Online multimethod platform for comprehensive characterization of monoclonal antibodies in cell culture fluid from injection of crude sample - Incorporation of middle-up and bottom-up workflows

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14310%2F25%3A00143559" target="_blank" >RIV/00216224:14310/25:00143559 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.sciencedirect.com/science/article/pii/S000326702500337X" target="_blank" >https://www.sciencedirect.com/science/article/pii/S000326702500337X</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.aca.2025.343943" target="_blank" >10.1016/j.aca.2025.343943</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Online multimethod platform for comprehensive characterization of monoclonal antibodies in cell culture fluid from injection of crude sample - Incorporation of middle-up and bottom-up workflows

Popis výsledku v původním jazyce

Background: Determination of critical quality attributes (CQAs) of pharmaceutical monoclonal antibodies (mAbs) is an essential part of quality control. Commonly, for each CQA, a separate analytical method and setup is required, making assessment of multiple CQAs time-consuming and labour-intensive. This typically involves offline purification and diverse protein digestion steps, in combination with multiple liquid-chromatographic modes. We developed an integrated, fully online multidimensional platform for direct analysis of mAbs in cell culture fluid (CCF) at an intact, subunit and peptide level from a single injection. Results: This paper focuses on the online middle-up and bottom-up workflows. The platform combines Protein A affinity chromatography (ProtA), immobilized enzyme reactors (IMERs), reversed-phase liquid chromatography (RPLC) and high-resolution mass spectrometry (MS) for characterization of mAbs. Online ProtA was used to isolate mAbs directly from CCF. Subsequent online digestion of isolated mAb was accomplished by IMERs featuring either the proteases IdeS or trypsin. Between ProtA and IMERs, buffer exchange and pH adjustment were achieved using a strong cation-exchange (SCX) trap column. RPLC-MS analysis of F(ab)'2 and Fc/2 fragments obtained after IdeS digestion provided information on mAb glycoform compositions and the potential presence of PTMs and subunit variants. RPLC-MS/MS analysis of trypsin-digested peptides provided over 95 % coverage of the mAb's amino acid sequence, but also identification and localization of modifications related toe. g. oxidation and deamidation. Comparisons with established offline methods were made. The overall capacity of the system to perform intact, middle-, and bottom-up analyses in parallel from a single injection is demonstrated for an industrially-relevant mAb in CCF. Significance: The developed multidimensional platform enables the simultaneous characterization of multiple fractions from a single ProtA-isolated mAb band at intact, middle-up, or bottom-up level using various LC modes at a substantially reduced analysis time.

Název v anglickém jazyce

Online multimethod platform for comprehensive characterization of monoclonal antibodies in cell culture fluid from injection of crude sample - Incorporation of middle-up and bottom-up workflows

Popis výsledku anglicky

Background: Determination of critical quality attributes (CQAs) of pharmaceutical monoclonal antibodies (mAbs) is an essential part of quality control. Commonly, for each CQA, a separate analytical method and setup is required, making assessment of multiple CQAs time-consuming and labour-intensive. This typically involves offline purification and diverse protein digestion steps, in combination with multiple liquid-chromatographic modes. We developed an integrated, fully online multidimensional platform for direct analysis of mAbs in cell culture fluid (CCF) at an intact, subunit and peptide level from a single injection. Results: This paper focuses on the online middle-up and bottom-up workflows. The platform combines Protein A affinity chromatography (ProtA), immobilized enzyme reactors (IMERs), reversed-phase liquid chromatography (RPLC) and high-resolution mass spectrometry (MS) for characterization of mAbs. Online ProtA was used to isolate mAbs directly from CCF. Subsequent online digestion of isolated mAb was accomplished by IMERs featuring either the proteases IdeS or trypsin. Between ProtA and IMERs, buffer exchange and pH adjustment were achieved using a strong cation-exchange (SCX) trap column. RPLC-MS analysis of F(ab)'2 and Fc/2 fragments obtained after IdeS digestion provided information on mAb glycoform compositions and the potential presence of PTMs and subunit variants. RPLC-MS/MS analysis of trypsin-digested peptides provided over 95 % coverage of the mAb's amino acid sequence, but also identification and localization of modifications related toe. g. oxidation and deamidation. Comparisons with established offline methods were made. The overall capacity of the system to perform intact, middle-, and bottom-up analyses in parallel from a single injection is demonstrated for an industrially-relevant mAb in CCF. Significance: The developed multidimensional platform enables the simultaneous characterization of multiple fractions from a single ProtA-isolated mAb band at intact, middle-up, or bottom-up level using various LC modes at a substantially reduced analysis time.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10406 - Analytical chemistry

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2025

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

ANALYTICA CHIMICA ACTA

ISSN

0003-2670

e-ISSN

1873-4324

Svazek periodika

1353

Číslo periodika v rámci svazku

June

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

12

Strana od-do

1-12

Kód UT WoS článku

001457130300001

EID výsledku v databázi Scopus

2-s2.0-105000573581