The Experts below are selected from a list of 315 Experts worldwide ranked by ideXlab platform
Jonathan Bones - One of the best experts on this subject based on the ideXlab platform.
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comparative elucidation of cetuximab heterogeneity on the intact protein level by Cation Exchange Chromatography and capillary electrophoresis coupled to mass spectrometry
Analytical Chemistry, 2020Co-Authors: Florian Fussl, Jonathan Bones, Anne Trappe, Sara Carillo, Craig JakesAbstract:Charge sensitive separation methods such as ion Exchange Chromatography (CEX) and capillary electrophoresis (CE) have recently been coupled to mass spectrometry to facilitate high resolution profiling of proteoforms present within the charge variant profile of complex biopharmaceuticals. Here we apply pH gradient Cation Exchange Chromatography and microfluidic capillary electrophoresis using the ZipChip platform for comparative characterization of the monoclonal antibody Cetuximab. Cetuximab harbors four glycans per molecule, two on each heavy chain, of which the Fab glycans have been reported to be complex and multiply sialylated. The presence of these extra glycosylation sites in the variable region of the molecule causes significant charge variant and glycan heterogeneity, which makes comprehensive analysis on the intact protein level challenging. Both pH gradient CEX-MS and CE-MS were found to be powerful for the separation of Cetuximab charge variants with eight major peaks being baseline resolved using both separation platforms. Informative native-like mass spectra were collected for each charge variant peak using both platforms that facilitated deconvolution and further analysis. The total proteoform coverage was exceptionally high with >100 isoforms identified and relatively quantified with CEX-MS, in case of CE-MS the proteoform coverage was >200. A deep insight into the heterogeneity of Cetuximab was unveiled, the high level of sensitivity achievable enables the implementation of the presented technologies even at early stages of the biopharmaceutical development platform, such as in developability assessment, process development and also for monitoring process consistency.
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charge variant analysis of monoclonal antibodies using direct coupled ph gradient Cation Exchange Chromatography to high resolution native mass spectrometry
Analytical Chemistry, 2018Co-Authors: Florian Fussl, Ken Cook, Kai Scheffler, Amy Farrell, Stefan Mittermayr, Jonathan BonesAbstract:Charge variant analysis (CVA) of monoclonal antibodies (mAbs) using Cation Exchange Chromatography is routinely used as a fingerprint of the distribution of posttranslational modifiCations present on the molecule. Traditional salt or pH based eluents are not suited for direct coupling to mass spectrometry due to nonvolatility or high ionic strength. This makes further analysis complicated when an alteration in the charge variant profile or the emergence of an additional peak is encountered. Here, the use of pH gradient elution using volatile, low ionic strength buffers is reported with direct coupling to high-resolution Orbitrap mass spectrometry. The development of a universal method based on pH elution was explored using a number of mAb drug products. Optimized methods facilitated the separation and identifiCation of charge variants including individual glycoforms of the mAbs investigated using the same buffer system but with tailored gradient slopes. The developed method represents an exciting advance ...
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charge variant analysis of monoclonal antibodies using direct coupled ph gradient Cation Exchange Chromatography to high resolution native mass spectrometry
Analytical Chemistry, 2018Co-Authors: Florian Fussl, Ken Cook, Kai Scheffler, Amy Farrell, Stefan Mittermayr, Jonathan BonesAbstract:Charge variant analysis (CVA) of monoclonal antibodies (mAbs) using Cation Exchange Chromatography is routinely used as a fingerprint of the distribution of posttranslational modifiCations present on the molecule. Traditional salt or pH based eluents are not suited for direct coupling to mass spectrometry due to nonvolatility or high ionic strength. This makes further analysis complicated when an alteration in the charge variant profile or the emergence of an additional peak is encountered. Here, the use of pH gradient elution using volatile, low ionic strength buffers is reported with direct coupling to high-resolution Orbitrap mass spectrometry. The development of a universal method based on pH elution was explored using a number of mAb drug products. Optimized methods facilitated the separation and identifiCation of charge variants including individual glycoforms of the mAbs investigated using the same buffer system but with tailored gradient slopes. The developed method represents an exciting advance for the characterization of biopharmaceuticals as intact entities through the combination of native charge variant separations with high-resolution native mass spectrometry.
Florian Fussl - One of the best experts on this subject based on the ideXlab platform.
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comparative elucidation of cetuximab heterogeneity on the intact protein level by Cation Exchange Chromatography and capillary electrophoresis coupled to mass spectrometry
Analytical Chemistry, 2020Co-Authors: Florian Fussl, Jonathan Bones, Anne Trappe, Sara Carillo, Craig JakesAbstract:Charge sensitive separation methods such as ion Exchange Chromatography (CEX) and capillary electrophoresis (CE) have recently been coupled to mass spectrometry to facilitate high resolution profiling of proteoforms present within the charge variant profile of complex biopharmaceuticals. Here we apply pH gradient Cation Exchange Chromatography and microfluidic capillary electrophoresis using the ZipChip platform for comparative characterization of the monoclonal antibody Cetuximab. Cetuximab harbors four glycans per molecule, two on each heavy chain, of which the Fab glycans have been reported to be complex and multiply sialylated. The presence of these extra glycosylation sites in the variable region of the molecule causes significant charge variant and glycan heterogeneity, which makes comprehensive analysis on the intact protein level challenging. Both pH gradient CEX-MS and CE-MS were found to be powerful for the separation of Cetuximab charge variants with eight major peaks being baseline resolved using both separation platforms. Informative native-like mass spectra were collected for each charge variant peak using both platforms that facilitated deconvolution and further analysis. The total proteoform coverage was exceptionally high with >100 isoforms identified and relatively quantified with CEX-MS, in case of CE-MS the proteoform coverage was >200. A deep insight into the heterogeneity of Cetuximab was unveiled, the high level of sensitivity achievable enables the implementation of the presented technologies even at early stages of the biopharmaceutical development platform, such as in developability assessment, process development and also for monitoring process consistency.
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charge variant analysis of monoclonal antibodies using direct coupled ph gradient Cation Exchange Chromatography to high resolution native mass spectrometry
Analytical Chemistry, 2018Co-Authors: Florian Fussl, Ken Cook, Kai Scheffler, Amy Farrell, Stefan Mittermayr, Jonathan BonesAbstract:Charge variant analysis (CVA) of monoclonal antibodies (mAbs) using Cation Exchange Chromatography is routinely used as a fingerprint of the distribution of posttranslational modifiCations present on the molecule. Traditional salt or pH based eluents are not suited for direct coupling to mass spectrometry due to nonvolatility or high ionic strength. This makes further analysis complicated when an alteration in the charge variant profile or the emergence of an additional peak is encountered. Here, the use of pH gradient elution using volatile, low ionic strength buffers is reported with direct coupling to high-resolution Orbitrap mass spectrometry. The development of a universal method based on pH elution was explored using a number of mAb drug products. Optimized methods facilitated the separation and identifiCation of charge variants including individual glycoforms of the mAbs investigated using the same buffer system but with tailored gradient slopes. The developed method represents an exciting advance ...
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charge variant analysis of monoclonal antibodies using direct coupled ph gradient Cation Exchange Chromatography to high resolution native mass spectrometry
Analytical Chemistry, 2018Co-Authors: Florian Fussl, Ken Cook, Kai Scheffler, Amy Farrell, Stefan Mittermayr, Jonathan BonesAbstract:Charge variant analysis (CVA) of monoclonal antibodies (mAbs) using Cation Exchange Chromatography is routinely used as a fingerprint of the distribution of posttranslational modifiCations present on the molecule. Traditional salt or pH based eluents are not suited for direct coupling to mass spectrometry due to nonvolatility or high ionic strength. This makes further analysis complicated when an alteration in the charge variant profile or the emergence of an additional peak is encountered. Here, the use of pH gradient elution using volatile, low ionic strength buffers is reported with direct coupling to high-resolution Orbitrap mass spectrometry. The development of a universal method based on pH elution was explored using a number of mAb drug products. Optimized methods facilitated the separation and identifiCation of charge variants including individual glycoforms of the mAbs investigated using the same buffer system but with tailored gradient slopes. The developed method represents an exciting advance for the characterization of biopharmaceuticals as intact entities through the combination of native charge variant separations with high-resolution native mass spectrometry.
Kazuhiko Tanaka - One of the best experts on this subject based on the ideXlab platform.
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influence of acidic eluent for retention behaviors of common anions and Cations by ion exclusion Cation Exchange Chromatography on a weakly acidic Cation Exchange resin in the h form
Journal of Chromatography A, 2006Co-Authors: Masanobu Mori, Kazuhiko Tanaka, Tatsuya Satori, Mikaru Ikedo, Hideyuki ItabashiAbstract:Influence of acidic eluent on retention behaviors of common anions and Cations by ion-exclusion/Cation-Exchange Chromatography (ion-exclusion/CEC) were investigated on a weakly acidic Cation-Exchange resin in the H(+)-form with conductivity. Sensitivities of analyte ions, especially weak acid anions (F(-) and HCOO(-)), were affected with degree of background conductivity level with pK(a1) (first dissociation constant) of acid in eluent. The retention behaviors of anions and Cations were related to that of elution dip induced after eluting acid to separation column and injecting analyte sample. These results were largely dependent on the natures of acid as eluent. Through this study, succinic acid as the eluent was suitable for simultaneous separation of strong acid anions (SO(4)(2-), Cl(-), NO(3)(-) and I(-)), weak acid anions (F(-), HCOO(-) and CH(3)COO(-)), and Cations (Na(+), K(+), NH(4)(+), Mg(2+) and Ca(2+)). The separation was achieved in 20 min under the optimum eluent condition, 20 mM succinic acid/2 mM 18-crown-6. Detection limits at S/N=3 ranged from 0.10 to 0.51 microM for strong acid anions, 0.20 to 5.04 microM for weak acid anions and 0.75 to 1.72 microM for Cations. The relative standard deviations of peak areas in the repeated chromatographic runs (n=10) were in the range of 1.1-2.9% for anions and 1.8-4.5% for Cations. This method was successfully applied to hot spring water containing strong acid anions, weak acid anions and Cations, with satisfactory results.
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simultaneous ion exclusion Cation Exchange Chromatography of anions and Cations in acid rain waters on a weakly acidic Cation Exchange resin by elution with sulfosalicylic acid
Journal of Chromatography A, 2000Co-Authors: Kazuhiko Tanaka, Kazutoku Ohta, James S Fritz, Akiyoshi Miyanaga, Paul R Haddad, Wenzhi Hu, Kiyoshi HasebeAbstract:A simple, selective, and sensitive method for the simultaneous determination of anions (sulfate, nitrate, and chloride) and Cations (sodium, ammonium, potassium, magnesium, and calcium) in acid rain waters was developed using ion-exclusion/Cation-Exchange Chromatography with conductimetric detection. A weakly acidic Cation-Exchange resin column (Tosho TSKgel OA-PAK-A) and a sulfosalicylic acid–methanol–water eluent was used. With a mobile phase comprising 1.25 mM sulfosalicylic acid in methanol–water (7.5:92.5) at 1.2 ml/min, simultaneous separation and detection of the above anions and Cations was achieved in about 30 min. Linear calibration plots of peak area versus concentration were obtained over the concentration ranges 0–1.0 mM for anions (R=0.9991) and 0–0.5 mM for Cations (R=0.9994). Detection limits calculated at S/N=3 ranged from 4.2 to 14.8 ppb for the anions and from 2.4 to 12.1 ppb for the Cations. The reproducibility of retention times was 0.14–0.15% relative standard deviation (RSD) for anions and 0.18–0.31% for Cations, and reproducibility of chromatographic peak areas was 1.22–1.75% RSD for anions and 1.81–2.10% for Cations. The method was applied successfully to the simultaneous determination of anions and Cations in aerosols transported from mainland China to central Japan, as determined by a meteorological satellite data analyzer.
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simultaneous determination of anions and Cations by ion exclusion Chromatography Cation Exchange Chromatography with tartaric acid 18 crown 6 as eluent
Journal of Chromatography A, 1999Co-Authors: Semog Kwon, Kazuhiko Tanaka, Kwangpill Lee, Kazutoku OhtaAbstract:Ion-exclusion Chromatography–Cation-Exchange Chromatography was developed for the simultaneous separation of common inorganic anions and Cations (Cl−, NO3− and SO42−; Na+, NH4+, K+, Mg2+ and Ca2+) on a weakly acidic Cation-Exchange column by elution with weak acid. Generally, the resolution among these monovalent Cations was only moderate, thereby hindering the determination of these analytes in natural-water samples. Therefore, 18-crown-6 was added to the eluent to improve the resolution. A good separation of these anions and Cations on a weakly acidic Cation-Exchange column was achieved in 30 min by elution with 5 mM tartaric acid/6 mM 18-crown-6/methanol–water (7.5:92.5). The ion-exclusion Chromatography–Cation-Exchange Chromatography method developed here was successfully applied to the separation of major anions and Cations in an environmental water sample.
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simultaneous ion exclusion Chromatography Cation Exchange Chromatography with conductimetric detection of anions and Cations in acid rain waters
Journal of Chromatography A, 1994Co-Authors: Kazuhiko Tanaka, Kazutoku Ohta, James S Fritz, Susumu Matsushita, Akiyoshi MiyanagaAbstract:Abstract A simple, selective and sensitive method was investigated for simultaneously determining anions (Cl−, NO3−, SO42−) and Cations (Na+, NH4+, K+, Mg2+, Ca2+) in acid rain and related environmental waters in central Japan. The method involves simultaneous ion-exclusion-Cation-Exchange Chromatography with conductimetric detection on a polyacrylate weakly acidic Cation-Exchange resin column with a weak-acid eluent. With the weak-acid eluent (tartaric acid) both anions and Cations were separated simultaneously, based on ion-exclusion and Cation-Exchange mechanism. Owing to the presence of H+ ions in the tartaric acid eluent, the detector response was positive for the anions and negative for the Cations. Using a 5 mM tartaric acid-7.5% methanol-water eluent, good simultaneous separation and detection were achieved in about 30 min. The results indicated an ionic balance of about 100% between the anions (including HCO3−) and the Cations (including H+).
Hisakuni Sato - One of the best experts on this subject based on the ideXlab platform.
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simultaneous determination of creatinine creatine and uv absorbing amino acids using dual mode gradient low capacity Cation Exchange Chromatography
Journal of Chromatography A, 2005Co-Authors: Yukio Yokoyama, Sachiyo Tsuji, Hisakuni SatoAbstract:Abstract A simple and versatile Cation-Exchange Chromatography technique for the simultaneous determination of urinary creatinine (Cre), creatine (Crn), methionine (Met), tyrosine (Tyr), phenylalanine (Phe), histidine (His), and tryptophan (Trp) was developed. A novel low-capacity Cation-Exchange column packed with a newly developed sulfoacylated hypercross-linked macroreticular polystyrene-divinylbenzene resin, referred to as TMR-A/75 (capacity: 75 μequiv/column), was successfully used with a binary dual-mode gradient eluting system. Two solvents, (A) 25 mM phosphoric acid–methanol (30:70, v/v) and (B) 25 mM disodium hydrogenphosphate–methanol (30:70, v/v) were pumped through the column by programming solvent delivery ratios as 0 to 5 min: A–B (55:45, pH 3.6); 5–21 min: A–B (49:51, pH 5.3); and 21–35 min: A–B (55:45, pH 3.6). The flow rate was simultaneously time-programmed to be 0.6 mL/min from 0 to 19 min and to be 1.0 mL/min from 19 to 35 min. This eluting system could permit the use of the UV detection at 210 nm. The analytes, Crn, Met, Tyr, His, Cre, Phe, and Trp, were well separated in this order in 27 min with minimum resolution of approximately 2, and the cycle time was about 35 min. Retention time of each analyte was very reproducible with relative standard deviations (RSDs) between 0.05 and 0.38% (n = 5). The peak area responses were also reproducible with RSDs between 0.74 and 2.24% (n = 5). Calibration lines based on area data were linear from 1 to 1000 μM with r2 values of 0.9998 (Crn), 0.9998 (Met), 0.9999 (Tyr), 0.9999 (His), 1.0000 (Cre), 1.0000 (Phe), and 0.9999 (Trp). The method was applicable to the screening and/or chemical diagnosis of inherited metabolic disorders such as phenylketonuria (PKU), tyrosinemia, and Lowe syndrome. The creatinine ratios of diagnostic markers (μM/μM Cre) were easily determined. The Phe/Cre ratios for five urines from patients with PKU ranged from 0.162 to 0.521, and the Tyr/Cre ratio for tyrosinemia was 0.147. The ratios of Tyr/Cre, Phe/Cre, and Trp/Cre for Lowe syndrome were 0.497, 0.321, and 0.495, respectively. In contrast, the creatinine ratios for healthy newborns showed one digit lower than those for patients did. The developed method is very practical and can provide useful information and results for the clinical or biomedical researches with low analytical run costs.
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simultaneous determination of urinary creatinine and uv absorbing amino acids using a novel low capacity Cation Exchange Chromatography for the screening of inborn errors of metabolism
Journal of Chromatography B, 2005Co-Authors: Yukio Yokoyama, Keiko Yamasaki, Hisakuni SatoAbstract:Abstract A simple and versatile low-capacity Cation-Exchange Chromatography system for the simultaneous determination of creatinine and UV-absorbing amino acids was developed. The separation column was packed with a newly developed low-capacity sulfoacylated macro-porous polystyrene-divinylbenzene resin selective for amino-acid Cations. Urinary creatinine, creatine, tyrosine, histidine, phenylalanine, and tryptophan were simultaneously separated and determined by an isocratic elution with phosphate/acetonitrile eluent in 25 min. Relative standard deviations (R.S.D.) of the retention times for the analytes were between 0.28 and 1.06%. R.S.D. of peak area responses for the analytes were between 0.75 and 3.51%. The r 2 values for the calibration lines were between 0.9994 and 0.9999. The method could provide the creatinine ratios for the analytes, and was applicable to the screening and/or chemical diagnosis of several inherited disorders of amino-acid metabolism such as phenylketonuria (PKU).
Kazutoku Ohta - One of the best experts on this subject based on the ideXlab platform.
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simultaneous ion exclusion Cation Exchange Chromatography of anions and Cations in acid rain waters on a weakly acidic Cation Exchange resin by elution with sulfosalicylic acid
Journal of Chromatography A, 2000Co-Authors: Kazuhiko Tanaka, Kazutoku Ohta, James S Fritz, Akiyoshi Miyanaga, Paul R Haddad, Wenzhi Hu, Kiyoshi HasebeAbstract:A simple, selective, and sensitive method for the simultaneous determination of anions (sulfate, nitrate, and chloride) and Cations (sodium, ammonium, potassium, magnesium, and calcium) in acid rain waters was developed using ion-exclusion/Cation-Exchange Chromatography with conductimetric detection. A weakly acidic Cation-Exchange resin column (Tosho TSKgel OA-PAK-A) and a sulfosalicylic acid–methanol–water eluent was used. With a mobile phase comprising 1.25 mM sulfosalicylic acid in methanol–water (7.5:92.5) at 1.2 ml/min, simultaneous separation and detection of the above anions and Cations was achieved in about 30 min. Linear calibration plots of peak area versus concentration were obtained over the concentration ranges 0–1.0 mM for anions (R=0.9991) and 0–0.5 mM for Cations (R=0.9994). Detection limits calculated at S/N=3 ranged from 4.2 to 14.8 ppb for the anions and from 2.4 to 12.1 ppb for the Cations. The reproducibility of retention times was 0.14–0.15% relative standard deviation (RSD) for anions and 0.18–0.31% for Cations, and reproducibility of chromatographic peak areas was 1.22–1.75% RSD for anions and 1.81–2.10% for Cations. The method was applied successfully to the simultaneous determination of anions and Cations in aerosols transported from mainland China to central Japan, as determined by a meteorological satellite data analyzer.
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simultaneous determination of anions and Cations by ion exclusion Chromatography Cation Exchange Chromatography with tartaric acid 18 crown 6 as eluent
Journal of Chromatography A, 1999Co-Authors: Semog Kwon, Kazuhiko Tanaka, Kwangpill Lee, Kazutoku OhtaAbstract:Ion-exclusion Chromatography–Cation-Exchange Chromatography was developed for the simultaneous separation of common inorganic anions and Cations (Cl−, NO3− and SO42−; Na+, NH4+, K+, Mg2+ and Ca2+) on a weakly acidic Cation-Exchange column by elution with weak acid. Generally, the resolution among these monovalent Cations was only moderate, thereby hindering the determination of these analytes in natural-water samples. Therefore, 18-crown-6 was added to the eluent to improve the resolution. A good separation of these anions and Cations on a weakly acidic Cation-Exchange column was achieved in 30 min by elution with 5 mM tartaric acid/6 mM 18-crown-6/methanol–water (7.5:92.5). The ion-exclusion Chromatography–Cation-Exchange Chromatography method developed here was successfully applied to the separation of major anions and Cations in an environmental water sample.
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simultaneous ion exclusion Chromatography Cation Exchange Chromatography with conductimetric detection of anions and Cations in acid rain waters
Journal of Chromatography A, 1994Co-Authors: Kazuhiko Tanaka, Kazutoku Ohta, James S Fritz, Susumu Matsushita, Akiyoshi MiyanagaAbstract:Abstract A simple, selective and sensitive method was investigated for simultaneously determining anions (Cl−, NO3−, SO42−) and Cations (Na+, NH4+, K+, Mg2+, Ca2+) in acid rain and related environmental waters in central Japan. The method involves simultaneous ion-exclusion-Cation-Exchange Chromatography with conductimetric detection on a polyacrylate weakly acidic Cation-Exchange resin column with a weak-acid eluent. With the weak-acid eluent (tartaric acid) both anions and Cations were separated simultaneously, based on ion-exclusion and Cation-Exchange mechanism. Owing to the presence of H+ ions in the tartaric acid eluent, the detector response was positive for the anions and negative for the Cations. Using a 5 mM tartaric acid-7.5% methanol-water eluent, good simultaneous separation and detection were achieved in about 30 min. The results indicated an ionic balance of about 100% between the anions (including HCO3−) and the Cations (including H+).