Cross-identification of N-Glycans by CE-LIF using two capillary coatings and three labeling dyes.

Recombinant protein biopharmaceuticals comprise a significant portion of the current drug development landscape. The glycosylation profile of these proteins is a key quality parameter as it can affect their safety, efficacy, and stability. However, glycan analysis is challenging because of the complexity of their structures. To overcome this challenge in achieving accurate glycan identification, cross-identification of N-Glycans by CE-LIF method using two capillary coatings and three labeling dyes was developed in this work. This work explored whether complementary separation capabilities can be achieved using homemade polyvinyl alcohol (PVA) coating and commercial Guarant™ (Guarant) coating in the analysis of N-glycans. Similar separation profiles were observed using the two capillary coatings, and hence the N-glycan GU databases generated by these coatings were comparable and complementary. The performance of cross-validation by labeling with three fluorescent dyes indicated that low covariance of APTS and Turquoise™ labeling can be obtained, and hence these two labeling mechanisms provided better accuracy for the identification of glycans. Superior reproducibility with RSDs less than 1% for all target glycan standards was achieved by the internal standards (IS) method using maltodextrin ladders as additives in the separation buffer. The developed CE-LIF analysis method was applied to the identification of N-glycans in IgG samples.

Detection of Endotoxins: From Inferring the Responses of Biological Hosts to the Direct Chemical Analysis of Lipopolysaccharides.

A significant portion of the scientific effort has been devoted to the detection of endotoxins in pharmaceutical solutions, as they pose major health threats as contaminants even in minute amounts. Conventional methods based on the biological response of endotoxins have been well-established, but as technology advances, many limitations surfaced in the recent years. As a result, information obtained by chemical analytical methods becomes valuable in crossvalidating these results. In addition to providing an overview on the main strategies for detecting the presence of endotoxins, the biological methods are compared with the chemical techniques. The review also investigates future advances aimed toward a more accurate, reliable, and convenient endotoxin test.

High throughput human plasma N-glycan analysis using DNA analyzer and multivariate analysis for biomarker discovery.

Carbohydrates form the majority of organic compounds found in nature and their presence on proteins influences many important bioactivities. Therefore, glycan profiling shows potential in clinical applications. This work demonstrates the use of a high-throughput GlycanAssure™ sample preparation technology and multi-capillary DNA analyzer for the analysis of the major N-linked glycans (N-glycans) found in human plasma. The application involves two biomarker studies: (1) in profiling patients with chronic kidney disease and (2) in differentiating heart disease patients with normal controls in response to an antiplatelet drug from hypo-responders. Due to complexity of the study data, bio-statistical methods were applied to data processing. 37 N-glycan peaks were observed from separation results, with confirmed structure for most glycans. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to build models to differentiate the patient groups. The percentages of correct classification of the models reached 95.45% for the chronic kidney disease dataset and 85.42% for the anti-platelet drug response dataset. Given that blood N-glycan profiles had been shown to reflect certain disease states, this high-throughput platform could potentially be used for the simultaneous screening of multiple glycan biomarkers, with as little as one drop of blood sample.

Extraction, separation and characterization of endotoxins in water samples using solid phase extraction and capillary electrophoresis-laser induced fluorescence.

This study focuses on one of the key environmental threats, endotoxins, also known as lipopolysaccharides (LPS). A capillary electrophoresis method in combination with laser induced fluorescence (LIF) detection was developed for the analysis of endotoxins from 16 different bacterial strains. LPSs were derivatized with the amino-reactive fluorescent dye, fluorescein isothiocyanate (FITC), separated by capillary zone electrophoresis (CZE) under the optimized conditions with the use of 50 mM sodium tetraborate buffer (pH 9.30), and detected by LIF detector. To improve the sensitivity of CZE-LIF detection for the determination of trace amounts of endotoxins and to remove possible interference materials in environmental samples, a solid phase extraction (SPE) pre-concentration technique was applied successfully. The SPE targeted at polysaccharide moieties of LPSs and showed LPS enrichment effects too. CE migration time could also reveal the O-antigen chain lengths of LPSs. This CE method and SPE pretreatment showed linearity at 99.84%, and repeatabilities at 8.44% and 11.0% for endotoxins from E. Coli O55:B5 and E. Coli O26:B6. The limit of detection (LOD) could reach around 5 ng/mL at optimized condition. The method was applied successfully to the determination of LPS levels in tap water and wastewater, and demonstrated sensitive, reproducible and reliable results.

Multiplexing N-glycan analysis by DNA analyzer.

Analysis of N-glycan structures has been gaining attentions over the years due to their critical importance to biopharma-based applications and growing roles in biological research. Glycan profiling is also critical to the development of biosimilar drugs. The detailed characterization of N-glycosylation is mandatory because it is a nontemplate driven process and that significantly influences critical properties such as bio-safety and bio-activity. The ability to comprehensively characterize highly complex mixtures of N-glycans has been analytically challenging and stimulating because of the difficulties in both the structure complexity and time-consuming sample pretreatment procedures. CE-LIF is one of the typical techniques for N-glycan analysis due to its high separation efficiency. In this paper, a 16-capillary DNA analyzer was coupled with a magnetic bead glycan purification method to accelerate the sample preparation procedure and therefore increase N-glycan assay throughput. Routinely, the labeling dye used for CE-LIF is 8-aminopyrene-1,3,6-trisulfonic acid, while the typical identification method involves matching migration times with database entries. Two new fluorescent dyes were used to either cross-validate and increase the glycan identification precision or simplify sample preparation steps. Exoglycosidase studies were carried out using neuramididase, galactosidase, and fucosidase to confirm the results of three dye cross-validation. The optimized method combines the parallel separation capacity of multiple-capillary separation with three labeling dyes, magnetic bead assisted preparation, and exoglycosidase treatment to allow rapid and accurate analysis of N-glycans. These new methods provided enough useful structural information to permit N-glycan structure elucidation with only one sample injection.

An efficient "off-on" carbon nanoparticle-based fluorescent sensor for recognition of chromium(vi) and ascorbic acid based on the inner filter effect.

A simple approach based on calcination treatment of diethylenetriaminepentaacetic acid (DTPA) was developed to prepare water-soluble nitrogen doped carbon nanoparticles (N-CNPs) with a high quantum yield of approximately 53.7%. The fluorescence of N-CNPs could be quickly and efficiently quenched by Cr(vi) rather than Cr(iii) based on an inner filter effect (IFE) process. The addition of ascorbic acid (AA) can recover the intensity of fluorescence of the N-CNP-Cr(vi) system through the reduction of Cr(vi) to Cr(iii) and inhibit the IFE process between N-CNPs and Cr(vi) (turn-on). Accordingly, an efficient N-CNP based fluorescent probe for sensitive and selective sensing of Cr(vi) ions and l-ascorbic acid (AA) has been established. The proposed fluorescence sensor displays excellent performance for Cr(vi) determination in the range from 0.5 to 160 µmol L-1 (R2 = 0.998) with a detection limit down to 0.15 µmol L-1. Moreover, the observed linear response concentration range was from 1 to 400 µmol L-1 for AA with a detection limit as low as 0.13 µmol L-1. The fluorescent probe was successfully applied to detect Cr(vi) concentration in different water samples and AA concentration in human serum samples.

Parallel analysis and orthogonal identification of N-glycans with different capillary electrophoresis mechanisms.

The deep involvement of glycans or carbohydrate moieties in biological processes makes glycan patterns an important direction for the clinical and medicine researches. A multiplexing CE mapping method for glycan analysis was developed in this study. By applying different CE separation mechanisms, the potential of combined parallel applications of capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC) and capillary gel electrophoresis (CGE) for rapid and accurate identification of glycan was investigated. The combination of CZE and MEKC demonstrated enhancing chromatography separation capacity without the compromises of sample pre-treatment and glycan concentration. The separation mechanisms for multiplexing platform were selected based on the orthogonalities of the separation of glycan standards. MEKC method exhibited promising ability for the analysis of small GU value glycans and thus complementing the unavailability of CZE. The method established required only small amount of samples, simple instrument and single fluorescent labelling for sensitive detection. This integrated method can be used to search important glycan patterns appearing in biopharmaceutical products and other glycoproteins with clinical importance.

Identification of functional elements of the GDP-fucose transporter SLC35C1 using a novel Chinese hamster ovary mutant.

The GDP-fucose transporter SLC35C1 critically regulates the fucosylation of glycans. Elucidation of its structure-function relationships remains a challenge due to the lack of an appropriate mutant cell line. Here we report a novel Chinese hamster ovary (CHO) mutant, CHO-gmt5, generated by the zinc-finger nuclease technology, in which the Slc35c1 gene was knocked out from a previously reported CHO mutant that has a dysfunctional CMP-sialic acid transporter (CST) gene (Slc35a1). Consequently, CHO-gmt5 harbors double genetic defects in Slc35a1 and Slc35c1 and produces N-glycans deficient in both sialic acid and fucose. The structure-function relationships of SLC35C1 were studied using CHO-gmt5 cells. In contrast to the CST and UDP-galactose transporter, the C-terminal tail of SLC35C1 is not required for its Golgi localization but is essential for generating glycans that are recognized by a fucose-binding lectin, Aleuria aurantia lectin (AAL), suggesting an important role in the transport activity of SLC35C1. Furthermore, we found that this impact can be independently contributed by a cluster of three lysine residues and a Glu-Met (EM) sequence within the C terminus. We also showed that the conserved glycine residues at positions 180 and 277 of SLC35C1 have significant impacts on AAL binding to CHO-gmt5 cells, suggesting that these conserved glycine residues are required for the transport activity of Slc35 proteins. The absence of sialic acid and fucose on Fc N-glycan has been independently shown to enhance the antibody-dependent cellular cytotoxicity (ADCC) effect. By combining these features into one cell line, we postulate that CHO-gmt5 may represent a more advantageous cell line for the production of recombinant antibodies with enhanced ADCC effect.

IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines.

A Tricistronic vector utilizing internal ribosome entry site (IRES) elements to express the light chain (LC), heavy chain (HC), and a neomycin phosphotransferase (NPT) selection marker from one transcript is designed for generation of mAb expressing CHO cell lines. As compared to the commonly used vectors, benefits of this design include: (1) minimized non-expressing clones, (2) enhanced stable mAb productivity without gene amplification, (3) control of LC and HC expression at defined ratios, and (4) consistent product quality. After optimization of the LC and HC arrangement and increasing selection stringency by weakening the NPT selection marker, this Tricistronic vector is able to generate stably transfected pools with specific productivity (qmAb) greater than 5pg/cell/day (pcd) and titers over 150mg/L. 5% of clones from these pools have qmAb greater than 20pcd and titers ranging from 300 to more than 500mg/L under non-optimized shake flask batch cultures using commercially available protein-free medium. The mAb produced by these clones have low aggregation and consistent glycosylation profiles. The entire process of transfection to high-expressing clones requires only 6 months. The IRES-mediated Tricistronic vector provides an attractive alternative to commonly used vectors for fast generation of mAb CHO cell lines with high productivity.

Rapid characterization of protein productivity and production stability of CHO cells by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The biopharmaceutical industry has been in pursuit of strategies which can isolate stable and high-producing cell lines. The whole cell mass spectrometry method by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) is a rapid and simple method for cell characterization based on the differences in the fingerprints of the mass spectra. This work describes how the method was evaluated for the application of screening for stable and high-producing clones from a panel of recombinant Chinese hamster ovary (CHO) cell lines. Detectable m/z values and their relative intensities were collected and processed by partial least squares (PLS). To reduce the errors introduced by the preparation method and spectra noise, high intensity preliminary data was selected and the number of variables introduced was validated by leave-one-out cross-validation. The differences in recombinant protein productivity and titer were revealed by PLS regression with promising results. Partial least-squares discriminant analysis (PLS-DA) was applied to differentiate stable and unstable cell lines as traditional stability testing would require several months involving numerous continuous passages. Results confirmed that the whole cell MALDI-TOF method can be a powerful method for routine monitoring of bioprocesses and study can be further developed by extending the number of the cell lines tested to establish a recombinant cell line database.

Rapid characterization of high/low producer CHO cells using matrix-assisted laser desorption/ionization time-of-flight.

An intact-cell mass spectrometry (ICM) method using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) was evaluated for the screening of stable recombinant Chinese hamster ovary (CHO) cell lines, an important mammalian cell line in bioprocessing. With rapid and simple cell pretreatments, viabilities of cells could be rapidly distinguished on the different fingerprints of mass spectra. Detectable m/z values on cell surfaces and their relative intensities were processed by two biostatistical methods, principle components analysis (PCA) and partial least squares (PLS), with promising results. Discrimination among cell lines with different expressed recombinant proteins or different productivities could be achieved. The ICM method has the advantage of providing multiple parameters simultaneously and possesses the potential to become a powerful method for routine monitoring of bioprocesses.

Simultaneous determination of 19 intracellular nucleotides and nucleotide sugars in Chinese Hamster ovary cells by capillary electrophoresis.

Twelve nucleotides and seven nucleotide sugars in Chinese Hamster ovary (CHO) cells were determined by capillary electrophoresis (CE). The CE operating conditions of buffer pH value, ion strength, capillary temperature, polymer additive and cell extraction method were investigated. Optimum separation was achieved with 40 mM sodium tetraborate buffer (pH 9.5) containing 1% (w/v) polyethylene glycol (PEG) at a capillary temperature of 22 degrees C. Acetonitrile and chloroform were used for intracellular extraction. This method can be used to monitor intracellular carbohydrate metabolism.

Floating resistivity detector for microchip electrophoresis.

A newly developed conductivity detector, the floating resistivity detector (FRD), for microchip electrophoresis was introduced in this work. The detector design permits decoupling of the detection circuit from the high separation voltage without compromising separation efficiency. This greatly simplifies the integration of microchip electrophoresis systems. Its method of detection relies on platinum electrodes being dipped in two buffer-filled branched detection probe reservoirs on the microchip device. In this way, analytes passing through the detection window will not pass through and subsequently adsorb onto the electrodes, alleviating problems of electrode fouling due to analyte contamination and surface reactions. A customized microchip design was proposed and optimized stepwise for the new FRD system. Each branched detection probe was determined to be 4.50 mm long with a 0.075 mm detection window gap between them. The distance between the detection window and buffer waste reservoir was determined to be 1.50 mm. The optimized microchip design was subsequently used in the analysis of four groups of analytes - inorganic cations, amino acids, aminoglycosides antibiotics, and biomarkers. Based on the preliminary results obtained, the detection limits were in the range of 0.4-0.7 mg/L for the inorganic cations and 1.5-15 mg/L for the amino compounds.

Rapid identification of purified enteropathogenic Escherichia coli by microchip electrophoresis.

In this work, the potential of PDMS-based microchip electrophoresis in the identifications and characterizations of microorganism was evaluated. Enteropathogenic E. coli (EPEC) was selected as the model microorganism. In this study, separation parameters such as applied voltage, concentrations of buffer and buffer modifier, injection voltage, and duration of injection had been investigated and optimized. Determination of EPEC bacteria could be completed within 2 min with good reproducibility. RSDs were less than 0.5 and 5% in migration time and peak area, respectively. Separation efficiency corresponding to plate number of more than 100,000 was achieved. In order to obtain reproducible separations, sample pretreatment was found to be essential. Microchip electrophoresis with LIF detection could potentially revolutionize certain aspects of microbiology involving diagnosis, profiling of pathogens, environmental analysis, and many other areas of study.

Immunoassay by capillary electrophoresis with quantum dots.

The application of quantum dots in capillary electrophoresis immunoassay was studied for the first time. Quantum dots were conjugated with antibody and subsequently tested by electrophoretic separation of free antibody and antibody-antigen complex. Antibody was fluorescently labeled by quantum dots via conjugation procedures and its electrophoretic characteristics were effectively modified due to the attachment of quantum dots. The determination of human IgM by direct CE based immunoassay could be easily achieved by simply changing the pH value of separation buffer. Polymer additive influenced the separation too but the effect was not as significant as buffer pH adjustment. Satisfactory separation of complex from free antibody could be achieved with 20mM sodium tetraborate as separation buffer, at pH 9.8. The immunoassay application of quantum dots in CE offers considerable advantages and can be readily applied to other large bio-molecules.

Rapid analysis of native neomycin components on a portable capillary electrophoresis system with potential gradient detection.

A simple method based on capillary electrophoresis with potential gradient detection was developed to separate and detect neomycin components within 4 min without a derivatization step. Satisfactory separation and good repeatability were obtained using a separation buffer composed of 1 mM ammonium citrate (pH 3.5). The linearity of the method ranged from 10 to 1000 ppm with a limit of detection for neomycin B of about 7 ppm. After a simple dilution and filtering pretreatment step, neomycin components in three real samples were successfully analyzed without any major interference. Due to its simplicity and reliability, this method could provide an excellent alternative to the assays currently listed in U.S. and European Pharmacopoeia. The experiments were performed on a portable capillary electrophoresis system and, hence, the method can be readily applied to field analysis and point-of-care testing. Figure Photo of portable CE-P2-PGD system.

Separation and determination of toxic pyrrolizidine alkaloids in traditional Chinese herbal medicines by micellar electrokinetic chromatography with organic modifier.

A simple and rapid micellar electrokinetic chromatography (MEKC) method was developed for the separation and determination of four toxic pyrrolizidine alkaloids (PAs) (senkirkine, senecionine, retrorsine, and seneciphylline) in two traditional Chinese herbal medicines (Qian liguang and Kuan donghua). Separation was performed in the running buffer consisting of 20 mM borate, 30 mM SDS, and 20% methanol at pH 9.1. With the optimized separation conditions, four PAs were separated in 17 min by a single run. The calibration curves showed good linearity with correlation efficiencies (R(2)) between 0.9940 and 0.9988. RSDs in migration time and peak area were 0.31, 0.40, 0.39, 0.48% and 3.28, 3.48, 4.16, 3.42% for senkirkine, senecionine, retrorsine, and seneciphylline, respectively. Limits of detection (S/N = 3) varied from 1.19 to 2.70 microg/mL. The proposed method was applied to determine the PAs extracted from Chinese herbal medicines (Qian liguang and Kuan donghua). PA of senkirkine in Kuan donghua was detected and the amount was found to be 79.1 microg/g. The results obtained indicate that the proposed MEKC method could potentially become an effective alternative tool for qualification control and quantitative analysis of herbal medicines in pharmaceutical industry.

Determination of the bacterial pathogen Edwardsiella tarda in fish species by capillary electrophoresis with blue light-emitting diode-induced fluorescence.

High-performance capillary electrophoresis (HPCE) has been applied to the identification, separation, and quantitation of intact bacteria. We demonstrate that a pathogen (Edwardsiella tarda) which causes systemic infection in commercially important fish species can be rapidly identified and determined (< 10 min) after direct injection into fish fluid by CE blue light-emitting diode (LED)-induced fluorescence. SYTO 13 (488 nm/509 nm), a cell-permeable green nucleic acid stain, was used to stain the cells. Remarkably high efficiency (> 1,200,000 theoretical plates/m) was achieved with this rapid and efficient CE method. It was found that proper sample vortexing (90 s) would be beneficial to disperse aggregated cells and facilitate the focusing of intact cells during electrophoresis. Ionization of the surface constituents of Edwardsiella tarda cells provided efficient surface charges for the intact cells to be separated from the EOF and damaged or lysed cells when the separation was performed in running buffer (3.94 mM Tris, 0.56 mM borate, 0.013 mM EDTA) at pH 10.5. The limit of detection (LOD) and recovery were found to be 4.2 x 10(4) cells/mL and 70.0%, respectively. This proposed CE method could become an effective tool for diagnosis and tracking of certain diseases caused by bacteria in fish species as well as in human beings.

On-chip potential gradient detection with a portable capillary electrophoresis system.

A portable chip-CE system with potential gradient detection (PGD) was developed and applied to the determinations of alkali metals and alkaloids. The separation efficiency appeared to be satisfactory and nonaqueous capillary electrophoresis (NACE) proved to be applicable to PGD or conductivity detection. The power supplies, separation and detection were built on a device of 3 kg in weight. A branch channel near the end of the separation channel was designed to perform PGD and make the application of relatively high field strength possible. The study is the first report on the application of PGD on the microchip platform. The design of the chip-CE system shows several advantages, such as simplicity, miniaturization and wide applicability.

Analysis of Chinese medicine preparations by capillary electrophoresis-mass spectrometry.

In Chinese medicines, herbs are usually prepared before use by patients. Since the preparation procedures convert the original component into one or more products, study of the procedures is usually complex and involves several compounds. On-line coupling of capillary electrophoresis (CE) to mass spectrometry (MS) allows both the efficient separation of CE and the specific and sensitive detection of MS to be achieved. In this study, CE-MS was applied to the determination of alkaloids in Maqianzi (the seed of Strychnos pierrian) and Wutou (aconite root, Radix aconiti praeparata) during the preparation procedure. With optimal CE-MS conditions, alkaloids in both prepared and unprepared Maqianzi were determined successfully in the total ion current (TIC) mode. However, single ion monitoring (SIM) had to be applied for the separation of aconitum alkaloids and their hydrolysis products. Quantification data indicated that MS detection under SIM mode is more sensitive than UV detection. Based on the CE-MS method developed, the hydrolysis of aconitum alkaloids in water and methanol was also studied.