Caroline Fernandes Jaegger Franco¹, Daniele Rocha¹, Anna Carolina Machado Marinho², Bárbara Cibelle Soares Farias Quintela², Luciana da Silva Madeira³, Marcelle Almeida Caminha³, Michel Batista⁴, Kelly Cavalcanti Machado⁴, Rodrigo Soares Caldeira Brant⁴, Eduardo M Schmidt¹

Introduction: Due to the inherent complexity of therapeutic mAb samples, regulatory agencies require comprehensive characterization of mAb features to ensure product quality, safety, and efficacy. Therefore, mass spectrometric (MS) analysis of antibodies at the protein, subunit and peptide levels have become essential techniques in mAb characterization throughout the development and production process. Multifaceted MS approaches have been showing significant benefits for greater primary sequence coverage and identification of low abundant PTM modifications.

Objective: Demonstrate solid and versatile BioPharma protocol for mAb characterization developed and assessed on the three main High-Resolution Accurate-Mass Orbitrap platforms. This protocol encompasses a complete analytical workflow, from sample preparation to MS analysis.
Methodology: HRAM: Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer, Orbitrap Exploris™ 240 Mass Spectrometer and Q Exactive™ Plus Mass Spectrometer, both equipped with BioPharma Option. MS settings: 1) Resolution: Intact Protein 240,000; Subunit: 180,000 (light chain) and 30,000 (heavy chain); Peptide mapping: 70,000. 2) Full MS mass range (m/z): Intact Protein 2200–5000; Subunit: 600–3000; Peptide mapping: 200–2000. Chromatography: Mobile phase A: Water with 0.1% formic acid (v/v) Mobile phase B: Acetonitrile with 0.1% formic. Intact protein and subunit, MAbPac™ RP column was used. Peptide mapping, Acclaim PepMap^TM RSLC C18 column. mAb sample: NISTmAb, Humanized IgG1κ Monoclonal Antibody Reference Material 8671 - 10 µg. µL^-1. Sample Preparation: 1) Intact Mass: dilution in formic acid 0.1%; 2) Subunit (heavy and light chain): denaturation and reduction with urea and DTT; 3) Peptide mapping: denaturation with urea, reduction with DTT, alkylation with IAA and digestion with trypsin. Data processing: Raw data files analyzed with Thermo Scientific™ BioPharma Finder™ 5.1 incorporating Sliding Window, ReSpect and Xtract algorithms. 

Results: Orbitrap Exploris™ 240 and Q Exactive™ Plus MS BioPharma, enable analysis of intact biotherapeutics under native-like and denatured conditions with an ion transmission and mass detection up to m/z 8,000. The BioPharma Option offers distinct operational modes that were optimized for the three characterization workflows: 1) Intact analysis with the High Pressure mode; 2) Subunit analysis with Low Pressure mode; 3) Peptide mapping with Standard mode. LC-MS analysis of the intact IgG1 sample under evaluation and subsequent data processing yielded high quality MS spectra and deconvoluted spectra, illustrating the high-resolution and mass accurate capabilities of the applied mass spectrometers. The average mass of the best set of results obtained upon deconvolution was determined to be 148199.70 Da (mass deviation <0.1ppm). For mass measurement of the reduced IgG1 subunits, average mass obtained upon deconvolution for light chain was 23124.41Da (mass deviation <0.1ppm) and 50908.31Da (mass deviation <0.1ppm) for heavy chain. Results are in agreement with theoretic average mass of this mAb. Amino acid sequence yielding 70% sequence coverage for both the mAb heavy and light chains. The protocol was successfully implemented on the high-end Orbitrap platform for protein characterization, the Orbitrap Fusion Lumos MS.

Conclusion: The high-resolution and accurate mass capabilities provided by Orbitrap-based BioPharma MS platform significantly supports the high level of confidence required for primary sequence determination and confirmation of therapeutic biomolecules. Mass spectrometry; Orbitrap technology, Monoclonal Antibodies characterization.

Agradecimentos: The Nova Analítica Application team would like to express its sincere gratitude to the Mass Spectrometry Platform at the Carlos Chagas Institute, Fiocruz Paraná; the Proteomics Platform at Fiocruz Ceará; and LATEA/DEDEP at Bio-Manguinhos, Fiocruz, for their invaluable support and collaboration in this research. We are particularly thankful for the trust and partnership extended by these institutions, whose contributions were instrumental to the success of this study.