Rapid high-resolution characterization of functionally important monoclonal antibody N-glycans by capillary electrophoresis

Characterization of the N-glycosylation present in the Fc region of therapeutic monoclonal antibodies requires rapid, high-resolution separation methods to guarantee product safety and efficacy during all stages of process development. Determination of fucosylated oligosaccharides is particularly important during clone selection, product characterization, and lot release as fucose has been shown to adversely affect the ability of mAbs to induce antibody dependent cellular cytotoxicity (ADCC). Here, we apply a general capillary electrophoresis optimization strategy to separate functionally relevant fucosylated and afucosylated glycans on mononclonal antibody products in the presence of several high mannose oligosaccharides. The N-glycans chosen represent those most commonly reported on CHO cell derived therapeutic antibodies. A rapid (<7 min) high-resolution separation of 12 commonly reported and functionally important IgG glycans was developed by systematically evaluating the effects of selectivity (boric acid) and efficiency (linear polyacrylamide) enhancing additives. The approach can be used to rapidly optimize capillary electrophoresis separation of other glycan mixtures. Following optimization, the method was applied to overnight sample processing for automated 96 well plate-based glycosylation analyses of two nonproprietary therapeutic monoclonal antibodies, demonstrating ruggedness and suitability for high-throughput process and product monitoring applications.     

Phospholipid bilayer coatings for the separation of proteins in capillary electrophoresis

The double-chained, zwitterionic phospholipid 1,2-dilauroyl-sn-phosphatidylcholine (DLPC, C12) was investigated for its use as a wall coating for the prevention of protein adsorption in capillary electrophoresis. DLPC forms a semipermanent coating at the capillary wall, which allows excess phospholipid to be removed from the capillary prior to electrophoretic separation. A DLPC-coated capillary allowed for the separation of both cationic and anionic proteins with efficiencies as high as 1.4 million plates/m. Migration time reproducibility was less than 1.3% RSD from run to run and less than 4.0% RSD from day to day. Protein recovery was as high as 93%. Cationic and anionic proteins could be separated over a pH range of 3-10, all yielding good efficiencies (N up to 1 million plates/m). The chain length of the phospholipid affected the performance of the wall coating. The C10 analogue of DLPC (DDPC) did not form a coating on the capillary wall while the C14 analogue of DLPC (DMPC) formed a stable coating that prevented protein adsorption to the same extent as its C12 counterpart.     

Glycoprotein characterization combining intact protein and glycan analysis by capillary electrophoresis-electrospray ionization-mass spectrometry

Glycosylated proteins play important roles in a large number of biological processes. Therefore, a complete characterization in terms of glycan structures and glycoform heterogeneity is needed. In this paper, a combined approach based on glycan and intact glycoprotein analysis by capillary zone electrophoresis-electrospray-mass spectrometry (CZE-ESI-MS) is presented. Based on a new capillary coating, a CZE-ESI-MS method for the separation and characterization of intact glycoproteins has been developed and compared to a method recently introduced for the characterization of erythropoietin. The excellent glycoform separation results in high-quality mass spectra, high dynamic range, and good sensitivity, allowing the correct characterization of minor glycan modifications. Additionally, a CZE-ESI-MS separation method for underivatized N-glycans has been developed. The separation of glycans differing in the degree of sialic acids and repeats of noncharged carbohydrates is achieved. The separation power of the method is demonstrated by obtaining mobility differences in glycans differing only by 16 Da. A time-of-flight mass spectrometer allowed the correct identification of the glycan composition based on high mass accuracy and resolution, identifying even minor modifications such as the exchange of "O" by "NH". An ion trap mass spectrometer provided structural information of the underivatized glycans from fragmentation spectra. The general applicability of both methods to glycoprotein analysis is illustrated for erythropoietin, fetuin, and alpha1-acid glycoprotein. The results obtained by the glycan analysis allowed an unequivocal glyco-assignment to the masses obtained for the intact proteins as long as the protein backbone is well characterized. Furthermore, modifications found for intact proteins can be attributed to differences in the glycostructure.     

Ultra performance liquid chromatographic profiling of serum N-glycans for fast and efficient identification of cancer associated alterations in glycosylation

Glycosylation is a diverse but critically important post-translational modification that modulates the physical, chemical and biological properties of proteins. Alterations in glycosylation have been noted in a number of diseases including cancer. The discovery of alterations in the glycosylation of serum glycoproteins which may offer potential as biomarkers is attracting considerable research interest. In the current study, the significant improvements in efficiency, selectivity, and analysis speed offered by ultra performance liquid chromatography (UPLC) profiling of fluorescently labeled N-linked oligosaccharides on a recently introduced sub-2 μm hydrophilic interaction (HILIC) based stationary phase are demonstrated to identify cancer associated alterations in the serum N-glycome of patients bearing stomach adenocarcinoma. The contribution of the glycosylation present on four highly abundant serum proteins namely, IgG, haptoglobin, transferrin, and α1-acid glycoprotein was evaluated. Alterations in the glycosylation present on these four proteins isolated from the pathologically staged cancer serum using either affinity purification or two-dimensional electrophoresis were then investigated as possible markers for stomach cancer progression. In agreement with previous reports, an increase in sialylation was observed on haptoglobin, transferrin, and α1-acid glycoprotein in the cancerous state. Increased levels of core fucosylated biantennary glycans and decreased levels of monogalactosylated core fucosylated biantennary glycans were present on IgG with increasing disease progression. The speed and selectivity offered by the sub-2 μm HILIC phase make it ideal for rapid yet highly efficient separation of complex oligosaccharide mixtures such as that present in the serum N-glycome.     

高效毛细管电泳/电导检测法测定饮用水中的阴离子

本实验采用四乙烯五胺作为毛细管电泳的电渗流抑制剂,以10mmol/L Tris(三羟甲基氨基甲烷)+10mmol/L H3BO3(硼酸)+0.2mmol/L EDTA(乙二胺四乙酸)+0.2%(V/V)四乙烯五胺为电泳运行液,负高压分离(-15kV),电导检测,对饮用水中的Cl-、NO3-、SO42-三种阴离子进行了直接分离测定,并考察了分离电压,pH值,电解质溶液的组成及浓度,电渗流抑制剂,有机添加剂组成等对分离的影响。建立高效、快速的饮用水无机阴离子高效毛细管电泳/电导检测的分析方法。     

Carbohydrate analysis of a chimeric recombinant monoclonal antibody by capillary electrophoresis with laser-induced fluorescence detection

A general method for the analysis of asparaginyl-linked (N-linked) carbohydrate moieties of an IgG1 monoclonal antibody is described here. The antibody, rituximab, is a mouse/human chimeric antibody to human CD20 antigen. The glycans present on rituximab are neutral complex biantennary oligosaccharides with zero, one, and two terminal galactose residues (G0, G1, and G2, respectively). To monitor the variation of the glycosylation during manufacture, the glycans were first enzymatically released from the antibody via digestion with peptide-N-glycosidase F, then derivatized with a charged fluorophore, 8-aminopyrene-1,3,6-trisulfonic acid and further separated by capillary electrophoresis with laser-induced fluorescence detection. All observed glycans were fully resolved, including the positional isomers of G1. The exact nature of the isomers in terms of the location of the terminal galactose was further characterized via multiple enzymatic digestion steps including mannosidase with activity toward specific Man(alpha 1,3) linkage. The optimization and several key parameters, i.e., enzymatic digestion and derivatization, in the assay development will be discussed. Moreover, to ensure that the assay can be used in routine lot release testing, the assay was validated and found to be accurate and precise. The analytical approach described is suitable for characterization as well as routine testing of the N-linked glycan content in any IgG1 monoclonal antibody and glycoproteins in general.     

A probe for the versatile analysis and characterization of N-linked oligosaccharides

A novel fluorescent probe, 3-(acetylamino)-6-aminoacridine (AA-Ac), has been synthesized and its applicability to the analysis of picomole levels of N-linked glycans investigated. AA-Ac was found to be an excellent derivatization reagent for N-linked glycans, giving at least twice the intensity of fluorescence as its predecessor 2-aminoacridone. AA-Ac-labeled glycans were analyzed by both normal and reversed-phase HPLC. They were also amenable to enzymatic sequencing and analysis by MALDI-TOF mass spectrometry, free zone capillary electrophoresis, and capillary electrophoresis/electrospray ionization mass spectrometry.     

Electrophoretic analysis of N-glycans on microfluidic devices

We report rapid and efficient electrophoretic separations of N-glycans on microfluidic devices. Using a separation length of 22 cm and an electric field strength of 750 V/cm, analysis times were less than 3 min, and separation efficiencies were between 400,000 and 655,000 plates for the N-glycans and up to 960,000 plates for other sample components. These high efficiencies were necessary to separate N-glycan positional isomers derived from ribonuclease B and linkage isomers from asialofetuin. Structural isomers of N-glycans derived from a blood serum sample of a cancer patient were also analyzed to demonstrate that clinically relevant, complex samples could be separated on-chip with efficiencies similar to those derived from model glycoproteins. In addition, we compared microchip and capillary electrophoresis under similar separation conditions, and the microchips performed as well as the capillaries. These results confirmed that the noncircular cross section of the microchannel did not hamper separation performance. For all experiments, the glycan samples were derivatized with 8-aminopyrene-1,3,6-trisulfonic acid to impart needed charge for electrophoresis and a fluorescent label for detection.     

毛细管区带电泳分离甲酚异构体的研究

本文系统地研究了邻、间、对甲酚三种异构体在毛细管区带电泳中的迁移行为。通过实验研究讨论了缓冲溶液类型、缓冲溶液的pH值、缓冲溶液的浓度和添加剂等因素对三种异构体分离的影响,获得了优化的分离条件。结果表明,在使用未涂层石英毛细管柱(50μm×50cm,有效长度为45 cm),检测波长225 nm,磷酸盐-环糊精-硼砂缓冲溶液浓度30 mmol/L,缓冲液pH值为11.60,分离电压为15 kV的条件下,甲酚三种异构体得到基线分离。     

Organically modified silica sol-mediated capillary electrophoresis

We describe in this paper the use of ormosil (organically modified silica) sols as additives to the run buffer for selectivity manipulations between solutes in capillary electrophoresis. CE systems that contain sol additives in the run buffer can be thought of as pseudocapillary electrochromatography. Three sols based on different types of silanes were studied. Methyltrimethoxysilane (MTMOS)-based sol was found to improve selectivities between various aromatic acids. Aminopropyltrimethoxysilane (APS) sol interacts differently with structural isomers of aromatic acids than does MTMOS. At low pH with APS sol in the run buffer, neutral solutes can be separated, as well. The separation of the neutral solutes seems to be facilitated by the formation of hydrogen bonds between the solutes and the APS sol. APS and N-[3-(trimethoxysilyl)propyl]-ethylenediamine (EDAS) affect the separation of the same compounds differently, thus indicating that even small changes of the functional groups of the sol have pronounced effect on the interactions between the sols and the solutes.     

Electrophoresis in organogels

A new matrix for electrophoresis, a low molecular weight organogel, is described. Dansylated amino acids and peptides were separated by planar and capillary electrophoresis in acetonitrile gels of trans-(1S,2S)-1,2-bis(dodecylamido)cyclohexane. The superior separation ability of the organogel over its corresponding buffer solution in capillary electrophoresis is illustrated. Organogels provide all the advantages associated with planar matrixes with 100% efficient recovery and transfer of the analytes to a mass spectrometer. We demonstrate that the planar gel can be liquefied and injected as is into an ESI-MS to identify the separands.     

Determination of lead in human saliva by combined cloud point extraction-capillary zone electrophoresis with indirect UV detection

A micelle-mediated phase separation without added chelating agents to preconcentrate trace levels of lead in human saliva as a prior step to its determination by capillary electrophoresis has been developed. The enrichment step is based on the cloud point extraction of lead with the non-ionic surfactant PONPE 7.5 in the absence of chelating agent. The surfactant-rich phase was diluted with acetonitrile and the resultant solution was injected directly into the CE instrument. Factors affecting the combined methodology such as surfactant-rich phase diluting agent, buffer pH and concentration, applied voltage, sample preparation and presence of additives were studied in detail. A BGE of 20 mM imidazole containing 30% acetonitrile, pH 6.20 was found to be optimal for the separation of lead from other saliva constituents. Indirect detection was performed at 205 nm. The detection limit value of lead for the preconcentration of 8 ml of saliva was 11.4 microg l(-1). The calibration graph using the preconcentration system was linear with a correlation coefficient of 0.997 at levels near the detection limits up to at least 400 microg l(-1). The reproducibility (R.S.D.) on the basis of migration time and peak area were better than 0.68 and 3.6%, respectively. The method was successfully applied to the determination of lead in human saliva.     

Performance of SU-8 microchips as separation devices and comparison with glass microchips

Effective analytical performance of native, all-SU-8 separation microdevices is addressed by comparing their performance to commercial glass microdevices in microchip zone electrophoresis accompanied by fluorescence detection. Surface chemistry and optical properties of SU-8 microdevices are also examined. SU-8 was shown to exhibit repeatable electroosmotic properties in a wide variety of buffers, and SU-8 microchannels were successfully utilized in peptide and protein analyses without any modification of the native polymer surface. Selected, fluorescent labeled, biologically active peptides were baseline resolved with migration time repeatability of 2.3-3.6% and plate numbers of 112,900-179,800 m(-1). Addition of SDS (0.1%) or SU-8 developer (1.0%) to the separation buffer also enabled protein analysis by capillary zone electrophoresis. Plate heights of 2.4-5.9 microm were obtained for fluorescent labeled bovine serum albumin. In addition, detection sensitivity through SU-8 microchannels was similar to that through BoroFloat glass, when fluorescence illumination was provided at visible wavelengths higher than 500 nm. On the whole, the analytical performance of SU-8 microchips was very good and fairly comparable to that of commercial glass chips as well as that of traditional capillary electrophoresis and chromatographic methods. Moreover, lithography-based patterning of SU-8 enables straightforward integration of multiple functions on a single chip and favors fully microfabricated lab-on-a-chip systems.     

Instrumentation for medium-throughput two-dimensional capillary electrophoresis with laser-induced fluorescence detection

In two-dimensional capillary electrophoresis, a sample undergoes separation in the first dimension capillary by sieving electrophoresis. Fractions are periodically transferred across an interface into a second dimension capillary, where components are further resolved by micellar electrokinetic capillary electrophoresis. Previous instruments employed one pair of capillaries to analyze a single sample. We now report a multiplexed system that allows separation of five samples in parallel. Samples are injected into five first-dimension capillaries, fractions are transferred across an interface to 5 second-dimension capillaries, and analyte is detected by laser-induced fluorescence in a five-capillary sheath-flow cuvette. The instrument produces detection limits of 940 +/- 350 yoctomoles for 3-(2-furoyl)quinoline-2-carboxaldehyde labeled trypsin inhibitor in one-dimensional separation; detection limits degrade by a factor of 3.8 for two-dimensional separations. Two-dimensional capillary electrophoresis expression fingerprints were obtained from homogenates prepared from a lung cancer (A549) cell line, on the basis of capillary sieving electrophoresis (CSE) and micellar electrophoresis capillary chromatography (MECC). An average of 131 spots is resolved with signal-to-noise greater than 10. A Gaussian surface was fit to a set of 20 spots in each electropherogram. The mean spot width, expressed as standard deviation of the Gaussian function, was 2.3 +/- 0.7 transfers in the CSE dimension and 0.46 +/- 0.25 s in the MECC dimension. The standard deviation in spot position was 1.8 +/- 1.2 transfers in the CSE dimension and 0.88 +/- 0.55 s in the MECC dimension. Spot capacity was 300.     

Reproducible two-dimensional capillary electrophoresis analysis of Barrett's esophagus tissues

We have constructed a high-speed, two-dimensional capillary electrophoresis system with a compact and high-sensitivity fluorescence detector. This instrument is used for the rapid and reproducible separations of Barrett's esophagus tissue homogenates. Proteins and biogenic amines are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde. Labeled biomolecules are separated sequentially in two capillaries. The first capillary employs capillary sieving electrophoresis using a replaceable sieving matrix. Fractions are successively transferred to a second capillary where they undergo additional separation by micellar electrokinetic capillary chromatography. The comprehensive two-dimensional separation requires 60 min. Within-day migration time reproducibility is better than 1% in both dimensions for the 50 most intense features. Between-day migration time precision is 1.3% for CSE and better than 0.6% for MECC. Biopsies were obtained from the squamous epithelium in the proximal tubular esophagus, Barrett's epithelium from the distal esophagus, and fundus region of the stomach from each of three Barrett's esophagus patients with informed consent. We identified 18 features from the homogenate profiles as biogenic amines and amino acids. For each of the patients, Barrett's biopsies had more than 5 times the levels of phenylalanine and alanine as compared to squamous tissues. The patient with high-grade dysplasia shows the highest concentrations for 13 of the amino acids across all tissue types. Concentrations of glycine are 40 times higher in squamous biopsies compared to Barrett's and fundal biopsies from the patient with high-grade dysplasia. These results suggest that two-dimensional capillary electrophoresis may be of value for the rapid characterization of endoscopic and surgical biopsies.     

Capillary zone electrophoresis in nonaqueous solvents in the presence of ionic additives

Capillary zone electrophoresis (CZE) in nonaqueous media and in the presence of ionic additives has been successfully applied to the determination of compounds that differ only slightly in their electrophoretic mobilities. Triazine herbicides of environmental interest were chosen as test compounds because they behave as very weak bases. CZE separation of these analytes (especially chlorotriazines) in aqueous solution is difficult due to the low pH required for their conversion into protonated cationic form (HA(+)). However, in mixed nonaqueous solvents, 50% (v/v) acetonitrile-methanol, the acid-base characteristics of these compounds are modified, yielding the protonated ionic species that is susceptible to migration when subjected to an electric field. A noteworthy increase in separation selectivity and resolution can be achieved by using ionic additives. Thus, in this mode of capillary zone electrophoresis, separation is based on ionic interactions between the charged analytes and the ionic additive present in the separation medium. These interactions contribute to enhancing mobility differences and to improving analyte separation. For the separation of chloro- and methylthiotriazines, 10 mM perchloric acid in 50% (v/v) acetonitrile-methanol and 20 mM SDS proved to be satisfactory, providing high resolution in short analysis times. The selectivity achieved was found to depend on the degree of association of the analyte with the ionic additive in the nonaqueous medium. This permits manipulation of the selectivity of the electrophoretic separations as a function of the type and concentration of the ionic additive and of the nature of the nonaqueous medium employed.     

Separation of DNA restriction fragments by capillary electrophoresis using coated fused silica capillaries.

The abilities of several different capillary electrophoresis techniques to separate DNA restriction fragments up to 23,000 bp were investigated. Methods employing electroosmotic flow in an untreated silica capillary were found to provide, at best, only partial resolution of the 23 fragments in a 1-kbp DNA ladder. By coating the inner walls of a silica capillary with poly(acrylamide) and filling these capillaries with buffers containing methylcellulose as a sleving medium, all fragments in the 1-kbp DNA ladder were separated. In addition, this technique facilitated the separation of the very large fragments in a lambda DNA-HindIII digest. Optimum resolution was obtained at low separation potentials using buffers containing at least 0.5% methylcellulose. The performance of this technique, i.e., resolution and quantitation, make capillary electrophoresis a powerful complement to slab gel electrophoresis and may make it a preferred alternative to both agarose gel electrophoresis and HPLC for applications such as the confirmation of plasmid integrity.     

A water-soluble tetraethylsulfonate derivative of 2-methylresorcinarene as an additive for capillary electrophoresis

A water-soluble tetraethylsulfonate derivative of 2-methylresorcinarene (TESMR), an aromatic-based, bowl-shaped macrocycle, was used as an additive in capillary electrophoresis. Several phenol derivatives are used as analytes to demonstrate the effect of this highly charged additive. TESMR is observed to interact differently with a mixture of positional isomers and other types of phenol derivatives. A comparative study of separations with two charged additives, TESMR and sulfobutyl ether-β-cyclodextrin (SBE-β-CD), provided insight into the selectivity exhibited by these additives. The influence of buffer pH, ionic strength, and organic modifier content on separation and peak shape is investigated. Peak asymmetry caused by the use of highly charged additives at lower pH is minimized by the addition of small amounts of a polar aprotic organic solvent to the run buffer. The effects of mixing a charged additive with a neutral additive are also discussed.     

Separation of recombinant human erythropoietin glycoforms by capillary electrophoresis using volatile electrolytes. Assessment of mass spectrometry for the characterization of erythropoietin glycoforms

The separation of the glycoforms of erythropoietin (EPO) by capillary electrophoresis (CE) was recently published as a monograph by the European Pharmacopoeia (European Pharmacopoeia 4 2002, 1316, 1123-1128). Although the experimental CE conditions employed a background electrolyte containing additives suitable for on-line UV-absorption detection, they were not appropriate for on-line mass spectrometry (MS) detection. In this work, an attempt was made to investigate experimental conditions employing volatile electrolyte systems to achieve the separation and characterization of EPO glycoforms using CE and ESI-MS methodologies. The influence of several operating conditions, such as the coating of the internal walls of the capillary as well as the composition, concentration, and the pH of the separation buffer were investigated. The results demonstrated that when the internal walls of the capillaries were permanently coated with Polybrene and a buffer electrolyte containing 400 mM of HAc-NH4Ac (acetic acid-ammonium acetate), pH 4.75, was used, a significantly reproducible separation was achieved for EPO glycoforms. Intact EPO was characterized by two mass spectrometry techniques: electrospray ionization (ESI-MS) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF-MS). The data demonstrated that MALDI-TOF-MS provided a good approximation to an average molecular mass of the EPO molecule. However, it was still necessary to carry out further separation of the intact EPO glycoforms in order to obtain molecular mass information when ESI-MS was used.     

On-line CE-LIF-MS technology for the direct characterization of N-linked glycans from therapeutic antibodies

Glycan characterization of therapeutic proteins is of utmost importance due to the role of carbohydrates in protein stability, half-life, efficacy and mechanism of action. The primary assay for characterization and lot release of N-linked glycans on glycoprotein products at Genentech, Inc., is a capillary electrophoresis (CE) based assay, wherein PNGase F-released, APTS-labeled glycans are separated by CE with laser induced fluorescence (LIF) detection. With the growing number of new molecular entities in the pipeline, a fast and direct characterization approach is of increasing importance. This paper describes the development of CE-MS technology with on-line LIF detection that allows identification of major and minor glycan species (1-5% of total glycans) by providing accurate mass information. Data is presented for therapeutic rMAbs which presented previously unidentified, minor peaks during routine CE-LIF analysis. CE-LIF-MS was then used to provide accurate mass on these species, identifying CE peaks corresponding to sialylated (G1 + NANA, G2 + NANA), afucosylated (G0-GlcNAc-fucose) and low-level isomers of major APTS-labeled glycans G0, G1, G1' and G2.