High-resolution Capillary Zone Electrophoresis Research Articles

High‐resolution capillary zone electrophoresis and mass spectrometry for distinction of undersialylated and hypoglycosylated transferrin glycoforms in body fluids

High-resolution capillary zone electrophoresis is used to distinguish transferrin glycoforms present in human serum, cerebrospinal fluid, and serum treated with neuraminidase and N-glycosidase F. The obtained data are compared to mass spectrometry data from the literature. The main focus is on the analysis of the various asialo-transferrin, monosialo-transferrin, and disialo-transferrin molecules found in these samples. The features of capillary zone electrophoresis and mass spectrometry are reviewed and highlighted in the context of the analysis of undersialylated and hypoglycosylated transferrin molecules. High-resolution capillary zone electrophoresis represents an effective tool to assess the diversity of transferrin patterns whereas mass spectrometry is the method of choice to elucidate structural identification about the glycoforms. Hypoglycosylated transferrin glycoforms present in sera of alcohol abusers and normal subjects are structurally identical to those in sera of patients with a congenital disorder of glycosylation type I. Asialo-transferrin, monosialo-transferrin and disialo-transferrin observed in sera of patients with a type II congenital disorder of glycosylation or a hemolytic uremic syndrome, in cerebrospinal fluid and after treatment of serum with neuraminidase are undersialylated transferrin glycoforms with two N-glycans of varying structure. Undersialylated disialo-transferrin is also observed in sera with high levels of trisialo-transferrin.

Complementary middle-down and intact monoclonal antibody proteoform characterization by capillary zone electrophoresis - mass spectrometry.

High-resolution capillary zone electrophoresis - mass spectrometry (CZE-MS) has been of increasing interest for the analysis of biopharmaceuticals. In this work, a combination of middle-down and intact CZE-MS analyses has been implemented for the characterization of a biotherapeutic monoclonal antibody (mAb) with a variety of post-translational modifications (PTMs) and glycosylation structures. Middle-down and intact CZE separations were performed in an acidified methanol-water background electrolyte on a capillary with a positively charged coating (M7C4I) coupled to an Orbitrap mass spectrometer using a commercial sheathless interface (CESI). Middle-down analysis of the IdeS-digested mAb provided characterization of PTMs of digestion fragments. High resolution CZE enabled separation of charge variants corresponding to 2X-deamidated, 1X-deamidated, and non-deamidated forms at baseline resolution. In the course of the middle-down CZE-MS analysis, separation of glycoforms of the FC /2 fragment was accomplished due to hydrodynamic volume differences. Several identified PTMs were confirmed by CZE-MS2 . Incorporation of TCEP-HCl reducing agent in the sample solvent resulted in successful analysis of reduced forms without the need for alkylation. CZE-MS studies on the intact mAb under denaturing conditions enabled baseline separation of the 2X-glycosylated, 1X-glycosylated, and aglycosylated populations as a result of hydrodynamic volume differences. The presence of a trace quantity of dissociated light chain was also detected in the intact protein analysis. Characterization of the mAb under native conditions verified identifications achieved via intact analysis and allowed for quantitative confirmation of proteoforms. Analysis of mAbs using CZE-MS represents a complementary approach to the more conventional liquid-chromatography - mass spectrometry-based approaches.

High-Resolution Capillary Zone Electrophoresis with Mass Spectrometry Peptide Mapping of Therapeutic Proteins: Improved Separation with Mixed Aqueous-Aprotic Dipolar Solvents (N,N-Dimethylacetamide and N,N-Dimethylformamide) as the Background Electrolyte.

Peptide mapping with mass spectrometry (MS) detection is a powerful technique routinely used for interrogating physicochemical properties of proteins. Peptide mapping benefits from an efficient front-end separation to increase selectivity and reduce complexity prior to MS detection. The most commonly used method for peptide mapping is based on reverse phase liquid chromatography with mass spectrometry. Capillary zone electrophoresis with mass spectrometry (CZE-MS) is an orthogonal technique with growing attention for peptide mapping of biotherapeutic proteins due to its high efficiency and sensitivity. However, that growth has been slow due to poorer peptide resolution and method robustness compared to RPLC. Here we present results from optimization of CZE-MS peptide mapping separation using mixed aqueous-aprotic dipolar solvent (N,N-dimethylacetamide (DMA) and N,N-dimethylformamide (DMF), as the background electrolyte (BGE) to improve the separation performance. Addition of DMA or DMF to the BGE impacts separation selectivity through differential change in pKa of the peptides. The CZE-MS peptide mapping method with the modified BGE produced significant improvement in resolution over the conventional CZE-MS methods. The method was evaluated with both sheathless and sheathflow CE-MS ion sources.

High-Resolution Capillary Zone Electrophoresis with Mass Spectrometry Peptide Mapping of Therapeutic Proteins: Peptide Recovery and Post-translational Modification Analysis in Monoclonal Antibodies and Antibody-Drug Conjugates.

Reversed phase liquid chromatography with mass spectrometry (RPLC-MS) peptide mapping is routinely used for interrogating molecular and structural attributes such as amino acid composition, sequence variants, and post-translational modifications (PTMs) in antibody-derived therapeutics. RPLC has some limitations that often impact the analysis of certain peptides including large hydrophobic peptides, hydrophilic di-/tripeptides and glycopeptides. Capillary zone electrophoresis with mass spectrometry (CZE-MS) has great potential for peptide mapping due to high efficiency and outstanding sensitivity. In this report we demonstrate the utility of CZE-MS as an orthogonal and complementary technique to RPLC-MS for peptide mapping analyses of antibody-drug conjugates (ADCs) and their parent antibodies. This work is based on high-resolution CZE-MS separation recently developed in our group, where a mixed aqueous-organic solvent system containing N,N-dimethylacetamide (DMA) or N,N-dimethylformamide (DMF) was used to improve the separation selectivity. The results described here show several advantages of CZE-MS for the analysis of small hydrophilic di-/tripeptides, large hydrophobic peptides, glycopeptides, and hydrophobic drug-linked peptides.

High Resolution CZE-MS Quantitative Characterization of Intact Biopharmaceutical Proteins: Proteoforms of Interferon-β1.

New and improved methods are required for the enhanced characterization of complex biopharmaceuticals, especially those with charge and glycan heterogeneity. High resolution separation and mass spectrometry (MS) analysis of intact proteoforms can contribute significantly to the characterization of such proteins, many of which are glycoproteins. Here, we report on capillary zone electrophoresis (CZE) coupled via a commercial CESI sheathless interface to an Orbitrap ELITE MS for the intact analysis of recombinant human interferon-β1 (Avonex, rhIFN-β1), a biopharmaceutical with complex glycosylation at a single N-linked site. Using a cross-linked polyethylenimine coating, column efficiencies between 350,000 and 450,000 plates were produced, allowing separation based on charge and subtle hydrodynamic volume differences. A total of 138 proteoforms were found, and 55 were quantitated. Charge species due to deamidation and sialylation were separated by CZE. Given the high column efficiency, isobaric positional isomers of a single sialic acid on biantennary glycan antennae were resolved. Further, triantennary isomers (antenna on α(1-3) or α(1-6) arms) were separated and confirmed by exoglycosidase digestion. Proteoforms of the N-terminal cleavage of methionine were detected by precursor molecular weight and top-down ETD and HCD analysis of the reduced protein. Quantitative analysis suggested potential correlations between the methionine loss with the relative amount of the deamidation, as well as the level of deamidation with glycan structure. We demonstrate that high resolution CZE separation of intact glycoprotein species coupled to MS has significant potential for the in-depth characterization and quantitative analysis of biopharmaceutical proteoforms.

Quantitative capillary zone electrophoresis method for the precise determination of charge differences arising from the manufacture of heparan-N-sulfatase

A rapid and reproducible high-resolution capillary zone electrophoresis (CZE) method capable of resolving the charge isoforms of intact heparan-N-sulfatase (HNS) has been developed to monitor the charge consistency across different batches of HNS. Separation was carried out using a bare fused silica capillary with a buffer system composed of 25mM Tris, pH 8.0. This CZE method allowed the separation and integration of 14 peaks, each arising from differences in the amount of sialic-acid and mannose-6-phosphate bearing glycoforms, which were confirmed using enzymatically modified samples. Standard conditioning and rinsing conditions of the capillary were used to achieve optimal repeatability. Excellent day-to-day precision was obtained for migration times of each peak relative to the electroosmotic flow marker with relative standard deviation (RSD)≤0.5%. The precision of the relative peak areas (peak area percentages) ranged from 0.6% to 2.8% RSD for the major isoforms (peaks 3–12), from 4.0% to 5.0% RSD for peaks 1 and 2, and from 7.4% to 23.2% RSD for peaks 13 and 14. The method was able to discriminate charge variation across different batches of HNS, including those with both significant and minor process changes.

Sensitivity investigation of high resolution capillary zone electrophoresis for serum proteins (CZE-HR SPE) for slight monoclonal bands as compared to Hevylite™

The purposes of this study were to evaluate whether there were changes in markers of cardiovascular and muscle damage and then to determine whether running economy (RE) was adversely affected due to those changes in a group of runners training for a marathon following a 26-km moderately paced outdoor long training run (LTR). Fifteen, healthy male participants (age: 35.2 ± 11.1 years; height = 171.2 ± 14.5 cm; body mass = 73.6 ± 11.9 kg; and VO2max = 63.6 ± 12.7 mL/kg/minute) completed the protocol. The LTR consisted of a 26-km run on a marked outdoor course with water stops and heart rate (HR) and rating of perceived exertion checkpoints every 5 km. Muscle damage [serum creatine kinase (CK)], delayed onset muscle soreness (DOMS), cardiorespiratory measures (HR, pulmonary ventilation, respiratory exchange ratio), muscle power, step rate (SR), and RE (oxygen consumption during treadmill running at 3.1, 3.6, and 4.0 m/second) were assessed before and 24, 48, and 72 hours after exercise. A repeated-measures analysis of variance showed that serum CK levels were significantly elevated at 24, 48, and 72 hours (145%, 112%, and 72% increase above baseline, respectively; p < 0.05). However, these significant increases in serum CK were not associated with an increase in DOMS. In addition, none of the RE measures or other dependent variables showed changes during the study. It was concluded that the muscle damage caused by a 26-km LTR was not reflected by changes in RE at submaximal speeds. Therefore, an LTR of this duration and intensity can be well tolerated in participants training for a marathon and routine training can be followed in the days after this run with few adverse consequences.

Microfluidic Picoliter-Scale Translational Spontaneous Sample Introduction for High-Speed Capillary Electrophoresis

A novel microfluidic picoliter-scale sample introduction approach was developed by combining the spontaneous injection approach with a capillary electrophoresis (CE) system based on a short capillary and slotted-vial array. A droplet splitting phenomenon at the capillary inlet end during the spontaneous sample introduction process was observed for the first time. On the basis of this phenomenon, a translational spontaneous injection approach was established to reduce sample injection volumes to the sub-100 pL range. A versatile high-speed capillary electrophoresis (HSCE) system was built on the basis of this sample injection approach with separation performance comparable to or even better than those reported in microfluidic chip-based CE systems. The HSCE system was composed of a short fused-silica capillary and an automated sample introduction system with slotted sample and buffer reservoirs and a computer-programmed translational stage. The translational spontaneous sample injection was performed by linearly moving the stage, allowing the capillary inlet first to enter the sample solution and then removing it. A droplet was left at the tip end and spontaneously drawn into the capillary by surface tension effect to achieve sample injection. The stage was continuously moved to allow the capillary inlet to be immersed into the buffer solution, and CE separation was performed by applying a high voltage between the buffer and waste reservoirs. With the use of the novel system, high-speed and efficient capillary zone electrophoresis (CZE) separation of a mixture of five fluorescein isothiocyanate (FITC) labeled amino acids was achieved within 5.4 s in a short capillary with a separation length of 15 mm, reaching separation efficiencies up to 0.40 microm plate height. Outstanding peak height precisions ranging from 1.2% to 3.7% RSD were achieved in 51 consecutive separations. By extension of the separation length to 50 mm, both high-speed and high-resolution CZE separation of eight FITC-labeled amino acids could be obtained in less than 21 s with theoretical plates ranging from 163,000 to 251,000 (corresponding to 0.31-0.20 microm plate heights). The present HSCE system also allowed fast chiral separations of FITC-labeled amino acids under micellar electrokinetic chromatography (MEKC) mode within 6.5 s.

Capillary zone electrophoresis determination of carbohydrate-deficient transferrin using the new CEofix reagents under high-resolution conditions

Capillary zone electrophoresis (CZE) with a dynamic double coating based on the new CEofix reagents is shown to provide high-resolution separations of serum transferrin (Tf) isoforms, a prerequisite for the monitoring of unusual and complex Tf patterns, including those seen with genetic variants and disorders of glycosylation. A 50 μm I.D. fused-silica capillary of 60 cm total length, an applied voltage of 20 kV and a capillary temperature of 30 °C results in 15 min CZE runs of high assay precision and thus provides a robust approach for the determination of carbohydrate-deficient transferrin (CDT, sum of asialo-Tf and disialo-Tf in relation to total Tf) in human serum. Except for selected samples of patients with severe liver diseases and sera with high levels of paraproteins, interference-free Tf patterns are detected. Compared with the use of the previous CEofix reagents for CDT under the same instrumental conditions, the resolution between disialo-Tf and trisialo-Tf is significantly higher (1.7 versus 1.4). The CDT levels of reference and patient sera are comparable, suggesting that the new assay can be applied for screening and confirmation analyses. The high-resolution CZE assay represents an attractive alternative to HPLC and can be regarded as a candidate of a reference method for CDT.

Rapid quantitative capillary zone electrophoresis method for monitoring the micro-heterogeneity of an intact recombinant glycoprotein

A simple high-resolution capillary zone electrophoresis (CZE) method capable of rapidly assessing the micro-heterogeneity of a 24 kDa molecular weight glycoprotein, has been developed. Separation is carried out using a bare silica capillary at a pH of 2.5 in a commercially available electrophoresis buffer system composed of triethanolamine and phosphoric acid. Over 30 peaks were detected within a run time of 15 min using a 27 cm capillary and approximately 60 peaks were detected using a 77 cm capillary. Although most of the peaks arise from differences in the oligosaccharide structures present on the one glycosylation site on this molecule, other forms of micro-heterogeneity due to the presence of the nonglycosylated form of this glycoprotein and various types of chemical degradation, e.g., deamidation, are also responsible for the multitude of peaks observed. Although the exact chemical identity of each peak in the resulting electropherogram of this glycoprotein is not known, useful information can be obtained for assessing comparability, stability, and batch consistency. Factors impacting the resolution, precision, accuracy, and robustness of the assay are also discussed along with inherent advantages and limitations associated with measuring the micro-heterogeneity of intact glycoproteins.

Capillary zone electrophoresis of neutral organic molecules in organic – aqueous solution

AbstractVery good separations of non‐ionic organic compounds have been achieved by capillary zone electrophoresis (CZE) using a tetrahep‐tylammonium salt as an additive in aqueous acetonitrile as solvent. A systematic study was undertaken to determine the effect of experimental parameters on electroosmotic mobility and electro‐phoretic mobility. It was found that pH, acetonitrile concentration, and the type and concentration of quaternary ammonium salt are important experimental variables. Under appropriate conditions, the separation window was enlarged and a broad range of electrically neutral organics, including very hydrophobic compounds (e. g. poly‐cyclic aromatic hydrocarbons) and fairly hydrophilic compounds were separated in a relatively short time. By adjusting the separation conditions, high resolution CZE of a specific group of neutral organic compounds could be achieved. A method for calculation of capacity factor was proposed and capacity factors for a variety of non‐lonic organic compounds were calculated.