and disposition of JNJ-10450232 (NTM-006) in rats, dogs, monkeys and humans.

JNJ-10450232 (NTM-006), a novel non-opioid, non-nonsteroidal anti-inflammatory drug with structural similarities to acetaminophen, demonstrated anti-pyretic and/or analgesic activities in preclinical models and humans and reduced potential to cause hepatotoxicity in preclinical species. Metabolism and disposition of JNJ-10450232 (NTM-006) following oral administration to rats, dogs, monkeys and humans are reported. Urinary excretion was the major route of elimination based on recovery of 88.6% (rats) and 73.7% (dogs) of oral dose. The compound was extensively metabolized based on low recovery of unchanged drug in excreta from rats (11.3%) and dogs (18.4%). Clearance is driven by O-glucuronidation, amide hydrolysis, O-sulfation and methyl oxidation pathways. The combination of metabolic pathways driving clearance in human is covered in at least one preclinical species despite a few species-dependent pathways. O-Glucuronidation was the major primary metabolic pathway of JNJ-10450232 (NTM-006) in dogs, monkeys and humans, although amide hydrolysis was another major primary metabolic pathway in rats and dogs. A minor bioactivation pathway to quinone-imine is observed only in monkeys and humans. Unchanged drug was the major circulatory component in all species investigated. Except for metabolic pathways unique to the 5-methyl-1H-pyrazole-3-carboxamide moiety, metabolism and disposition of JNJ-10450232 (NTM-006) are similar to acetaminophen across species.

Modifying last layer in polyelectrolyte multilayer coatings for capillary electrophoresis of proteins.

Protein adsorption on the inner wall of the fused silica capillary wall is an important concern for capillary electrophoresis (CE) analysis since it is mainly responsible for separation efficiency reduction. Successive Multiple Ionic-polymer Layers (SMIL) are used as capillary coatings to limit protein adsorption, but even low residual adsorption strongly impacts the separation efficiency, especially at high separation voltages. In this work, the influence of the chemical nature and the PEGylation of the polyelectrolyte deposited in the last layer of the SMIL coating was investigated on the separation performances of a mixture of four model intact proteins (myoglobin (Myo), trypsin inhibitor (TI), ribonuclease a (RNAse A) and lysozyme (Lyz)). Poly(allylamine hydrochloride) (PAH), polyethyleneimine (PEI), ε-poly(L-lysine) (εPLL) and α-poly(L-lysine) (αPLL) were compared before and after chemical modification with polyethyleneglycol (PEG) of different chain lengths. The experimental results obtained by performing electrophoretic separations at different separation voltages allowed determining the residual retention factor of the proteins onto the capillary wall via the determination of the plate height at different solute velocities and demonstrated a strong impact of the polycationic last layer on the electroosmotic mobility, the separation efficiency and the overall resolution. Properties of SMIL coatings were also characterized by quartz microbalance and atomic force microscopy, demonstrating a glassy structure of the films.

A simple and reliable method for separation of mineral oil/polychlorobiphenyl mixtures.

Polychlorinated biphenyls (PCBs) were broadly applied worldwide as electrical insulators in transformers and power capacitors, due to their high dielectric constant and non-flammability. They were often added to mineral oils (MOs) and used as dielectric fluids, which are nowadays classified as hazardous waste. Indeed, the Stockholm Convention aims to eliminate the use of equipment with PCB content greater than 0.005 wt-% (=50 ppm) by 2025. Accurate identification and quantification of small traces of PCBs contained in MO thus represent a great analytical challenge. To achieve this goal, a simple, cost-effective and fast chromatographic process was developed to separate PCBs from MO, allowing to obtain reliable data to determine the concentration of PCBs, reduced to 2-3 ppm. Experimental and analytical methods, such as thin layer chromatography, column chromatography as well as gas chromatography coupled with mass spectroscopy, were applied to acquire a high level of qualitative and quantitative determination of PCBs in transformer MOs.

Taylor Dispersion Analysis to support lipid-nanoparticle formulations for mRNA vaccines.

Lipid nanoparticles (LNPs) are currently the most advanced non-viral clinically approved messenger ribonucleic acid (mRNA) delivery systems. The ability of a mRNA vaccine to have a therapeutic effect is related to the capacity of LNPs to deliver the nucleic acid intact into cells. The role of LNPs is to protect mRNA, especially from degradation by ribonucleases (RNases) and to allow it to access the cytoplasm of cells where it can be translated into the protein of interest. LNPs enter cells by endocytosis and their size is a critical parameter impacting their cellular internalization. In this work, we studied different formulation process parameters impacting LNPs size. Taylor dispersion analysis (TDA) was used to determine the LNPs size and size distribution and the results were compared with those obtained by Dynamic Light Scattering (DLS). TDA was also used to study both the degradation of mRNA in the presence of RNases and the percentage of mRNA encapsulation within LNPs.

Successive Multiple Ionic-Polymer Layer Coatings for Intact Protein Analysis by Capillary Zone Electrophoresis-Mass Spectrometry: Application to Hemoglobin Analysis.

Adsorption of analytes, e.g., proteins, often interfere with separation in CE, due to the relatively large surface of the narrow capillary. Coatings often are applied to prevent adsorption and to determine the electroosmotic flow (EOF), which is of major importance for the separation in CE. Successive multiple ionic-polymer layer (SMIL) coatings are frequently used for protein analysis in capillary electrophoresis resulting in high separation efficiency and repeatability. Here, the coating procedure of a five-layer SMIL coating is described using quaternized diethylaminoethyl dextran (DEAEDq) as polycation and poly(methacrylic acid) (PMA) as polyanion. Depending on the analyte, different polyions may be used to increase separation efficiency. However, the coating procedure remains the same.To demonstrate the applicability of SMIL coatings in CE-MS, human hemoglobin was measured in a BGE containing 2 M acetic acid. DEAEDq-PMA coating was found to be the most suitable for hemoglobin analysis due to relatively low reversed electroosmotic mobility leading to increased electrophoretic resolution of closely related proteoforms. Thereby, not only alpha and beta subunit of the hemoglobin could be separated, but also positional isoforms of glycated and carbamylated species were separated within 24 min.

Polyelectrolyte Multilayers in Capillary Electrophoresis.

Capillary electrophoresis (CE) has been proven to be a performant analytical method to analyze both small and macro molecules. Indeed, it is capable of separating compounds of the same nature according to differences in their charge to size ratios, particularly proteins, monoclonal antibodies and peptides. However, one of the major obstacles to reach high separation efficiency remains the adsorption of solutes on the capillary wall. Among the different coating approaches used to control and minimize solute adsorption, polyelectrolyte multilayers can be applied to CE as a versatile approach. These coatings are made up of alternating layers of polycations and polyanions, and may be used in acidic, neutral or basic conditions depending on the solutes to be analyzed. This Review provides an overview of Successive Multiple Ionic-polymer Layer (SMIL) coatings used in CE, looking at how different parameters induce variations on the electro-osmotic flow (EOF), separation efficiency and coating stability, as well as their promising applications in the biopharmaceutical field.

Determination of ultrahigh molar mass of polyelectrolytes by Taylor dispersion analysis.

Taylor dispersion analysis (TDA) was successfully applied to obtain broadly distributed, ultrahigh molar masses of industrial anionic polyacrylamides (IPAMs) up to 25 × 106 g/mol, far beyond the limits of Size Exclusion Chromatography (SEC) (about 7.3 × 106 g/mol for anionic polyacrylamides standards (APAM)). Two protocols of TDA differing in capillary surface and rinsing procedure were employed: (i) bare fused silica capillaries under intensive between-run rinsing with 1 M NaOH, and (ii) fused silica capillaries coated with polyelectrolyte multilayers composed of polydiallyldimethylammonium chloride polycation and sodium polystyrenesulfonate polyanion under simple rinsing with background electrolyte. Both cases led to similar results and in agreement with those obtained by static light scattering, the rinsing capillary step being much shorter in the second case (8 min instead of 30 min). The data processing of the obtained taylorgrams was realized using multiple-Gaussian fitting of the overall taylorgrams, by separating the contribution of low molar mass impurities from the polymeric profiles, and by determining the mean hydrodynamic radii and diffusion coefficients of the polymers. The molar masses of ultra-high molar mass industrial anionic polyacrylamides (IPAM) were derived from the hydrodynamic radii according to logRh versus logMw linear correlation established with APAM standards. Compared to capillary gel electrophoresis for which the size separation was only feasible up to Mw ∼ 10×106 g/mol due to field induced polymer aggregation, TDA largely extended the range of accessible molar mass with easy-to-run and time saving assays.

Size and Charge Characterization of Lipid Nanoparticles for mRNA Vaccines.

Messenger RNA vaccines have come into the spotlight as a promising and adaptive alternative to conventional vaccine approaches. The efficacy of mRNA vaccines relies on the ability of mRNA to reach the cytoplasm of cells, where it can be translated into proteins of interest, allowing it to trigger the immune response. However, unprotected mRNA is unstable and susceptible to degradation by exo- and endonucleases, and its negative charges are electrostatically repulsed by the anionic cell membranes. Therefore, mRNA needs a delivery system that protects the nucleic acid from degradation and allows it to enter into the cells. Lipid nanoparticles (LNPs) represent a nonviral leading vector for mRNA delivery. Physicochemical parameters of LNPs, including their size and their charge, directly impact their in vivo behavior and, therefore, their cellular internalization. In this work, Taylor dispersion analysis (TDA) was used as a new methodology for the characterization of the size and polydispersity of LNPs, and capillary electrophoresis (CE) was used for the determination of LNP global charge. The results obtained were compared with those obtained by dynamic light scattering (DLS) and laser Doppler electrophoresis (LDE).

Separation of three strains of polio virus by capillary zone electrophoresis and study of their interaction with aluminum oxyhydroxide.

The development of combination vaccines is essential to reduce the number of injections, shorten vaccination schedules and increase vaccination coverage. Vaccine adjuvants are used to modulate and enhance the immune response induced by the antigens. To support the development of combination vaccines, the study of antigen-adjuvant interactions in the final vaccine formulations is required as interaction competitions may take place between the different antigens. In the present work, a capillary zone electrophoresis (CZE) methodology was firstly optimized on six model proteins, namely bovine serum albumin, ß-lactoglobulin, myoglobin, ribonuclease A, cytochrome C and lysozyme. A cationic dynamic coating (polybrene) and a zwitterionic amino acid additive (ß-alanine) in the background electrolyte were used to reduce the phenomena of protein adsorption on the inner wall of the capillary and thus optimize the separation efficiency of the proteins. The developed methodology was then used to separate three strains from inactivated polio virus, each strain being a whole virus composed of copies of 4 viral proteins and study their interaction with aluminum oxyhydroxide. The antigen-adjuvant interactions could be modulated by addition of phosphate ions playing the role of competitors for the poliovirus.

Generation and characterization of air micro-bubbles in highly hydrophobic capillaries.

The generation of air microbubbles in microfluidic systems or in capillaries could be of great interest for transportation (single cell analysis, organite transportation) or for liquid compartmentation. The physicochemical characterization of air bubbles and a better understanding of the process leading to bubble generation during electrophoresis is also interesting in a theoretical point of view. In this work, the generation of microbubbles on hydrophobic Glaco™ coated capillaries has been studied in water-based electrolyte. Air bubbles were generated at the detection window and the required experimental parameters for microbubbles generation have been identified. Generated bubbles migrated against the electroosmotic flow, as would do strongly negatively charged solutes, under constant electric field. They have been characterized in terms of dimensions, electrophoretic mobility, and apparent charge.

Chemoprevention with a tea from hawthorn (Crataegus oxyacantha) leaves and flowers attenuates colitis in rats by reducing inflammation and oxidative stress.

The purpose of the study was to determine the effects of a tea from the leaves and flowers of Crataegus oxyacantha in rats with colitis. Colitis was induced by administration of 2,4,6-trinitrobenzene sulfonic acid. Hawthorn tea (HT) (100 mg/kg) was given via gavage for 21 days and the mesalamine drug (100 mg/kg) was administrated during the period of disease onset. HT was rich in total phenolic compounds (16.5%), flavonoids (1.8%), and proanthocyanidins (1.5%); vitexin-2-O-rhamnoside was the main compound detected. Mesalamine and the HT diminished the length of the lesions formed in the colon, in addition to reducing the levels of myeloperoxidase and interleukin-1ß. Mesalamine was able to significantly reverse the body weight loss, while HT improved the activity of glutathione reductase and catalase. Histological scoring was not changed by the interventions, but it was highly correlated with the necrotic area. HT given at 100 mg/kg can be effective against colitis.