Protein analysis using capillary electrophoresis coupled to mass spectrometry through vibrating sharp‐edge spray ionization DOI
Makenzie T. Witzel,

Lindsay M. Veltri,

Marius M. Kostelic

et al.

Electrophoresis, Journal Year: 2024, Volume and Issue: unknown

Published: April 5, 2024

Abstract Capillary electrophoresis (CE) interfaced to mass spectrometry (MS) with electrospray ionization typically incorporates acidic additives or organic solvents assist in ionization. Vibrating sharp‐edge spray (VSSI) is a voltage‐free method interface CE and MS that does not require these additives, making it appealing for protein analyses. CE–VSSI nanoflow sheath separations are performed low ionic strength aqueous solutions the reduce suppression. Serine also included analyte adduction. Proteins detected 2.5–10 µM range, corresponding an injected range of 0.1–1.2 ng. The anionic proteins β‐lactoglobulin transferrin resolved using unmodified fused silica capillary because they do exhibit nonspecific surface adsorption. Conversely, cationic cytochrome c , ribonuclease A, α‐chymotrypsinogen A background electrolytes overcome Alternatively, semipermanent coating comprised self‐assembled lipids overcomes adsorption at neutral pH. Separations zwitterionic hybrid coatings complete within 15 6 min, respectively. dimeric form triosephosphate isomerase was observed 60 µM, 19 ng, by dropping temperature inlet.

Language: Английский

Enabling icIEF Peak Identification of AAV Capsid Proteins by Fractionation on MauriceFlex and Subsequent Analysis by LC–MS DOI Creative Commons

Will McElroy,

Sisi Huang, Xiaoping He

et al.

Electrophoresis, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 20, 2025

ABSTRACT A significant limitation of imaged capillary electric focusing (icIEF) is the inability to identify and characterize specific species in electropherogram. This has led development complementary ion‐exchange chromatography (IEX)‐based methods that are amenable either fraction collection subsequent characterization or online IEX coupled mass spectrometry. To overcome this while maintaining use icIEF, novel approaches, including an icIEF separation fractionation technology (MauriceFlex, ProteinSimple), have been developed. approach enables various peaks, which can then be characterized by spectrometry confirm identity separated charged species. Herein, MauriceFlex was applied adeno‐associated viral (AAV) gene therapy products, contain a DNA transgene packaged into protein capsid shown tremendous therapeutic potential recent years. Utilizing system, we developed for from AAV proteins (VP) liquid (LC–MS). When applying same sample preparation, charge profiles on instrument were demonstrated consistent with those original Maurice platform, industrial gold standard. Optimization VP method required low concentration samples, optimization mobilization conditions, enhancement recovery, maintenance stability post fractionation. able successfully collect charge‐separated samples subsequently analyze them MS analysis. In addition, workflow based downstream LC–MS established confirmation additional heterogeneity.

Language: Английский

Citations

2
Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications—Updated and completely revised edition DOI Creative Commons
Finja Krebs,

Holger Zagst,

M. Stein

et al.

Electrophoresis, Journal Year: 2023, Volume and Issue: 44(17-18), P. 1279 - 1341

Published: Aug. 3, 2023

Abstract This review is in support of the development selective, precise, fast, and validated capillary electrophoresis (CE) methods. It follows up a similar article from 1998, Wätzig H, Degenhardt M, Kunkel A. “Strategies for electrophoresis: method validation pharmaceutical biological applications,” pointing out which fundamentals are still valid at same time showing enormous achievements last 25 years. The structures both reviews widely similar, order to facilitate their simultaneous use. Focusing on applications, successful use CE now demonstrated by more than 600 carefully selected references. Many those recent reviews; therefore, significant overview about field provided. There extra sections sample pretreatment related microchip CE, completely revised section protein analytes biomolecules general. general strategies summed with regard selectivity, efficiency, precision, analysis time, limit detection, requirements, validation.

Language: Английский

Citations

30
Capillary electrophoresis‐mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018–March 2023) DOI Creative Commons
Katarína Maráková, Martina Opetová, Radovan Tomašovský

et al.

Journal of Separation Science, Journal Year: 2023, Volume and Issue: 46(15)

Published: May 26, 2023

Capillary electrophoresis is recognized as a valued separation technique for its high efficiency, low sample consumption, good economic and ecological aspects, reproducibility, complementarity to traditional liquid chromatography techniques. experiments are generally performed utilizing optical detection, such ultraviolet or fluorescence detectors. However, in order provide structural information, capillary hyphenated highly sensitive selective mass spectrometry has been developed overcome the limitations of detections. electrophoresis‐mass increasingly popular protein analysis, including biopharmaceutical biomedical research. It frequently applied determination physicochemical biochemical parameters proteins, offers excellent performance in‐depth characterizations biopharmaceuticals at various levels also already proven promising tool biomarker discovery. In this review, we focus on possibilities analysis their intact level. Various modes interfaces, well approaches prevent adsorption enhance loading capacity, discussed recent (2018–March 2023) developments applications field summarized.

Language: Английский

Citations

27
Multi‐dimensional technology – Recent advances and applications for biotherapeutic characterization DOI Creative Commons
Thomas Bouvarel, Julien Camperi, Davy Guillarme

et al.

Journal of Separation Science, Journal Year: 2024, Volume and Issue: 47(5)

Published: March 1, 2024

This review provides an overview of the latest advancements and applications in multi‐dimensional liquid chromatography coupled with mass spectrometry (mD‐LC‐MS), covering aspects such as inter‐laboratory studies, digestion strategy, trapping column, multi‐level analysis. The shift from offline to online workflow reduces sample processing artifacts, analytical variability, analysis time, labor required for data acquisition. Over past few years, this technique has demonstrated sufficient maturity application across a diverse range complex products. Moreover, there is potential strategy evolve into integrated process technology tool real‐time monitoring monoclonal antibody quality. also identifies emerging trends, including its new modalities, possibility evaluating biological activity within mD‐LC set‐up, consideration capillary electrophoresis alternative mD‐LC. As mD‐LC‐MS continues integrate it holds shape next generation tools, offering exciting possibilities enhanced characterization biopharmaceutical

Language: Английский

Citations

8
What is the role of current mass spectrometry in pharmaceutical analysis? DOI Creative Commons

Maria A. Khalikova,

Jakub Jireš, Ondřej Horáček

et al.

Mass Spectrometry Reviews, Journal Year: 2023, Volume and Issue: 43(3), P. 560 - 609

Published: July 28, 2023

Abstract The role of mass spectrometry (MS) has become more important in most application domains recent years. Pharmaceutical analysis is specific due to its stringent regulation procedures, the need for good laboratory/manufacturing practices, and a large number routine quality control analyses be carried out. MS is, therefore, very different throughout whole drug development cycle. While it dominates within discovery phase, control, minor indispensable only selected applications. Moreover, case small molecule pharmaceuticals biopharmaceuticals. Our review explains current both small‐molecule chemical drugs Important features MS‐based technologies being implemented, method requirements, related challenges are discussed. differences analytical procedures biopharmaceuticals pointed single or set methods usually sufficient often not indispensable, panel including extensive use must used Finally, expected future trends outlined.

Language: Английский

Citations

20
Mass Spectrometry-Based Charge Heterogeneity Characterization of Therapeutic mAbs with Imaged Capillary Isoelectric Focusing and Ion-Exchange Chromatography as Separation Techniques DOI
Gang Wu, Chuanfei Yu, Wenbo Wang

et al.

Analytical Chemistry, Journal Year: 2023, Volume and Issue: 95(4), P. 2548 - 2560

Published: Jan. 19, 2023

Imaged capillary isoelectric focusing (icIEF) and ion-exchange chromatography (IEX) are two essential techniques that routinely used for charge variant analysis of therapeutic monoclonal antibodies (mAbs) during their development in quality control. These separate mAb variants based on different mechanisms IEX have been developed as front-end separation online mass spectrometry (MS) detection, which is robust intact protein identification. Recently, an innovative, coupled icIEF-MS technology has constructed our laboratory. In this study, strong cation exchange (SCX)-MS were optimized heterogeneity characterization a diverse mAbs results compared methodological validation. It was found outperformed SCX-MS study by demonstrating outstanding sensitivity, low carryover effect, accurate identification, higher resolution although contributed to throughput. Ultimately, integrating novel icIEF-HRMS with the more common can provide promising comprehensive strategy accelerating complex therapeutics.

Language: Английский

Citations

19
What should next-generation analytical platforms for biopharmaceutical production look like? DOI
Anurag S. Rathore, Deepika Sarin

Trends in biotechnology, Journal Year: 2023, Volume and Issue: 42(3), P. 282 - 292

Published: Sept. 27, 2023

Language: Английский

Citations

18
Capillary isoelectric focusing – The role of markers of isoelectric point and recent applications in the field DOI
Filip Duša,

Anna Kubešová,

Jiří Šalplachta

et al.

TrAC Trends in Analytical Chemistry, Journal Year: 2023, Volume and Issue: 162, P. 117018 - 117018

Published: March 11, 2023

Language: Английский

Citations

17
Optimization of the Imaged cIEF Method for Monitoring the Charge Heterogeneity of Antibody-Maytansine Conjugate DOI Creative Commons
Ayat Abbood

Journal of Analytical Methods in Chemistry, Journal Year: 2023, Volume and Issue: 2023, P. 1 - 10

Published: June 2, 2023

The aim of this study was to develop a whole-column imaging-detection capillary isoelectric focusing (icIEF) method for the analytical characterization charge heterogeneity novel humanized anti-EphA2 antibody conjugated maytansine derivative. In addition time, sample composition optimized: pH range, percent carrier ampholytes, concentration, and urea concentration. A good separation isoforms obtained with 4% ampholytes large (3-10) narrow range (8-10.5) (1 : 1 ratio), concentration (0.3-1 mg/ml) linearity (R2: 0.9905), 2 M 12 minute focusing. optimized icIEF demonstrated interday repeatability RSD values: <1% (pI), <8% (% peak area), 7% (total areas). useful as an tool assess charged isoform profile discovery batch studied maytansinoid-antibody conjugate in comparison its naked antibody. It exhibited pI (7.5-9.0), while showed (8.9-9.0). conjugate, 2% had same isoforms.

Language: Английский

Citations

15
Two-Dimensional Capillary Zone Electrophoresis-Mass Spectrometry: Intact mAb Charge Variant Separation Followed by Peptide Level Analysis Using In-Capillary Digestion DOI
Johannes Schlecht, Kevin Jooß, Bernd Moritz

et al.

Analytical Chemistry, Journal Year: 2023, Volume and Issue: 95(8), P. 4059 - 4066

Published: Feb. 17, 2023

Characterization of charge heterogeneity is an essential pillar for pharmaceutical development and quality control therapeutic monoclonal antibodies (mAbs). The highly selective commonly applied capillary zone electrophoresis (CZE) method containing high amounts ε-aminocaproic acid (EACA) provides a detailed robust profile intact mAb variants. Nevertheless, the exact location protein modifications within these profiles remains ambiguous. Electrospray ionization mass spectrometry (ESI-MS) promising tool this purpose; however, EACA incompatible with electrospray. In context, we present two-dimensional CZE-CZE-MS system to combine efficient variant separation mAbs subsequent peptide analysis after in-capillary digestion selected first dimension based on generic CZE(EACA) in fused silica capillary. second dimension, neutral-coated used reduction Tris(2-carboxyethyl)phosphine (TCEP) pepsin, followed by CZE MS/MS-characterization resulting peptides. setup demonstrated using stressed nonstressed where peaks basic, main, acidic variants were transferred heart-cut fashion, digested, characterized level. Sequence coverages more than 90% obtained heavy chain (HC) light (LC) four different mAbs, including low-abundance (<2% main peak). Frequently observed (deamidation, oxidation, etc.) could be detected localized. This study demonstrates proof-of-concept identification localization from and, way, expected support testing pharmaceuticals.

Language: Английский

Citations

13