An integrated protocol based on workflows of imaged capillary isoelectric focusing (icIEF) for in-depth protein heterogenous characterization: High-efficient fractionation and online mass spectrometry detection
We present an integrated protocol for studying the charge heterogeneity of therapeutic proteins combining two workflows: imaged capillary isoelectric focusing (icIEF) with fractionation and online mass spectrometry (MS) detection on a single platform. This protocol enables both intact MS-based prote...
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| Format: | Article |
| Language: | English |
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Elsevier
2025-03-01
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| Series: | Green Analytical Chemistry |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2772577425000011 |
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| author | Teresa Kwok She Lin Chan Mike Zhou Tong Chen Niusheng Xu Victor Li Tiemin Huang Tao Bo |
| author_facet | Teresa Kwok She Lin Chan Mike Zhou Tong Chen Niusheng Xu Victor Li Tiemin Huang Tao Bo |
| author_sort | Teresa Kwok |
| collection | DOAJ |
| description | We present an integrated protocol for studying the charge heterogeneity of therapeutic proteins combining two workflows: imaged capillary isoelectric focusing (icIEF) with fractionation and online mass spectrometry (MS) detection on a single platform. This protocol enables both intact MS-based protein charge variant characterization and in-depth peptide mapping of collected fractions via high-performance liquid chromatography (HPLC) tandem mass spectrometry. Through systematic methodology validation, the platform is demonstrated to be robust, with step-by-step method development, standardized operating procedures (SOPs), exceptional reproducibility, and high sensitivity. Notably, this protocol facilitates icIEF-UV separation followed by either a fractionation scheme or MS online detection within a single platform, providing simplified workflows with reduced reagent consumption compared to traditional techniques. Embracing the ''Green Chemistry'' concept, the protocol addresses key challenges in biopharmaceutical discovery, quality control, and manufacturing, promoting sustainability throughout the process. |
| format | Article |
| id | doaj-art-b86f7d007b6b43e3af8816902f2bf2a8 |
| institution | Kabale University |
| issn | 2772-5774 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Green Analytical Chemistry |
| spelling | doaj-art-b86f7d007b6b43e3af8816902f2bf2a82025-01-16T04:29:25ZengElsevierGreen Analytical Chemistry2772-57742025-03-0112100204An integrated protocol based on workflows of imaged capillary isoelectric focusing (icIEF) for in-depth protein heterogenous characterization: High-efficient fractionation and online mass spectrometry detectionTeresa Kwok0She Lin Chan1Mike Zhou2Tong Chen3Niusheng Xu4Victor Li5Tiemin Huang6Tao Bo7Advanced Electrophoresis Solutions Ltd, 380 Jamieson Parkway, Unit 7 and 8, Cambridge, ON, N3C 4N4, CanadaAdvanced Electrophoresis Solutions Ltd, 380 Jamieson Parkway, Unit 7 and 8, Cambridge, ON, N3C 4N4, CanadaAdvanced Electrophoresis Solutions Ltd, 380 Jamieson Parkway, Unit 7 and 8, Cambridge, ON, N3C 4N4, CanadaAdvanced Electrophoresis Solutions Ltd, 380 Jamieson Parkway, Unit 7 and 8, Cambridge, ON, N3C 4N4, CanadaAdvanced Electrophoresis Solutions Ltd, 380 Jamieson Parkway, Unit 7 and 8, Cambridge, ON, N3C 4N4, CanadaAdvanced Electrophoresis Solutions Ltd, 380 Jamieson Parkway, Unit 7 and 8, Cambridge, ON, N3C 4N4, CanadaAdvanced Electrophoresis Solutions Ltd, 380 Jamieson Parkway, Unit 7 and 8, Cambridge, ON, N3C 4N4, CanadaCorresponding author; Advanced Electrophoresis Solutions Ltd, 380 Jamieson Parkway, Unit 7 and 8, Cambridge, ON, N3C 4N4, CanadaWe present an integrated protocol for studying the charge heterogeneity of therapeutic proteins combining two workflows: imaged capillary isoelectric focusing (icIEF) with fractionation and online mass spectrometry (MS) detection on a single platform. This protocol enables both intact MS-based protein charge variant characterization and in-depth peptide mapping of collected fractions via high-performance liquid chromatography (HPLC) tandem mass spectrometry. Through systematic methodology validation, the platform is demonstrated to be robust, with step-by-step method development, standardized operating procedures (SOPs), exceptional reproducibility, and high sensitivity. Notably, this protocol facilitates icIEF-UV separation followed by either a fractionation scheme or MS online detection within a single platform, providing simplified workflows with reduced reagent consumption compared to traditional techniques. Embracing the ''Green Chemistry'' concept, the protocol addresses key challenges in biopharmaceutical discovery, quality control, and manufacturing, promoting sustainability throughout the process.http://www.sciencedirect.com/science/article/pii/S2772577425000011Imaged capillary isoelectric focusingIntegrated protocolProtein heterogeneityFractionationMass spectrometry online |
| spellingShingle | Teresa Kwok She Lin Chan Mike Zhou Tong Chen Niusheng Xu Victor Li Tiemin Huang Tao Bo An integrated protocol based on workflows of imaged capillary isoelectric focusing (icIEF) for in-depth protein heterogenous characterization: High-efficient fractionation and online mass spectrometry detection Green Analytical Chemistry Imaged capillary isoelectric focusing Integrated protocol Protein heterogeneity Fractionation Mass spectrometry online |
| title | An integrated protocol based on workflows of imaged capillary isoelectric focusing (icIEF) for in-depth protein heterogenous characterization: High-efficient fractionation and online mass spectrometry detection |
| title_full | An integrated protocol based on workflows of imaged capillary isoelectric focusing (icIEF) for in-depth protein heterogenous characterization: High-efficient fractionation and online mass spectrometry detection |
| title_fullStr | An integrated protocol based on workflows of imaged capillary isoelectric focusing (icIEF) for in-depth protein heterogenous characterization: High-efficient fractionation and online mass spectrometry detection |
| title_full_unstemmed | An integrated protocol based on workflows of imaged capillary isoelectric focusing (icIEF) for in-depth protein heterogenous characterization: High-efficient fractionation and online mass spectrometry detection |
| title_short | An integrated protocol based on workflows of imaged capillary isoelectric focusing (icIEF) for in-depth protein heterogenous characterization: High-efficient fractionation and online mass spectrometry detection |
| title_sort | integrated protocol based on workflows of imaged capillary isoelectric focusing icief for in depth protein heterogenous characterization high efficient fractionation and online mass spectrometry detection |
| topic | Imaged capillary isoelectric focusing Integrated protocol Protein heterogeneity Fractionation Mass spectrometry online |
| url | http://www.sciencedirect.com/science/article/pii/S2772577425000011 |
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