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 to e.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.

Agilent Technologies R and D and Marketing GmbH Hewlett Packard Strasse 8 76337 Waldbronn Germany

Centre for Analytical Sciences Amsterdam the Netherlands; Analytical Chemistry Group Van't Hoff Institute for Molecular Sciences University of Amsterdam PO Box 94720 1090 GE Amsterdam the Netherlands

Department of Chemistry Faculty of Science Masaryk University Kamenice 5 62500 Brno Czech Republic

Division of Bioanalytical Chemistry Department of Chemistry and Pharmaceutical Sciences Amsterdam Institute of Molecular and Life Sciences Vrije Universiteit Amsterdam De Boelelaan 1085 1081 HV Amsterdam the Netherlands; Centre for Analytical Sciences Amsterdam the Netherlands

Polpharma Biologics Utrecht B 5 Yalelaan 46 3584 CM Utrecht the Netherlands

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