Characterization of critical quality attributes (CQAs) for bispecific antigen- binding biotherapeutic (BABB) through comprehensive analysis using separation techniques and footprinting approaches coupled with mass spectrometry DOI Open Access
Arnik Shah

Published: Jan. 1, 2023

Bispecific antigen-binding biotherapeutics (BABB) are a novel class of therapeutics developed to combat various disease conditions. Identifying critical quality attributes (CQAs) for is important in establishing by design (QbD) achieve consistent product ensure safety and efficacy patients. BABB molecules produced at low concentrations due their high potency compared traditional monoclonal antibodies. This creates challenges characterizing the based on analytical approaches. Mass spectrometry-based methods have recently emerged as powerful tools characterize post-translation modifications (PTMs) biotherapeutics. In this work, we explored different separation techniques footprinting approaches coupled with mass spectrometry identify molecules. first chapter, describe how utilized microfluidic-based capillary zone electrophoresis fragmentation sites therapeutic. second overview native workflow was present molecule, offering rapid unbiased characterization predominant abundance PTMs more third explore applications covalent labeling subtle changes molecule under two states (native vs. stressed) that can impact quality. These workflows provide in-depth conformation amino acid level molecule.Application such stages drug development aid CQAs related other modalities protein therapeutics. --Author's abstract

Language: Английский

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
Capillary electrophoresis‐mass spectrometry for protein analyses under native conditions: Current progress and perspectives DOI Creative Commons
Ann‐Katrin Schwenzer,

Lena Kruse,

Kevin Jooß

et al.

PROTEOMICS, Journal Year: 2023, Volume and Issue: 24(3-4)

Published: June 13, 2023

Abstract Native mass spectrometry is a rapidly emerging technique for fast and sensitive structural analysis of protein constructs, maintaining the higher order structure. The coupling with electromigration separation techniques under native conditions enables characterization proteoforms highly complex mixtures. In this review, we present an overview current CE‐MS technology. First, status described capillary zone electrophoresis (CZE), affinity (ACE), isoelectric focusing (CIEF), as well their chip‐based formats, including essential parameters such electrolyte composition coatings. Further, required ESI‐MS (large) instrumental QTOF Orbitrap systems, requirements interfacing are presented. On basis, methods applications different modes summarized discussed in context biological, medical, biopharmaceutical questions. Finally, key achievements highlighted concluded, while remaining challenges pointed out.

Language: Английский

Citations

26
Applications of capillary electrophoresis for biopharmaceutical product characterization DOI
Ramesh Kumar, András Guttman, Anurag S. Rathore

et al.

Electrophoresis, Journal Year: 2021, Volume and Issue: 43(1-2), P. 143 - 166

Published: Sept. 30, 2021

Capillary electrophoresis (CE), after being introduced several decades ago, has carved out a niche for itself in the field of analytical characterization biopharmaceutical products. It does not only offer fast separation, high resolution miniaturized format, but equally importantly represents an orthogonal separation mechanism to high-performance liquid chromatography. Therefore, it is surprising that CE-based methods can be found all major pharmacopoeias and are recommended analysis products during process development, characterization, quality control, release testing. Different formats CE, such as capillary gel electrophoresis, isoelectric focusing, zone widely used size charge heterogeneity well purity stability testing therapeutic proteins. Hyphenation CE with MS emerging promising bioanalytical tool assess primary structure proteins along any impurities. In this review, we confer latest developments critical attributes covering past 6 years (2015-2021). Monoclonal antibodies, due their significant share market, have been given prioritized coverage.

Language: Английский

Citations

33
An improved capillary isoelectric focusing-mass spectrometry method for high-resolution characterization of monoclonal antibody charge variants DOI
Tian Xu,

Linjie Han,

Alayna M. George Thompson

et al.

Analytical Methods, Journal Year: 2021, Volume and Issue: 14(4), P. 383 - 393

Published: Dec. 17, 2021

We developed an automated cIEF-MS method with good stability and high resolution for characterizing charge variants of mAbs by bettering capillary neutral coating, reducing catholyte pH to 10, optimizing the cIEF separation conditions.

Language: Английский

Citations

27
Concepts and recent advances in microchip electrophoresis coupled to mass spectrometry: Technologies and applications DOI
Elahe Naghdi,

Griffin E. Moran,

Marika Ejby Reinau

et al.

Electrophoresis, Journal Year: 2022, Volume and Issue: 44(1-2), P. 246 - 267

Published: Aug. 17, 2022

The online coupling of microchip electrophoresis (ME) as a fast, highly efficient, and low-cost miniaturized separation technique to mass spectrometry (MS) an information-rich sensitive characterization results in ME-MS attractive tool for various applications. In this paper, we review the basic concepts latest advances technology ME coupled MS during period 2016-2021, covering materials, structures, fabrication techniques, interfacing electrospray ionization (ESI)-MS matrix-assisted laser desorption/ionization-MS. Two critical issues ESI-MS include electrical connection used define electrophoretic field strength along channel generation detection, well as, ESI-tip. recent commercialization zone isoelectric focusing modes has led widespread application these techniques academia industry. Here summarize applications detection antibodies, proteins, peptides, carbohydrates, metabolites, so on. Throughout paper are discussed context benefits limitations comparison alternative techniques.

Language: Английский

Citations

18
CE-MS/MS and CE-timsTOF to separate and characterize intramolecular disulfide bridges of monoclonal antibody subunits and their application for the assessment of subunit reduction protocols DOI Creative Commons
Jasmin Schairer, Jennifer Römer, Dietmar Lang

et al.

Analytical and Bioanalytical Chemistry, Journal Year: 2024, Volume and Issue: 416(7), P. 1599 - 1612

Published: Feb. 1, 2024

Abstract Characterization at the subunit level enables detailed mass spectrometric characterization of posttranslational modifications (PTMs) monoclonal antibodies (mAbs). The implemented reduction often leaves intramolecular disulfide bridges intact. Here, we present a capillary electrophoretic (CE) method based on neutral-coated for separation immunoglobulin G-degrading enzyme Streptococcus pyogenes (IdeS) digested and reduced mAb subunits followed by spectrometry (MS), MS/MS identification, trapped ion mobility (timsTOF). Our CE approach (i) different moieties, (ii) various states, (iii) positional isomers these partly moieties. location remaining can be determined middle-down electron transfer higher energy collisional dissociation (EThcD) experiments. All CE-separated variants show differences in timsTOF measurements. Applying presented CE-MS/MS method, parameters such as use chaotropic salts were studied. For investigated antibodies, urea improved significantly, whereas guanidine hydrochloride (GuHCl) leads to multiple signals same separation. CE-MS is powerful tool disulfide-variant mAbs level. It understanding bridge processes potentially other proteins.

Language: Английский

Citations

3
Greenness of proteomic sample preparation and analysis techniques for biopharmaceuticals DOI Creative Commons
Katarína Maráková, Radovan Tomašovský, Martina Opetová

et al.

TrAC Trends in Analytical Chemistry, Journal Year: 2023, Volume and Issue: 171, P. 117490 - 117490

Published: Dec. 19, 2023

Proteins are efficient therapeutic agents and attractive potential new biomarkers due to their physiological pathological functions. Further, biopharmaceuticals feature complex protein architectures that must be assayed quality controlled. Capillary electrophoresis (CE) has become one of the most powerful tools for proteomic analysis. CE is an eco-friendlier alternative traditional liquid chromatography; however, its true greenness environmental impact not very often confirmed by green metrics. Here, we assessed bottom-up, middle-up, top-down sample preparation analytical approaches analysis proteins using AGREEprep AGREE metric systems, respectively. The combined use both can help identify weaknesses overall procedure point toward greener alternatives. results showed trends in workflows should focused mainly on miniaturization devices applying in-line on-line preparation.

Language: Английский

Citations

7
Characterization of Therapeutic Antibody Charge Heterogeneity Under Stress Conditions by Microfluidic Capillary Electrophoresis Coupled with Mass Spectrometry DOI
Zhijie Wu, Hongxia Wang, Xueqing Zhao

et al.

Journal of Pharmaceutical Sciences, Journal Year: 2024, Volume and Issue: 113(8), P. 2170 - 2177

Published: May 23, 2024

Language: Английский

Citations

2
Antibodies, repertoires and microdevices in antibody discovery and characterization DOI Creative Commons
Luca Johannes Schlotheuber, Ines Lüchtefeld, Klaus Eyer

et al.

Lab on a Chip, Journal Year: 2024, Volume and Issue: 24(5), P. 1207 - 1225

Published: Jan. 1, 2024

Therapeutic antibodies are paramount in treating a wide range of diseases, particularly auto-immunity, inflammation and cancer, novel antibody candidates recognizing vast array antigens needed to expand the usefulness applications these powerful molecules. Microdevices play an essential role this challenging endeavor at various stages since many general requirements overall process overlap nicely with advantages microfluidics. Therefore, microfluidic devices rapidly taking over steps new candidate isolation, such as characterization discovery workflows. Such technologies can allow for improvements time-lines incorporate conservative stability assays, but most prominently screenings functional within integrated workflows due high throughput standardized First, we aim provide overview challenges developing therapeutic candidates, their repertoires requirements. Afterward, review focuses on using systems, technological aspects micro small-scale protein selection, well integration implementation into We close future developments detection isolation principles field general.

Language: Английский

Citations

1
Characterization of Monoclonal Antibody Charge Variants under Near-Native Separation Conditions using Nanoflow Sheath Liquid Capillary Electrophoresis-Mass Spectrometry DOI Creative Commons
Annika A. M. van der Zon, Alisa Höchsmann, Tijmen S. Bos

et al.

Published: May 3, 2024

Monoclonal antibodies (mAbs) undergo multiple post-translational modifications (PTMs), e.g., charge variants, oxidation, etc., during production and storage, necessitating evaluation of the resulting PTMs as critical quality attributes (CQA) for protein safety. Charge variants can be separated using capillary zone electrophoresis (CZE). The CZE EACA method developed by He et al. (2011) with UV detection is applied routinely in pharmaceutical industry analyzing variants. However, cannot directly hyphenated mass spectrometry (MS), preventing direct identification due to non-volatile background electrolyte (BGE), which hinders reliable variant identification. This study presents a CZE-UV/MS neutral static coating hydroxypropyl methylcellulose combined volatile BGE at pH 5.0 allow MS-compatible mAb separations. effect several parameters, including concentration BGE, voltage, injected concentrations terms separation performance on panel mAbs was investigated. final tested IgG subclasses (IgG1 IgG4) different pI (7.4-9.2), degrees heterogeneity. Basic acidic were from parent 50 mM acetic acid adjusted 5.0. A linear correlation relative abundance obtained between our method. CZE-MS coupling done nanoCEasy, low-flow sheath liquid interface, allowed quantitation various low-abundance (<10% respect main compound). uses buffers operates closer non-denaturing conditions (pH 5.0), allowing flexibility hyphenation MS. useful tool in-depth characterization mAbs.

Language: Английский

Citations

1