Rapid multi-attribute characterization of intact bispecific antibodies by a microfluidic chip-based integrated icIEF-MS technology

Rapid, direct identification and quantitation of protein charge variants, and assessment of critical quality attributes with high sensitivity are important drivers required to accelerate the development of biotherapeutics. We describe the use of an enhanced microfluidic chip-based integrated imaged capillary isoelectric focusing-mass spectrometry (icIEF-MS) technology to assess multiple quality attributes of intact antibodies in a single run. Results demonstrate comprehensive detection of multiple charge variants of an aglycosylated knob-into-hole bispecific antibody. Upfront, on-chip separation by icIEF coupled to MS provides the orthogonal separation required to resolve and identify acidic posttranslational modifications including difficult-to-detect deamidation and glycation events at the intact protein level. In addition, on-chip UV detection enables pI determination and relative quantitation of charge isoforms. Six charge variant peaks were resolved by icIEF, mobilized toward the on-chip electrospray tip and directly identified by in-line icIEF-MS using a connected quadrupole time-of-flight mass spectrometer. In addition to acidic charge variants, basic variants were identified as C-terminal lysine, N-terminal cyclization, proline amidation, and the combination of modifications (not typically identified by other intact methods), including lysine and one or two hexose additions. Nonspecific chain cleavages were also resolved, along with their acidic charge variants, demonstrating highly sensitive and comprehensive intact antibody multi-attribute characterization within a 15-min run time.     

Dual-detection approach for a charge variant analysis of monoclonal antibody combination products using imaged capillary isoelectric focusing

The clinical benefits of treatments with a combination of two or more therapeutic monoclonal antibodies (mAbs) have emerged in recent years. Imaged capillary isoelectric focusing is a frequently used technology in the biopharmaceutical industry for charge variant analysis of protein therapeutics. However, with the wide concentration ranges of combination products, one component may fall within the linear detection range, whereas the other does not. Here, we report a novel methodology to explore charge variants of mAb mixtures using multiple detection techniques simultaneously. We use ultraviolet absorbance to monitor the charge variants of the high-concentration component and native fluorescence (FL) to monitor the variants of the low-concentration one. Charge variants of mixtures that span 40-fold in ratio differences can be accurately quantified with this approach. In contrast to the conventional methods, it is not necessary to prepare and analyze two samples at different concentrations and combine the results for combination product testing. Additionally, the use of FL detection enables the charge variant analysis of highly potent/low abundant mAbs in a mixture. This methodology is more quality-control friendly and efficient for the charge variant analysis of combination products with wide ratios.     

Off-line capillary electrophoresis/fully automated nanoelectrospray chip quadrupole time-of-flight mass spectrometry and tandem mass spectrometry for glycoconjugate analysis

Implementation and optimization of an off-line capillary electrophoresis (CE)/(−)nanoESIchip-quadrupole time-of-flight (QTOF) mass spectrometric (MS) and tandem MS system for compositional mapping and structural investigation of components in complex carbohydrate mixtures is described. The approach was developed for glycoscreening and applied to O-glycosylated peptides from urine of a patient suffering from α-N-acetylhexosaminidase deficiency, known as Schindler's disease. The fundamental issue of sensitivity, previously representing a serious drawback of the off-line CE/MS analysis, could be positively addressed by the off-line conjunction of CE with automated chip-based ESI-QTOF-MS to provide flexibility for CE/chip MS coupling and enhance structural elucidation of single components in heterogeneous mixtures. Copyright © 2004 John Wiley & Sons, Ltd.     

Simultaneous determination of antidepressants by non-aqueous capillary electrophoresis-time of flight mass spectrometry

Simultaneous determination of 20 antidepressants in plasma samples was carried out by non-aqueous capillary electrophoresis with time of flight mass spectrometry via electrospray ionization, where a mixture of 60 mM ammonium acetate and 1M acetic acid in acetonitrile, and water, as well as methanol (100:1:0.5, v/v/v) was selected as the background electrolyte. By using time of flight mass spectrometry, accurate mass information was obtained and the background noise was dramatically decreased, thus causing a great improvement in qualitative ability. As for the plasma sample, solid phase extraction with Oasis HLB was used. The limits of detection and quantification were in the range of 0.5-1 and 1-5 ng/ml, respectively. The sensitivity of the present method was found better, i.e. approximately 10-60 folds compared to that using photo diode array detectors because the analyte peak could be clearly distinguished from the background derived from the plasma. The present method was found very useful and practical as regards to routine analysis of plasma samples.     

Determination of urinary androgen glucuronides by capillary electrophoresis with electrospray tandem mass spectrometry

Capillary electrophoresis–electrospray tandem mass spectrometry (CE‐ESI/MS/MS) is a simple and highly sensitive method for quantifying seven urinary androgen glucuronides. The urine samples were diluted and filtered through a membrane filter, and the filtrate was injected into a CE‐MS/MS system without further sample preparation steps such as extraction and derivatization. The calibration ranges were 0.01–5 µg/mL for glucuronides of androsterone and 11β‐OHAn‐3G, and 5–500 ng/mL for glucuronides of 11‐ketoAn, DHEA, testosterone, epitestosterone and DHT. The linearity of the method was 0.992–0.998, and the limits‐of‐detection at a signal‐to‐noise ratio of 3 were 5–10 ng/mL. The coefficients of variation were in the range of 4.0–9.0% for intra‐day assay and 4.1–9.8% for inter‐day assay. The proposed method may be applicable to metabolic profiling in both quantitative and qualitative analysis. Copyright © 2008 John Wiley & Sons, Ltd.     

Quantitative analysis of microcystin variants by capillary electrophoresis mass spectrometry with dynamic pH barrage junction focusing

Dynamic pH junction is an online focusing method in CE based on the electrophoretic mobility difference of analytes in the sample matrix and the background electrolyte. An advantage of this method over the conventional CE is that the sensitivity can be significantly improved. By injecting a long sample plug in the capillary and focusing the analytes at the pH boundary between the background electrolyte and sample matrix, the LOD can be improved by 10–100 folds. The dynamic pH junction method can be easily coupled with ESI‐MS. In this work, we used this method for the analysis of microcystins (MCs). The detection limits and dynamic ranges were studied. The separation was optimized by adjusting the injection time, and concentrations and pH values of the background electrolyte. The optimization of analyte focusing leads to enhanced detection response compared to conventional injections, achieving 200–400 fold higher averaged peak heights for four microcystin (MC) variants. More importantly, this method was successfully used for the quantitative analysis of microcystins (MCs) in crude algae samples from natural water bodies, making it promising for practical applications.     

Analysis of six active components in Radix tinosporae by nonaqueous capillary electrophoresis with mass spectrometry

Nonaqueous capillary electrophoresis with mass spectrometry has advantages for the analysis of active components in herbs. Here, a rapid nonaqueous capillary electrophoresis with mass spectrometry method was developed to separate, identify, and quantify palmatin, columbin, cepharanthine, menisperine, magnoflorine, and 20‐hydroxyecdysone in Radix tinosporae . Electrospray ionization MS^1‐3 spectra of the six components were collected and possible cleavage pathways of main fragment ions were elucidated. The conditions that could affect separation, such as the composition of running buffer and applied voltage, were studied, and the conditions that could affect the mass spectrometry detection, such as the composition and flow rate of sheath liquid, the pressure of nitrogen gas, and the temperature and flow rate of the dry gas, were also optimized. Under the optimized conditions, the correlation coefficient was >0.99. The relative standard deviations of migration time and peak areas were <10%. The recoveries were calculated to be 99.31–107.80% in real samples. It has been demonstrated that the proposed method has good potential to be applied to determine the six bioactive components in Radix tinosporae .     

Characterization of industrial alkylpolyphosphonates by infusion electrospray ionization-ion trap mass spectrometry with identification of the impurities by tandem capillary zone electrophoresis

Technical grade diethylene-triaminepentakis(methylenephosphonic acid) (I), dihexamethylene-triaminepentakis(methylenephosphonic acid) (II), ethylene-diaminetetrakis(methylenephosphonic acid) (III), hexamethylene-diaminetetrakis(methylenephosphonic acid) (IV), amino-tris(methylenephosphonic acid) (V), hydroxyethyl-aminobis(methylenephosphonic acid) (VI), 1-hydroxyethylidene-1,1-diphosphonic acid (VII), and 2-phosphonobutane-1,2,4-tricarboxylic acid (VIII) were characterized by ion trap mass spectrometry with electrospray ionization (ESI-ITMS). Using the negative ion mode and acid and alkaline media, peak series corresponding to the nominal compounds and to impurities with a lower number of phosphonate groups were distinguished in I-V. Each series was constituted by [M - nH + (n - 1)Na](-) peaks and peaks produced from them by losses of water, H(3)PO(3)(or water plus HPO(2)), and combined losses. For each [M - nH + (n - 1)Na](-) peak, the number of losses coincided with the number of phosphonate groups not bound to sodium ions minus one (the group bearing the charge). Owing to the hydroxyethyl group, the spectrum of VI was dominated by the formation of intermolecular esters, with both losses and gains of water according to [nM - H +/- mH(2)O](-). A series of [M - nH + (n - 1)Na](-) peaks were observed for VII and VIII, showing in the latter case that the carboxylate groups may also form adducts with sodium ions. Losses of water and H(3)PO(3)were observed in VII, whereas losses of water, CO(2), and HPO(3) were seen in VIII. The reaction pathways leading to the production of the observed ions are described. The nominal compounds and the impurities were also separated and identified by capillary electrophoresis with ESI-ITMS detection.     

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.     

High-throughput glycosylation analysis of intact monoclonal antibodies by mass spectrometry coupled with capillary electrophoresis and liquid chromatography

The analysis of monoclonal antibodies glycosylation is a crucial quality control attribute of biopharmaceutical drugs. High throughput screening approaches for antibody glycoform analysis are required in various stages of process optimization. Here, we present high throughput screening suitable mass spectrometry-based workflows for the analysis of intact antibody glycosylation out of cell supernatants. Capillary electrophoresis and liquid chromatography were coupled with quadrupole time-of-flight mass spectrometry or Orbitrap mass spectrometry. Both separation methods offer fast separation (10–15 min) and the capability to prevent the separated cell supernatant matrix to enter the mass spectrometry by post-separation valving. Both mass spectrometry instruments provide comparable results and both are sufficient to determine the glycosylation pattern of the five major glycoforms of the measured antibodies. However, the Orbitrap yields higher sensitivity of 25 μg/mL (CE-nanoCEasy-Orbitrap mass spectrometry) and 5 μg/mL (liquid chromatography-Orbitrap mass spectrometry). Data processing was optimized for a faster processing and easier detection of low abundant glycoforms based on averaged charge-deconvoluted mass spectra. This approach combines a non-target glycoform analysis while yielding the same glycosylation pattern as the traditional approach based on extracted ion traces. The presented methods enable the high throughput screening of the glycosylation pattern of antibodies down to low μg/mL-range out of cell supernatant without any sample preparation.     

Accurate mass measurement and tandem mass spectrometry of intact globin chains identify the low proportion variant hemoglobin Lepore-Boston-Washington from the blood of a heterozygote

Within a mixture of proteins, minor polymorphic components are difficult to identify using a conventional proteomic approach. Their identification generally requires multi-dimensional separation steps, before or after proteolytic cleavage, followed by sequence analysis of the proteolytic products. In this study, we investigated the potential of tandem mass spectrometry for protein characterization by identifying the delta-beta hybrid human hemoglobin variant Lepore-Boston-Washington using electrospray ionization tandem mass spectrometry. Hemoglobin Lepore-Boston-Washington occurs mainly in heterozygotes, where it comprises approximately 10% of the total non-alpha-chains, the dominant non-alpha-chain being the normal beta (approximately 90%). Furthermore, Hemoglobin Lepore-Boston-Washington has an average molecular mass (15,865.23 Da) that is only 2 Da lower than that of the normal beta-chain (15,867.24 Da). Consequently, it cannot be resolved from the normal beta-chain by mass spectrometry. Here we show how Hemoglobin Lepore-Boston-Washington was identified directly from the diluted blood of a heterozygote by analyzing the product ions from the Lepore-Boston-Washington and normal beta-chain ions without prior separation of the individual chains. This study shows the potential of the tandem mass spectrometry for identifying a minor component in an unseparated mixture of proteins.     

Characterization of therapeutic mAb charge heterogeneity by iCIEF coupled to mass spectrometry (iCIEF–MS)

Imaged capillary isoelectric focusing (iCIEF) has emerged as an important technique for therapeutic monoclonal antibody (mAb) charge heterogeneity analysis in the biopharmaceutical context, providing imaged detection and quantitation by UV without a mobilization step. Besides quantitation, the characterization of separated charge variants ideally directly by online electrospray ionization–mass spectrometry (ESI–MS) is crucial to ensure product quality, safety, and efficacy. Straightforward direct iCIEF–MS coupling combining high separation efficiency and quantitative results of iCIEF with the characterization power of MS enables deep characterization of mAb charge variants. A short technical setup and optimized methodical parameters (30 nl/min mobilization rate, 2%–4% ampholyte concentration, 0.5–2 mg/ml sample concentration) allow successful mAb charge variant peak assignment from iCIEF to MS. Despite a loss of separation resolution during the transfer, separated intact mAb charge variants, including deamidation as well as major and minor glycoforms even from low abundant charge variants, could be characterized by online ESI–MS with high precision. The presented setup provides a large potential for mAb charge heterogeneity characterization in biopharmaceutical applications.     

Simultaneous detection of zinc dimethyldithiocarbamate and zinc ethylenebisdithiocarbamate in cabbage leaves by capillary electrophoresis with inductively coupled plasma mass spectrometry

We report a simple and highly sensitive method for the simultaneous detection of trace zinc dimethyldithiocarbamate and zinc ethylenebisdithiocarbamate by capillary electrophoresis with inductively coupled plasma mass spectrometry. Zinc dimethyldithiocarbamate and zinc ethylenebisdithiocarbamate were chelated with trans‐1,2‐diaminocyclohexane‐N,N,N′,N′‐tetraacetic acid to form a macromolecule complex. Then, these two compounds were separated by α‐cyclodextrin‐modified capillary electrophoresis within 12 min at a separation voltage of 15 kV and measured by inductively coupled plasma mass spectrometry. The developed method is sensitive with detection limit of 1.9 and 3.0 ng Zn/mL for zinc dimethyldithiocarbamate and zinc ethylenebisdithiocarbamate, respectively. By means of ultrasound‐assisted extraction methods, zinc dimethyldithiocarbamate and zinc ethylenebisdithiocarbamate spiked into cabbage leaves were successfully extracted and determined with a relative standard deviation (n  = 5) ≤ 6% and a recovery of 95–107%.     

毛细管电泳-电感耦合等离子体质谱法测定藻类中6种不同形态的砷化合物

建立了一种利用毛细管电泳与电感耦合等离子体质谱联用技术(CE-ICP-MS)分析检测6种不同形态砷化合物的方法。详细研究了缓冲溶液的种类、pH值和浓度,分离电压以及进样时间等因素对6种砷化合物的分离度、灵敏度和重现性等的影响。结果表明,在最佳条件下,三价砷(As3+)、一甲基砷(MMA)、二甲基砷(DMA)、五价砷(As5+)、砷胆碱(AsC)和砷甜菜碱(AsB)6种化合物在25 min内得到完全分离。6次平行测定中,6种砷化合物峰面积的相对标准偏差(RSD)为3%～5%,检出限(以As计)(3倍信噪比)为0.08～0.12 μg/L。应用该方法成功地对海带中6种砷化合物进行了分析,回收率为90%～103%。该方法具有耗时短、灵敏度高、样品消耗量少、稳定性好等优点,可用于藻类样品中不同形态砷化合物的分析。     

Identification of anthraquinone coloring matters in natural red dyes by electrospray mass spectrometry coupled to capillary electrophoresis

Capillary electrophoresis with UV/visible diode-array detection (DAD) and electrospray mass spectrometric (ESI-MS) detection were used for the identification of anthraquinone color components of cochineal, lac-dye and madder, natural red dyestuffs often used by ancient painters. For the purpose of such analysis, ESI-MS was found to be a much more appropriate detection technique than DAD one owing to its higher sensitivity (detection limits in the range 0.1-0.5 micro g ml(-1)) and selectivity. The method developed made it possible to identify unequivocally carminic acid and laccaic acids A, B and E as coloring matters in the examined preparations of cochineal and lac-dye, respectively. In madder, European Rubia tinctorum, alizarin and purpurin were found. The method allows the rapid, direct and straightforward identification and quantification of components of natural products used in art and could be very helpful in restoration and conservation procedures.     

Chip electrospray mass spectrometry for carbohydrate analysis

Currently two types of chip systems are used in conjunction with MS: out-of-plane devices, where hundreds of nozzles, nanospray emitters are integrated onto a single silicon substrate from which electrospray is established perpendicular to the substrate, and planar microchips, embedding a microchannel at the end of which electrospray is generated in-plane, on the edge of the microchip. In the last two years, carbohydrate research greatly benefited from the introduction and implementation of the chip-based MS. In two laboratories the advantages of the chip electrospray in terms of ionization efficiency, sensitivity, reproducibility, quality of data in combination with high mass accuracy, and resolution of detection were systematically explored for several carbohydrate classes: O- and N-glycopeptides, oligosaccharides, gangliosides and glycoprotein-derived O- and N-glycans, and glycopeptides. The current state-of-the-art in interfacing the chip electrospray devices to high-performance MS for carbohydrate analysis, and the particular requirements for method optimization in both positive and negative ion modes are reviewed here. The recent applications of these miniaturized devices and their general potential for glycomic-based surveys are highlighted.     

Capillary electrophoresis-electrospray ionization mass spectrometry using uncoated fused-silica capillaries and alkaline buffer solution for the analysis of small carboxylic acids

A simple and cost-effective capillary electrophoresis/mass spectrometric (CE/MS) method for the analysis of small carboxylic acids including succinate, malate, tartarate, maleinate and citrate, is described. All CE/MS experiments were performed with uncoated fused-silica capillaries and with alkaline volatile buffer solution (ammonium formate buffer, pH 10). Since sheath liquids have significant effects on the sensitivity in typical CE/MS applications, the effects of type and flow rate of the sheath liquids on the sensitivity of carboxylic acids were investigated. As the result, the best sensitivity was obtained with the alkaline sheath liquid (5 mM ammonium hydroxide in water/methanol (50/50, v/v) solution) at 6 μl min⁻¹. With the alkaline volatile buffer solution, sufficient electroosmotic flow (EOF) to carry all small carboxylic acids toward the cathode (MS side) was obtained, although all analytes had different electrophoretic mobilities toward the anode (the CE inlet). Taking advantage of the relatively higher EOF velocity, several carboxylic acids could be detected by MS in ESI-negative mode with a short analysis time. The R.S.D. values (n=5) for the migration time and the peak area of the carboxylic acids tested were less than 0.6 and 4.2%, respectively. The method was applied to the CE/MS analysis of carboxylic acids in apple juice to demonstrate the applicability to real samples.     

A new interface used to couple capillary electrophoresis with an inductively coupled plasma mass spectrometry for speciation analysis

In this work, a novel and high-efficiency interface has been developed in coupling CE with inductively coupled plasma MS (ICPMS). The interface completely avoids laminar flow in CE capillary caused by the suction of nebulizer, and can be easily and stably operated at room temperature with high analyte transport efficiency to ICPMS. The new interface has a liquid dead volume smaller than 5 nL, which was much smaller than those (65-2500 microL) reported previously for other interfaces. All above features led to a higher sensitivity and a better electrophoretic resolution for CE-ICPMS coupled with this new interface. With the help of this new interface, we have successfully separated and determined five species of arsenic, As(III), As(V), monomethylarsonic acid, dimethylarsinic acid and p-aminophenylarsonic acid using CE-ICPMS within 11 min with a detection limit of 0.046-0.075 ng/mL and an RSD of 2-6% (n=6).     

采用实时直接分析质谱法原位快速鉴别茶叶

采用近年来发展迅速的常温常压离子化技术——实时直接分析质谱法,建立了对茶叶中主要成分如茶氨酸、咖啡碱等的快速测定方法,通过特征的质谱信号离子,实现了对不同茶叶的快速鉴别。实时直接分析质谱法在大气压下进行,无需对茶叶进行任何的样品处理,大大缩短了分析时间,实现了原位、快速、准确且高通量的检测。     

Development of a new hybrid technique for rapid speciation analysis by directly interfacing a microfluidic chip-based capillary electrophoresis system to atomic fluorescence spectrometry

This paper represents the first study on direct interfacing of microfluidic chip-based capillary electrophoresis (chip-CE) to a sensitive and selective detector, atomic fluorescence spectrometry (AFS) for rapid speciation analysis. A volatile species generation technique was employed to convert the analytes from the chip-CE effluent into their respective volatile species. To facilitate the chip-CE effluent delivery and to provide the necessary medium for subsequent volatile species generation, diluted HCl solution was introduced on the chip as the makeup solution. The chip-CE-AFS interface was constructed on the basis of a concentric "tube-in-tube" design for introducing a KBH4 solution around the chip effluent as sheath flow and reductant for volatile species generation as well. The generated volatile species resulting from the reaction of the chip-CE effluent and the sheath flow were separated from the reaction mixture in a gas-liquid separator and swept into the AFS atomizer by an argon flow for AFS determination. Inorganic mercury (Hg(II)) and methylmercury (MeHg(I)) were chosen as the targets to demonstrate the performance of the present technique. Both mercury species were separated as their cysteine complexes within 64 s. The precision (relative standard deviation, RSD, n = 5) of migration time, peak area, and peak height for 2 mg.L(-1) Hg(II) and 4 mg.L(-1) MeHg(I) (as Hg) ranged from 0.7 to 0.9%, 2.1 to 2.9%, and 1.5 to 1.8%, respectively. The detection limit was 53 and 161 microg.L(-1) (as Hg) for Hg(II) and MeHg(I), respectively. The recoveries of the spikes of mercury species in four locally collected water samples ranged from 92 to 108%.