Two-Dimensional Capillary Zone Electrophoresis-Mass Spectrometry: Intact mAb Charge Variant Separation Followed by Peptide Level Analysis Using In-Capillary Digestion

Analytical Chemistry, Год журнала: 2023, Номер 95(8), С. 4059 - 4066

Опубликована: Фев. 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.

Язык: Английский

---

Buffering agents and additives for the background electrolyte solutions used for peptide and protein capillary zone electrophoresis

TrAC Trends in Analytical Chemistry, Год журнала: 2023, Номер 164, С. 117080 - 117080

Опубликована: Май 6, 2023

The background electrolyte solution (BGE) has a strong impact on the separation performance of peptides and proteins in all techniques governed by electrophoretic principles, due to its considerable influence mechanism physicochemical properties both sample inner surface device, which mostly consists fused-silica capillary tube. This review article examines discusses effects composition pH BGE electrokinetic phenomena taking place at solid liquid interface within capillaries, generating electroosmotic flow (EOF), zone electrophoresis (CZE), is most used mode for these biopolymers. paper reviews literature relevant explain how pH, ionic strength, conductivity employed peptide protein CZE efficiency, selectivity resolution other factors affecting separation, including flow. Also examined discussed compatibility with ionization sources on-line hyphenation mass spectrometry two popular preconcentration methods field-amplified stacking (FASS) dynamic junction.

Язык: Английский

---

Peptide mapping of proteins by capillary electromigration methods

Journal of Separation Science, Год журнала: 2022, Номер 45(23), С. 4245 - 4279

Опубликована: Окт. 6, 2022

This review article provides a wide overview of important developments and applications capillary electromigration methods in the area peptide mapping proteins period 1997-mid-2022, including articles on this topic. It deals with all major aspects by methods: i) precleavage sample preparation involving purification, preconcentration, denaturation, reduction alkylation protein(s) to be analyzed, ii) generation fragments off-line or on-line enzymatic and/or chemical cleavage protein(s), iii) postcleavage generated mixture for separation, iv) separation complex mixtures one-, two- multidimensional coupled mass spectrometry detection, v) large application variable purposes, such as qualitative analysis monoclonal antibodies other protein biopharmaceuticals, monitoring posttranslational modifications, determination primary structure investigation function biochemical clinical research, characterization origin well identification proteomic peptidomic studies.

Язык: Английский

---

Rapid identification of antibody impurities in size-based electrophoresis via CZE-MS generated spectral library

Scientific Reports, Год журнала: 2024, Номер 14(1)

Опубликована: Авг. 30, 2024

Methods for the reliable and effective detection identification of impurities are crucial to ensure quality safety biopharmaceutical products. Technical limitations constrain accurate individual impurity peaks by size-based electrophoresis separations followed mass spectrometry. This study presents a electrophoretic method detecting identifying in antibody production. A hydrogen sulfide-accelerated degradation was employed generate known products observed bioreactors that forms basis size calibration. LabChip GXII channel enabled rapid (< 1 min) based on size, while capillary zone electrophoresis-mass spectrometry (CZE-MS) facilitated their identification. We combine these techniques examine resulting from cell culture harvest conditions forced assess stability. To mimic impact degradation, we subjected samples cathepsin at different pH buffers or exposed them high temperature. Our demonstrated feasibility broad applicability using CZE-MS generated spectral library unambiguously assign throughput (i.e., GXII) with identifications likely impurity. Overall, this strategy combines utility as high-resolution separation methods typically used detect (not identify) during discovery development therapeutics.

Язык: Английский

---

Lipid‐Based Nanoparticles

Опубликована: Фев. 16, 2024

Lipid nanoparticles (LNPs) have gained significant attention in developing vaccines due to their unique properties, including biocompatibility, biodegradability, and the ability encapsulate hydrophobic hydrophilic molecules. They been used enhance efficacy of by improving antigen delivery presentation facilitating targeted specific cells tissues. Additionally, LNPs shown adjuvant effects, which can further immune responses. These properties make promising tools for safe effective against infectious diseases cancers. The chapter overviews LNPs, types, synthesis methods, characteristics, vaccine applications. two main types discussed are solid lipid nanostructured carriers. Various methods also presented, high-pressure homogenization, solvent emulsification– evaporation, microfluidics. characteristics size shape, surface charge, encapsulation efficiency, stability, discussed. then delves into applications vaccines, enhancing responses, delivery, effects. Finally, challenges future directions explored, safety toxicity concerns, stability issues, scale-up production challenges, opportunities research.

Язык: Английский

---

Recent advances in computational and experimental protein-ligand affinity determination techniques

Expert Opinion on Drug Discovery, Год журнала: 2024, Номер 19(6), С. 649 - 670

Опубликована: Май 7, 2024

Modern drug discovery revolves around designing ligands that target the chosen biomolecule, typically proteins. For this, evaluation of affinities putative is crucial. This has given rise to a multitude dedicated computational and experimental methods are constantly being developed improved.

Язык: Английский

---

Current PAT Landscape in the Downstream Processing of Biopharmaceuticals

Analytical Science Advances, Год журнала: 2025, Номер 6(1)

Опубликована: Май 29, 2025

ABSTRACT Protein‐based therapeutics have revolutionized modern medicine, addressing complex diseases with unprecedented specificity and efficacy. The rising demand for biologics has driven the evolution of biomanufacturing practices to ensure consistent quality operational efficiency. Traditional batch testing, its inherent limitations, is being replaced by design (QbD) frameworks process analytical technology (PAT). PAT facilitates real‐time monitoring control integrating advanced tools data‐driven methodologies optimize downstream processing (DSP). This review highlights recent advancements in tools, including spectroscopy, chromatography biosensors. Spectroscopic techniques provide rapid, non‐invasive measurements, while biosensors offer high critical attributes. Additionally, integration chemometric modelling digital twins enables predictive analytics enhances control, paving way release (RTR) product. Despite challenges regulatory compliance integration, innovations automation machine learning are bridging these gaps, accelerating transition intelligent manufacturing systems. article provides a comprehensive evaluation emerging technologies strategic insights into their aiming support biopharmaceutical industry's shift towards robust, continuous adaptive paradigms.

Язык: Английский

---

An Integrated Electrochemiluminescence Microfluidic Device For Detecting Ultra-low Concentration of Ceftriaxone

Biosensors and Bioelectronics, Год журнала: 2025, Номер unknown, С. 117657 - 117657

Опубликована: Июнь 1, 2025

Язык: Английский

---

Global intercompany assessment of ICIEF platform comparability for the characterization of therapeutic proteins

Electrophoresis, Год журнала: 2022, Номер 43(9-10), С. 1050 - 1058

Опубликована: Март 4, 2022

An international team spanning 19 sites across 18 biopharmaceutical and in vitro diagnostics companies the United States, Europe, China, along with one regulatory agency, was formed to compare precision robustness of imaged CIEF (ICIEF) for charge heterogeneity analysis National Institute Standards Technology (NIST) mAb a rhPD-L1-Fc fusion protein on iCE3 Maurice instruments. This information has been requested help better understand how these instruments transition ICIEF methods from instrument. The different laboratories performed NIST both using analytical specifically developed each molecules. After processing electropherograms, statistical evaluation data determine consistencies within between laboratory outlying information. apparent isoelectric point (pI) generated, based two-point calibration, main isoform showed high laboratories, RSD values less than 0.3% SDs charged variants percent peak area are 1.02% laboratories. These results validate appropriate use industry support process development submissions biotherapeutic Further, comparability illustrates that platform is next-generation replacement provides comparable data.

Язык: Английский

---

On-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry for the analysis of protein biomarkers in biological fluids and food: A tutorial

Advances in Sample Preparation, Год журнала: 2023, Номер 7, С. 100082 - 100082

Опубликована: Июль 4, 2023

The analysis by capillary electrophoresis (CE) of low abundant proteins in complex samples, such as biological fluids and food, is especially challenging, due to the poor concentration sensitivity microscale separation techniques sample matrix complexity. In order overcome these major drawbacks, microextraction preparation based on on-line solid-phase extraction (SPE-CE) are regarded an excellent alternative for clean-up analyte preconcentration with minimum handling. this study, we present, a tutorial, valve-free aptamer affinity electrophoresis-mass spectrometry (AA-SPE-CE-MS) method purification, preconcentration, separation, detection, characterization intact protein biomarkers food using cases study α‑synuclein (α‑syn), concanavalin A (Con A), β-lactoglobulin (β-LG), which related Parkinson's disease allergy, respectively. This tutorial not limited description analytical method, but it also provides ready-to-use procedures sorbent devices, introduces strategies undesired effects, allowing straightforward implementation optimum performance AA-SPE-CE-MS, platform develop further applications.

Язык: Английский

---