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
The result's identifiers
Result code in 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>
Result on the web
<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>
Alternative languages
Result language
angličtina
Original language name
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
Original language description
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.
Czech name
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Czech description
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Classification
Type
J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database
CEP classification
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OECD FORD branch
10406 - Analytical chemistry
Result continuities
Project
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Continuities
I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace
Others
Publication year
2025
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů
Data specific for result type
Name of the periodical
ANALYTICA CHIMICA ACTA
ISSN
0003-2670
e-ISSN
1873-4324
Volume of the periodical
1353
Issue of the periodical within the volume
June
Country of publishing house
NL - THE KINGDOM OF THE NETHERLANDS
Number of pages
12
Pages from-to
1-12
UT code for WoS article
001457130300001
EID of the result in the Scopus database
2-s2.0-105000573581