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.

Method development for quantitative monitoring of monoclonal antibodies in upstream cell-culture process samples with limited sample preparation - An evaluation of various capillary coatings.

Monoclonal antibodies (mAbs) have become an important class of biopharmaceuticals used for the treatment of various diseases. Their quantification during the manufacturing process is important. In this work, a capillary zone electrophoresis (CZE) method was developed for the monitoring of the mAb concentration during cell-culture processes. CZE method development rules are outlined, particularly discussing various capillary coatings, such as a neutral covalent polyvinyl alcohol coating, a dynamic successive multiple ionic-polymer coating, and dynamic coatings using background electrolyte additives such as triethanolamine (T-EthA) and triethylamine. The dynamic T-EthA coating resulted in most stable electro-osmotic flows and most efficient peak shapes. The method is validated over the range 0.1-10 mg/ml, with a linear range of 0.08-1.3 mg/ml and an extended range of 1-10 mg/ml by diluting samples in the latter concentration range 10-fold in water. The intraday precision and accuracy were 2%-12% and 88%-107%, respectively, and inter-day precision and accuracy were 4%-9% and 93%-104%, respectively. The precision and accuracy of the lowest concentration level (0.08 mg/ml) were slightly worse and still well in scope for monitoring purposes. The presented method proved applicable for analysing in-process cell-culture samples from different cell-culture processes and is possibly well suited as platform method.

Recent (2018-2020) development in capillary electrophoresis.

Development of new capillary electrophoresis (CE) methodology and instrumentation, as well as application of CE to solve new problems, remains an active research area because of the attractive features of CE compared to other separation techniques. In this review, we focus on the representative works about sample preconcentration, separation media or capillary coating development, detector construction, and multidimensional separation in CE, which are judiciously selected from the papers published in 2018-2020.

Novel materials as capillary coatings for in-tube solid-phase microextraction for bioanalysis.

In-tube solid-phase microextraction with a capillary column as extraction device can be directly coupled with high-performance liquid chromatography systems (HPLC). The in-tube solid-phase microextraction technique has been continuously developed since it was introduced in 1997. New couplings have also been evaluated on the basis of state-of-the-art HPLC instruments. Different types of capillaries (wall-coated open tubular, porous layer open tubular, sorbent-packed, porous monolithic rods, or fiber-packed) with selective stationary phases (monoliths, magnetic nanoparticles, conducting polymers, restricted access materials, ionic liquids, carbon, deep eutectic solvents, and hybrid materials) have been developed to boost in-tube solid-phase microextraction performance (sorption capacity and selectivity). This technique has been successfully applied to analyze biological samples (serum, plasma, whole blood, hair, urine, milk, skin, and saliva) for therapeutic drug monitoring, to study biomarkers, to detect illicit drugs, to conduct metabolomics studies, and to assess exposure to drugs. This review describes current advances in in-tube solid-phase microextraction extraction devices and their application in bioanalysis.

Hybrid phospholipid bilayer coatings for separations of cationic proteins in capillary zone electrophoresis.

Protein separations in CZE suffer from nonspecific adsorption of analytes to the capillary surface. Semipermanent phospholipid bilayers have been used to minimize adsorption, but must be regenerated regularly to ensure reproducibility. We investigated the formation, characterization, and use of hybrid phospholipid bilayers (HPBs) as more stable biosurfactant capillary coatings for CZE protein separations. HPBs are formed by covalently modifying a support with a hydrophobic monolayer onto which a self-assembled lipid monolayer is deposited. Monolayers prepared in capillaries using 3-cyanopropyldimethylchlorosilane (CPDCS) or n-octyldimethylchlorosilane (ODCS) yielded hydrophobic surfaces with lowered surface free energies of 6.0 ± 0.3 or 0.2 ± 0.1 mJ m(-2) , respectively, compared to 17 ± 1 mJ m(-2) for bare silica capillaries. HPBs were formed by subsequently fusing vesicles comprised of 1,2-dilauroyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphocholine to CPDCS- or ODCS-modified capillaries. The resultant HPB coatings shielded the capillary surface and yielded reduced electroosmotic mobility (1.3-1.9 × 10(-4) cm(2) V(-1) s(-1) ) compared to CPDCS- and ODCS-modified or bare capillaries (3.6 ± 0.2 × 10(-4) cm(2) V(-1) s(-1) , 4.8 ± 0.4 × 10(-4) cm(2) V(-1) s(-1) , and 6.0 ± 0.2 × 10(-4) cm(2) V(-1) s(-1) , respectively), with increased stability compared to phospholipid bilayer coatings. HPB-coated capillaries yielded reproducible protein migration times (RSD ≤ 3.6%, n ≥ 6) with separation efficiencies as high as 200 000 plates/m.

A novel diazoresin/poly(N-vinyl aminobutyric acid) covalent capillary coating for the analysis of proteins by capillary electrophoresis.

A novel method for the preparation of covalently linked capillary coatings of poly(N-vinyl aminobutyric acid) (PVAA) obtained from hydrolyzed polyvinylpyrrolidone was demonstrated using photosensitive diazoresin (DR) as a coupling agent. A layer-by-layer self-assembled film of DR and PVAA based on ionic bonding was first fabricated on the inner wall of capillary, then ionic bonding was converted into covalent bonding after treatment with UV light through a unique photochemical reaction of DR. The covalently bonded coatings suppressed protein adsorption on the inner surface of the capillary, and thus a baseline separation of lysozyme, cytochrome c, BSA, amyloglucosidase, and myoglobin was achieved using CE. Compared with bare capillary or noncovalently bonded DR/PVAA coatings, the covalently linked DR/PVAA capillary coatings not only improved the CE separation performance for proteins, but also exhibited good stability and repeatability. Due to the replacement of the highly toxic and moisture-sensitive silane coupling agent by DR in the covalent coating preparation, this method may provide a green and easy way to make covalently coated capillaries for CE.

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.

Modulation of the electroosmotic mobility using polyelectrolyte multilayer coatings for protein analysis by capillary electrophoresis.

Successive multiple ionic-polymer layers (SMIL) coatings have been often used in capillary electrophoresis due to their simplicity to implement and regenerate. However, the performances of the separation are strongly dependent on the nature of the polyelectrolyte partners used to build the SMIL coating. In this work, we investigate new couples of polyelectrolytes that were not tested before: namely, polybrene (PB), quaternized diethylaminoethyl dextran (DEAEDq) and ε-poly(lysine) (ε-PLL), as polycations, in combination with poly(acrylic acid), dextran sulfate, poly(styrenesulfonate), poly(methacrylic acid) and poly(l-lysine citramide), as polyanions. Systematic study of intra- and inter-capillaries repeatabilities/reproducibilities was performed based on the determination of migration time, separation efficiency and electroosmotic mobility. Interestingly, the electroosmotic flow was found to vary with the nature of the polycation on a broad range of electroosmotic mobility decreasing in magnitude in the order of PB>ε-PLL > DEAEDq, whatever the polyanion associated. Application of the coatings to the separation of proteins is illustrated in a 0.5 M acetic acid BGE, including CE-MS separation of ribonuclease B-glycoforms of the same mass (positional or structural isomers).

Using ZIF-8 as stationary phase for capillary electrophoresis separation of proteins.

Recently, the separation of proteins has received much attention, although many techniques require expensive instrumentation and trained analysts. In this work, a low-cost, effective, and environmental friendship capillary electrophoresis (CE) for proteins separation was first time introduced. The ZIF-8 with outstanding properties of large surface area, and accessible tunnels and cages were coated the inner surface of silica capillary as a separation media by electrostatic interaction. The fast baseline separation of Lys, CC, BSA and RNase A can be obtained within 10 min using the ZIF-8 nanocrystals coated capillary column under the optimum separation conditions. Meanwhile, this system showed good reproducibility and stability. Using L-glutamic acid as the selector ligand, the D- and L-phenylalanine were successfully separated by the ZIF-8 nanocrystals coated capillary column. Furthermore, the method was also applied to separate egg white proteins, and three main proteins were separated in a single run.

Analysis and characterization of aluminum chlorohydrate oligocations by capillary electrophoresis.

Aluminum chlorohydrates (ACH) are the active ingredients used in most antiperspirant products. ACH is a water soluble aluminum complex which contains several oligomeric polycations of aluminum with degrees of polymerization up to Al13 or Al30. The characterization and quantification of ACH oligo-cations remain a challenging issue of primary interest for developing structure/antiperspirant activity correlations, and for controlling the ACH ingredients. In this work, highly repeatable capillary electrophoresis (CE) separation of Al3+, Al13 and Al30 oligomers contained in ACH samples was obtained at pH 4.8, owing to a careful choice of the background electrolyte counter-ion and chromophore, capillary I.D. and capillary coating. This is the first reported separation of Al13 and Al30 oligomers in conditions that are compatible with the aluminum speciation in ACH solution or in conditions of antiperspirant application/formulation. Al13 and Al30 effective charge numbers were also determined from the sensitivity of detection in indirect UV detection mode. The relative mass proportion of Al13 compared to Al13+Al30 could be determined in different aluminum chlorohydrate samples. Due to its simplicity, repeatability/reproducibility, minimal sample preparation and mild analytical conditions, CE appears to be a promising analytical separation technique for the characterization of ACH materials and for the study of structure/antiperspirant activity correlations.

Self-assembled and covalently linked capillary coating of diazoresin and cyclodextrin-derived dendrimer for analysis of proteins by capillary electrophoresis.

Self-assembled and covalently linked capillary coatings of cyclodextrin-derived (CD) dendrimer were prepared using photosensitive diazoresin (DR) as a coupling agent. Layer by layer (LBL) self-assembled DR/CD-dendrimer coatings based on ionic bonding was fabricated first on the inner surface of capillary, and subsequently converted into covalent bonding after treatment with UV light through a unique photochemistry reaction of DR. Protein adsorption on the inner surface of capillary was suppressed by the DR/CD-dendrimer coating, and thus a baseline separation of lysozyme (Lys), myoglobin (Mb), bovine serum albumin (BSA) and ribonuclease A (RNase A) was achieved using capillary electrophoresis (CE). Compared with the bare capillary, the DR/CD-dendrimer covalently linked capillary coatings showed excellent protein separation performance with good stability and repeatability. Because of the replacement of highly toxic and moisture sensitive silane coupling agent by DR in the covalent coating preparation, this method may provide an environmentally friendly and simple way to prepare the covalently coated capillaries for CE.

Influence of the ionic strength of acidic background electrolytes on the separation of proteins by capillary electrophoresis.

The ionic strength is one of the key parameters for optimizing CE separations. However, only a few data are available in the literature about the ionic strength effect on the separation of proteins. The effect of ionic strength on separation performances is rather complex since many different parameters are involved: such as the protein effective mobility, the electroosmotic mobility, the separation efficiency via the electromigration dispersion, as well as the viscosity and temperature of the background electrolyte. In the present work, the influence of ionic strength on the electrophoretic separation of five model proteins has been investigated in acidic conditions, on successive multi-ionic layers coated capillary, in counter-electroosmotic mode with anodic electroosmotic flow. The decrease in effective and electroosmotic mobilities with increasing ionic strength were compared using the slope-plot approach, which is very helpful for understanding the observed changes in apparent selectivity and resolution. The relative decrease of the protein effective mobility was about 30-40% of the mobility determined at 5mM ionic strength per ionic strength decade. It was found that relatively low ionic strength (∼5-10mM) was preferable to optimize the overall separation of the five model proteins.

Polyelectrolyte multilayer coatings for the separation of proteins by capillary electrophoresis: Influence of polyelectrolyte nature and multilayer crosslinking.

The present work aims at studying the influence of the nature of the polyelectrolytes used in successive multiple ionic polymers on the performances of protein separation in acetic acid volatile background electrolyte. A broad library of polyelectrolyte multilayers was compared on the basis of 9 different weak/strong polyanions and 8 different weak/strong polycations. More than 20 couples of different polyelectrolytes were investigated. The separation efficiencies (expressed as the N/l ratio, where N is the plate number and l is the capillary effective length) were systematically compared for the separation of a protein test mixture. The coating stability was evaluated by the relative standard deviation of the migration times. For weak polyelectrolyte multilayers, the influence of the polymer crosslinking on the coating stability and separation efficiency has been studied. Intra-day repeatability of 100 successive runs, and capillary-to-capillary reproducibility were tested on coatings of each category (crosslinked and non crosslinked). The main (not obvious) result rising from this study is that the nature of the polyanion constituting the multilayers is of primary importance for the performance in terms of separation efficiency and stability, even when the mulilayers finish with a polycation.

Charge-based characterisation of high-molecular-weight glutenin subunits from common wheat by capillary isoelectric focusing.

In this study, the capillary isoelectric focusing (CIEF) method for the separation and charge characterisation of the heterogeneity of high molecular-weight-glutenin subunits (HMW-GS) in common wheat (Triticum aestivum L.) using linear polyacrylamide (LPA) and polyvinyl alcohol (PVA) coated capillaries was developed. Particularly good repeatability and well-resolved charge isoform profiles were obtained by introducing a mixture of carrier ampholytes (pH 3-10 and pH 5-8), a high concentration of urea (6M) and SB3-12 as detergent in a sample solution during separation in a PVA-coated capillary. One major and one or two minor isoforms were observed for the individual HMW-GS. These isoforms were satisfactorily separated using a pH gradient into two groups: y-type isoforms and x-type isoforms encoded by the Glu-B1 locus with shorter migration times and remaining x-type isoforms with longer times. The method produced from eight to twelve isoforms of wheat HMW-GS with pI points in the range of 4.72-6.98. Generally, the minor isoforms were more acidic compared with the major isoform. The y-type subunits had an approximately neutral character (pI 6.70-6.98); however, x-types showed a weakly acidic character (pI 4.72-5.23), with the exception of subunits encoded by the Glu-B1 locus. The isoelectric point peak profiles were compared with capillary zone electrophoresis (CZE) electropherograms. Generally, the number of detected isoforms for the particular HMW-GS detected using both methods were similar.

Capillary electrophoresis-based assessment of nanobody affinity and purity.

Drug purity and affinity are essential attributes during development and production of therapeutic proteins. In this work, capillary electrophoresis (CE) was used to determine both the affinity and composition of the biotechnologically produced "nanobody" EGa1, the binding fragment of a heavy-chain-only antibody. EGa1 is an antagonist of the epidermal growth factor receptor (EGFR), which is overexpressed on the surface of tumor cells. Using a background electrolyte (BGE) of 50mM sodium phosphate (pH 8.0) in combination with a polybrene-poly(vinylsulfonic acid) capillary coating, CE analysis of EGa1 showed the presence of at least three components. Affinity of the EGa1 components towards the extracellular domain of EGFR was assessed by adding different concentrations (0-12 nM) of the receptor to the BGE while measuring the effective electrophoretic mobility of the respective EGa1 components. Binding curves obtained by plotting electrophoretic mobility shifts as a function of receptor concentration, yielded dissociation constants (Kd) of 1.65, 1.67, and 1.75 nM for the three components, respectively; these values were comparable to the Kd of 2.1 nM obtained for the bulk EGa1 product using a cellular assay. CE with mass spectrometry (MS) detection using a BGE of 25 mM ammonium acetate (pH 8.0) revealed that the EGa1 sample comprised of significant amounts of deamidated, bisdeamidated and N-terminal pyroglutamic acid products. CE-MS using a BGE of 100mM acetic acid (pH 2.8) in combination with a polybrene-dextran sulfate-polybrene capillary coating demonstrated the additional presence of minor products related to incomplete removal of the signal peptide from the produced nanobody. Combining the results obtained from affinity CE and CE-MS, it is concluded that the EGa1 nanobody product is heterogeneous, comprising highly-related proteins that exhibit very similar affinity towards EGFR.