Introduction of a Capillary Gel Electrophoresis-Based Workflow for Biotherapeutics Characterization: Size, Charge, and N-Glycosylation Variant Analysis of Bamlanivimab, an Anti-SARS-CoV-2 Product.

Coronavirus Disease 2019 (COVID-19) is a major public health problem worldwide with 5-10% hospitalization and 2-3% global mortality rates at the time of this publication. The disease is caused by a betacoronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The receptor-binding domain (RBD) of the Spike protein expressed on the surface of the virus plays a key role in the viral entry into the host cell via the angiotensin-converting enzyme 2 receptor. Neutralizing monoclonal antibodies having the RBD as a target have the ability to inhibit angiotensin-converting enzyme 2 (ACE2) receptor binding, therefore, prevent SARS-CoV-2 infection, represent a promising pharmacological strategy. Bamlanivimab is the first anti-spike neutralizing monoclonal antibody, which got an emergency use authorization from the FDA for COVID-19 treatment. Albeit, bamlanivimab is primarily a neutralizing mAb, some of its effector function related activity was also emphasized. The effector function of antibody therapeutics is greatly affected by their N-linked carbohydrates at the conserved Fc region, possibly influenced by the manufacturing process. Various capillary gel electrophoresis methods are widely accepted in the biopharmaceutical industry for the characterization of therapeutic antibodies. In this paper we introduce a capillary gel electrophoresis based workflow for 1) size heterogeneity analysis to determine the presence/absence of the non-glycosylated heavy chain (NGHC) fragment (SDS-CGE); 2) capillary gel isoelectric focusing for possible N-glycosylation mediated charge heterogeneity determination, e.g., for excess sialylation and finally, 3) capillary gel electrophoresis for N-glycosylation profiling and sequencing. Our results have shown the presence of negligible amount of non-glycosylated heavy chain (NGHC) while 25% acidic charge variants were detected. Comprehensive N-glycosylation characterization revealed the occurrence of approximately 8.2% core-afucosylated complex and 17% galactosylated N-linked oligosaccharides, suggesting the possible existence of antibody dependent cell mediated cytotoxicity (ADCC) effector function in addition to the generally considered neutralizing effect of this particular therapeutic antibody molecule.

Single bubble in-tube microextraction coupled with capillary electrophoresis.

Headspace (HS) extraction is a sample pretreatment technique for volatile and semivolatile organic compounds in a complex matrix. Recently, in-tube microextraction (ITME) coupled with CE using an acceptor plug placed in the capillary inlet was developed as a simple but powerful HS extraction method. Here, we present single bubble (SB) ITME using a bubble hanging to the capillary inlet immersed in a sample donor solution as a HS of submicroliter volume (∼200 nL). The analytes evaporated to the bubble were extracted into the acceptor phase through the capillary opening, then electrophoresis of the enriched extract was carried out. Since the bubble volume was much smaller than a conventional HS volume (∼1 mL), it was filled with the evaporated analytes rapidly and the analytes could be enriched much faster compared to conventional HS-ITME. Owing to the high surface-to-volume ratio of the SB, 5 min SB-ITME yielded the enrichment factor values similar to those of 10 min HS-ITME. When 5 min SB-ITME at room temperature was applied to a tap water sample, the enrichment factors of 2,4,6-trichlorophenol (TCP), 2,3,6-TCP, and 2,6-dichlorophenol were 53, 41, and 60, respectively, and the LOQs obtained by monitoring the absorbance at 214 nm were 5.6-8.3 ppb, much lower than 200 ppb, the World Health Organization guideline for the maximum permissible concentration of 2,4,6-TCP in drinking water.

Facile and highly efficient three-phase single drop microextraction in-line coupled with capillary electrophoresis.

A high-performance version of in-line, three-phase direct immersion-single drop microextraction (DI-SDME) coupled with capillary electrophoresis (CE) was demonstrated using a commercial CE instrument, and all the major and minor details were described to provide an easy-to-follow and user-friendly protocol. The excellent sample cleanup and enrichment power of this method was demonstrated with nonsteroidal anti-inflammatory drugs (NSAIDs) in human urine. The only preparation of urine samples was the addition of HCl to acidify the urine sample to pH 2. The acidic NSAIDs in the acidified urine sample were extracted into a basic acceptor drop covered with a thin organic layer attached to the inlet tip of a capillary immersed in the sample. A simple but powerful DI-SDME-CE method could be carried out automatically without any modification of the existing CE instrument. For improved performance, sample agitation and heating were employed by installing a microstirrer and a thermostating jacket in the sample tray. With 10 min of DI-SDME at 35°C with stirring, NSAIDs such as ketoprofen, ibuprofen, and naproxen in urine were enriched 340-970-fold with intraday and interday RSDs of 0.8-2.4% and 1.1-3.6%, respectively. The LODs obtained with in-line coupled CE/UV were 10-50 nM (2-10 µg/L). The performance of DI-SDME-CE/UV was also demonstrated by determining the naproxen level in human urine collected 24 h after taking a single oral dose of the drug. The spike recovery of naproxen from a single-point standard addition to the urine sample was 80%. Our high-performance three-phase DI-SDME-CE method is quite promising for the analysis of ionizable trace analytes in a complex sample matrix.

Application of capillary electrophoresis-nano-electrospray ionization-mass spectrometry for the determination of N-nitrosodimethylamine in pharmaceuticals.

After a presence of highly hepatotoxic and potentially carcinogenic N-nitrosodimethylamine was detected in certain lots of sartan, ranitidine, metformin, and other pharmaceuticals, local regulatory authorities issued recalls of suspected products, and concerns of the pharmacotherapy safety were widely discussed. Since then, testing of a representative sample of each produced lot of these pharmaceuticals is required as a part of quality control processes. Hence, an interface-free CE-nanoESI system coupled with MS detection was employed for the development of a simple and economical method for quantitative detection of this contaminant in the valsartan drug substances and finished formulations used as model matrices. In this arrangement, a fused-silica capillary was used as both a separation column and a nanoESI emitter providing high ionization efficiency and sensitivity. The optimized procedure was found to have sufficient selectivity, linearity, accuracy, and precision. The established LOD and LOQ values were 0.3 and 1.0 ng/mL, respectively. The practical applicability of the method was tested by analyses of commercially available Valsacor® tablets. The results obtained prove that the developed procedure represents a promising alternative to currently available GC- and LC-based methods. Furthermore, after an adjustment of the separation conditions, the CE-nanoESI/MS system can be conceptually used for the determination of NDMA in other suspected pharmaceuticals.

Applications of deep eutectic solvents to quantitative analyses of pharmaceuticals and pesticides in various matrices: a brief review.

Pharmaceuticals and pesticides are important analytes of interest in clinical, environmental, and food analyses for ensuring public health. Sample pretreatment steps are often prerequisites for the quantitative analysis of these compounds, which are generally present in low concentrations in samples with complex matrices. In compliance with the current trend towards green analytical chemistry, the replacement of conventional toxic organic solvents with ecofriendly and safe solvents has been pursued in developing sample pretreatment methods. Subsequent to several reports in 2017, deep eutectic solvents (DESs) have been increasingly applied as desirable alternative solvents in numerous types of sample pretreatment methods for the analysis of pharmaceuticals and pesticides. The present review summarizes analytical methods involving DESs as extraction solvents and as the reaction media or functional materials for preparing adsorbents to quantify pharmaceuticals and pesticides in various matrices.

Miniaturized LC in Molecular Omics.

Miniaturized LC has evolved at an exponential rate over the last 50 years. In the past decade, it has received considerable attention in the field of bioanalytical separation science and technology due to the need to measure different classes of biomolecules present in a variety of matrixes on a global scale to gain a deeper understanding of complex biological processes. This field has become a dominant area underpinning the molecular omics research (e.g., proteomics, metabolomics, lipidomics, and foodomics), allowing key insights into the function and mechanism of small to very large biomolecules on a molecular level. This Feature highlights the recent advances in molecular omics focusing on miniaturized LC technology combined with mass spectrometry-based platforms, with a particular emphasis on the strategies adopted and applications using new and sensitive nanoscale analytical methodologies.

Analyte focusing by micelle collapse for liquid extraction surface analysis coupled with capillary electrophoresis of neutral analytes on a solid surface.

Liquid extraction surface analysis (LESA) has an advantage of directly sampling analytes on a surface, thus avoiding unnecessary dilution by homogenization of the bulk sample commonly practiced in solid sample analysis. By combining LESA with CE, the additional advantage of separating analytes before detection can be accomplished. For neutral molecules, MEKC needs to be used. Since the detection sensitivity of CE in general suffers from the small capillary dimension, analyte focusing by micelle collapse was employed for enhanced extraction in LESA and sample preconcentration for MEKC. In addition, using a commercial CE instrument, the LESA process was performed much faster and more reliably compared to our first demonstration of LESA-CE using a homemade CE setup. Three neutral water-insoluble pesticides sprayed on an apple skin were directly extracted, preconcentrated, and analyzed by the automated LESA-analyte focusing by micelle collapse-MEKC with high sensitivity in 10 min. The relative standard deviations of the migration times and peak heights were 0.8-2.1 and 1.2-3.0%, respectively when ametryn was used as an internal standard. The limits of detection obtained with UV absorbance at 200 nm were 1.8-6.4 ppb.

Synergistic coupling of in-line single-drop microextraction and on-line large-volume sample stacking for capillary electrophoresis/mass spectrometry.

Single-drop microextraction (SDME) and large-volume sample stacking using an electroosmotic flow pump (LVSEP) were coupled with capillary electrophoresis/mass spectrometry (CE/MS) for sample cleanup and preconcentration. Without filtration or centrifugation of a soil sample containing debris, SDME using a pentanol acceptor drop was directly applied to the sample. After SDME, a large volume of the enriched pentanol extract was injected and further concentrated by LVSEP. For the drop formation in SDME and the sample matrix removal in LVSEP, a run buffer vial was temporarily placed to the electrospray tip, without any physical modification of the CE/MS interface. This method enabled the double preconcentration by SDME and LVSEP, achieving 600~1300-fold enrichments of anionic analytes including pesticide and herbicide compounds to provide limits of detection in the range of 0.4~0.8 ppb in soil. Graphical abstract ᅟ.

A molecular basis behind heterophylly in an amphibious plant, Ranunculus trichophyllus.

Ranunculus trichophyllus is an amphibious plant that produces thin and cylindrical leaves if grown under water but thick and broad leaves if grown on land. We found that such heterophylly is widely controlled by two plant hormones, abscisic acid (ABA) and ethylene, which control terrestrial and aquatic leaf development respectively. Aquatic leaves produced higher levels of ethylene but lower levels of ABA than terrestrial leaves. In aquatic leaves, their distinct traits with narrow shape, lack of stomata, and reduced vessel development were caused by EIN3-mediated overactivation of abaxial genes, RtKANADIs, and accompanying with reductions of STOMAGEN and VASCULAR-RELATED NAC-DOMAIN7 (VDN7). In contrast, in terrestrial leaves, ABI3-mediated activation of the adaxial genes, RtHD-ZIPIIIs, and STOMAGEN and VDN7 established leaf polarity, and stomata and vessel developments. Heterophylly of R.trichophyllus could be also induced by external cues such as cold and hypoxia, which is accompanied with the changes in the expression of leaf polarity genes similar to aquatic response. A closely-related land plant R. sceleratus did not show such heterophyllic responses, suggesting that the changes in the ABA/ethylene signaling and leaf polarity are one of key evolutionary steps for aquatic adaptation.

Sensitive arsenic speciation by capillary electrophoresis using UV absorbance detection with on-line sample preconcentration techniques.

The World Health Organization (WHO) guideline states that the total arsenic concentration in drinking water must not exceed 10 ppb. However, arsenic toxicity varies significantly, with inorganic arsenic species being more toxic than organic species. Arsenic speciation is therefore important for evaluating the health risks from arsenic-contaminated drinking water. Capillary electrophoresis provides the necessary high performance separation to determine arsenic species in water, but its sensitivity with absorbance detection is far below than needed. Using a coated capillary, several on-line sample preconcentration techniques such as large volume sample stacking with an electroosmotic flow pump, field amplified sample injection (FASI), transient isotachophoresis (tITP), electrokinetic supercharging (EKS) combining FASI and tITP, and counter flow (CF)-EKS, were therefore investigated. With CF-EKS using phosphate and N-cyclohexyl-2-aminoethanesulfonate as leading and terminating electrolytes, respectively, standard samples of arsenite, arsenate, monomethylarsonic acid, and dimethylarsinic acid were preconcentrated from 6,300- to 45,000-fold. The limits of detection obtained with UV absorbance detection were 0.08-0.3 ppb As. For a spring water sample spiked with the four arsenic species, LODs of 2-9 ppb As were obtained, which are lower than the WHO guideline of 10 ppb total As.

Headspace in-tube microextraction coupled with micellar electrokinetic chromatography of neutral aromatic compounds.

Headspace (HS) extraction can be carried out easily and aptly via single drop microextraction coupled with capillary electrophoresis (CE). However, one drawback is the difficulty of keeping the single drop stably at the capillary tip. To solve this problem, we have recently demonstrated HS in-tube microextraction (ITME) of acidic compounds such as chlrophenols in an acidic sample using a basic run buffer plug in the separation capillary for CE as an acceptor phase. In this report, an organic acceptor plug in a capillary was used to extract neutral organic volatile pollutants such as BTEX (benzene, toluene, ethylbenzene, and m-xylene). After extraction, the analytes enriched in the organic acceptor plug were analyzed with micellar electrokinetic chromatography (MEKC). The enrichment factors for BTEX in a standard solution were up to 350 under an optimal condition of 25°C for 20 min. As an application, BTEX spiked into bottled water were analyzed with HS-ITME-MEKC, and the enrichment factors for BTEX were up to 320. The limits of detections were 1-4 ppb, which are at least 200 times lower than the US Environmental Protection Agency guidelines for drinking water, except benzene. The entire procedure of HS-ITME-MEKC was carried out automatically using a commercial CE instrument.

Rotational-State-Dependent Dispersion of Molecules by Pulsed Optical Standing Waves.

We report on the rotational-state-dependent, transverse acceleration of CS_{2} molecules affected by pulsed optical standing waves. The steep gradient of the standing wave potential imparts far stronger dipole forces on the molecules than propagating pulses do. Moreover, large changes in the transverse velocities (i.e., up to 80 m/s) obtained with the standing waves are well reproduced in numerical simulations using the effective polarizability that depends on the molecular rotational states. Our analysis based on the rotational-state-dependent effective polarizability can therefore serve as a basis for developing a new technique of state selection for both polar and nonpolar molecules.

In-line coupling of single-drop microextraction with capillary electrophoresis-mass spectrometry.

Single-drop microextraction (SDME) was in-line coupled with capillary electrophoresis-mass spectrometry to provide sample cleanup and enrichment simultaneously. Since there is no outlet vial in a conventional capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) configuration, it is not easy to hang a single drop in the capillary inlet for extraction. We overcame the difficulty of coupling SDME and CE-MS by using a temporary outlet reservoir. Basic drugs such as methamphetamine, amphetamine, phenethylamine, methoxyphenamine, and mephentermine were extracted from a basic sample solution to an acidic acceptor drop covered with a thin octanol layer formed at the capillary inlet tip. Compared to the CE-MS method in the multiple reaction monitoring (MRM) mode, the in-line SDME-CE-MS/MS technique showed 130∼150-fold enrichment in 10 min. The relative standard deviations (RSDs) of peak height ranged from 9 to 13 %. RSDs can be reduced from 4 to 6 % using mephentermine as an internal standard. We examined the pretreatment of sample with and without SDME from human urine under the full-scan mode, which confirmed that many metabolites were cleaned up by the selective extraction method of SDME. Even if the analytes from human urine were analyzed under the MRM mode used as a mass filter, there was an isobaric compound causing a disturbance to the analysis. However, in-line SDME-CE-MS/MS made it possible to perform a sample cleanup as well as sample enrichment. The research is extremely advantageous in that it is rapid, convenient, and highly sensitive for the analysis of biological samples using a commercially available instrument.

Liquid extraction surface analysis in-line coupled with capillary electrophoresis for direct analysis of a solid surface sample.

A surface-sampling technique of liquid extraction surface analysis (LESA) was in-line coupled with capillary electrophoresis (CE) to expand the specimen types for CE to solid surfaces. The new direct surface analysis method of LESA-CE was applied to the determination of organophosphorus pesticides, including glufosinate-ammonium, aminomethylphosphonic acid, and glyphosate on the external surface of a fruit such as apple. Without any sample pretreatment, the analytes sprayed on the surface of a half apple were directly extracted into a liquid microjunction formed by dispensing the extractant from the inlet tip of a separation capillary. After extraction, the analytes were derivatized in-capillary with a fluorophore 4-fluoro-7-nitro-2,1,3-benzoxadiazole and analyzed with CE-laser induced fluorescence (LIF). The limits of detection for glufosinate-ammonium, aminomethylphosphonic acid, and glyphosate were 2.5, 1, and 10ppb, respectively, which are at least 20 times lower than the tolerance limits established by the U.S. Environmental Protection Agency. Thus, we demonstrated that LESA-CE is a quite sensitive and convenient method to determine analytes on a solid surface avoiding the dilution from sample pretreatment procedures including homogenization of a bulk sample.

Novel and simple headspace in-tube microextraction coupled with capillary electrophoresis.

In liquid phase microextraction, high enrichment factors can be obtained using an acceptor phase of small volume. By hanging an acceptor drop at the separation capillary tip, single drop microextraction (SDME) can be in-line coupled with capillary electrophoresis (CE). The small surface-to-volume ratio of the drop enables high enrichment factors to be obtained in a short time. One practical issue in SDME is how to keep the drop attached to the capillary stable. Here, we present novel but extremely simple in-tube microextraction (ITME) using the liquid inside the capillary as an acceptor phase, without forming a drop at the capillary tip. As a first example, ITME has been combined with headspace (HS) extraction. Simply by placing a capillary filled with a basic run buffer in the HS above an acidic donor solution, volatile acidic analytes were extracted into the acceptor phase in the capillary. After extraction, electrophoresis of the extracts in the capillary was carried out. Owing to the robust nature of the acceptor phase, the extraction temperature and time ranges of HS-ITME can be extended significantly, compared to HS-SDME. The enrichment factors for chlorophenols in a standard solution were up to 1100 under an optimal HS-ITME condition of 80°C for 15min and the limits of detections (LODs) obtained by monitoring the absorbance at 214nm were about 4nM. The whole procedures of HS-ITME-CE were carried out automatically using built-in programs of a commercial CE instrument.

Highly sensitive analysis of catecholamines by counter-flow electrokinetic supercharging in the constant voltage mode.

Electrokinetic supercharging is one of the most powerful sample-stacking methods that combines field amplified sample injection and transient ITP. In counter-flow electrokinetic supercharging, a constant counter pressure is applied during sample injection in order to counterbalance the movement of the injected sample zone. As a result, there will be a pronounced increase in the amount of sample injected and the portion of the capillary available for electrophoresis. In this report, counter-flow electrokinetic supercharging optimization factors such as the electric field application in the constant voltage and constant current modes, the magnitude of counter pressure, and the terminating electrolyte concentrations were investigated. The enrichments obtained with a 30 min injection of 10 nM catecholamines in 5 mM terminating electrolyte solution in the constant voltage mode applying a counter pressure of 1.3 psi were 41,000-fold for dopamine, 50,000-fold for norepinephrine, and 32,000-fold for epinephrine, yielding detection limits of 1.3, 1.4, and 1.2 nM, respectively, with absorbance detection at 200 nm.

On-chip immunoassay of a cardiac biomarker in serum using a polyester-toner microchip.

An on-chip immunoassay to detect C-reactive protein (CRP) was performed using a polyester-toner (PT) microchip. CRP is a highly conserved plasma protein responding to inflammation and is used for clinical purposes to diagnose an inflammatory state. For rapid analysis and specific interactions in immunoassays, extensive studies using microfluidic chips have been carried out. Recently, a simple technique to fabricate a disposable PT microchip by a direct printing process was developed and several applications were introduced. One major drawback of the PT microchip, however, is the poor separation performance due to the quality of the microfluidic structures. This problem for a PT microchip can be overcome using a cleavable tag immunoassay, which requires minimal separation performance. After analytes are conjugated onto antibodies which are immobilized on the surface of microbeads placed on the PT microchip, a second group of fluorescently tagged antibodies are added and complexed with the analytes. The tag is then cleaved and the solution containing the cleaved tag is analyzed by electrophoresis. The time needed for the complete analysis to be carried out on a PT microchip was less than 35 min. The dynamic range of the CRP in 10-fold diluted serum was 0.3-100 mg/L and the limit of detection was 0.3 mg/L, which demonstrated the possibility of a quantitative analysis of CRP in serum in clinical trials.

Headspace-single drop microextraction with a commercial capillary electrophoresis instrument.

Automated coupling of headspace-single drop microextraction (HS-SDME) and CE has been demonstrated using a commercial CE instrument. When a drop hanging at the inlet tip of a capillary for CE is used as the acceptor phase, HS-SDME becomes a simple but powerful sample pretreatment technique for CE before injection to facilitate sample cleanup and enrichment. By combining HS-SDME with an on-line sample preconcentration technique, large volume sample stacking using an electroosmotic flow pump, the sensitivity can be improved further. The overall enrichment factors for phenolic compounds were from 1900 to 3400. HS-SDME large volume sample stacking using an electroosmotic flow pump was successfully applied to a red wine sample to obtain an LOD of 4 nM (0.8 ppb) for 2,4,6-trichlorophenol which is a precursor for 2,4,6-trichloroanisole causing the foul odor in wine called cork taint.

Near infrared dye indocyanine green doped silica nanoparticles for biological imaging.

Indocyanine green (ICG) is an FDA-approved near infrared (NIR) fluorescent dye used in clinical imaging. However, its applications remain limited due to its short half-life, nonspecific plasma binding, optical instability, and poor aqueous stability. Dye doped silica nanoparticles provide an effective barrier in keeping the dye away from the surrounding environment, but ICG cannot be encapsulated into silica easily by conventional methods. In this study, ICG molecules ion-paired with a cationic polymer polyethylenimine (PEI) were successfully encapsulated into a silica matrix to form ICG doped silica nanoparticles by using the Stöber method. Pairing with PEI reduced self-quenching of fluorescence by preventing the aggregation of ICG molecules in silica nanoparticles. Dye leakage was also reduced to the level of 3-6% loss in 5 days. NIR fluorescence images of ICG doped silica NPs below a 2.0 cm thick porcine muscle sample illuminated by NIR light were obtained.