Mariana Santos de Freitas¹, Maria Catarina Teles do Nascimento Gomes¹, Matheus Ferreira de Aguiar¹, Bárbara Cibelle Soares Farias Quintela¹, Anna Carolina Machado Marinho¹

The multi-attribute method (MAM), based on liquid chromatography–mass spectrometry (LC-MS), is a powerful analytical strategy for characterizing and monitoring multiple critical quality attributes in biotherapeutics, particularly monoclonal antibodies (mAbs). Among MAM approaches, middle-up/down analysis is gaining traction as a complementary technique to intact and bottom-up analyses. By focusing on large subunits generated through specific enzymatic cleavage, the middle-up strategy offers improved mass accuracy and simplified spectra compared to intact analysis, while maintaining structural context lost in bottom-up methods. In this study, the molecular weights of individual subunits of a monoclonal IgG mAb (~150 kDa) were determined using a middle-up LC-MS approach. The mAb was enzymatically cleaved at the hinge region with 1 μL of IdeS (50 U/μL), yielding two Fc/2 fragments and one F(ab’)₂ fragment. The digestion was performed at 37 °C for 2 hours. Samples were treated with 8 M guanidine and 50 mM DTT, followed by incubation at 56 °C for 45 minutes to denature the F(ab’)₂ fragment and reduce disulfide bonds, resulting in the release of the light chain (LC) and Fd fragment. Samples were then dried in a SpeedVac at room temperature for 30 minutes and reconstituted to 1 μg/μL in 0.1% formic acid. LC separation was performed using a reversed-phase Dionex Ultimate™ 3000 RSLCnano system equipped with a MabPac™ RP column (4 μm, 1500 Å, 2.1 × 100 mm). A 1 μL injection (full loop) was run at 250 μL/min using a 28-minute linear gradient from 25% to 80% solvent B (acetonitrile with 0.1% formic acid), followed by a column wash at 80% B and re-equilibration to 25% B. Mass spectrometry was conducted using an Orbitrap instrument operating in Full MS mode with a resolution of 280,000, extended mass range (600–4000 m/z), high-mass-range (HMR) positive mode, AGC target of 3 × 10^6, and maximum injection time of 200 ms. Ion source parameters included a spray voltage of 4.2 kV, a source temperature of 75 °C, and an S-lens RF level of 60 eV. Mass deconvolution was performed using Biopharma Finder 5.1 (Intact Mass Analysis module) with the Xtract™ algorithm, considering a 20 ppm mass tolerance and post-translational modifications (PTMs) such as glycosylation, C-terminal lysine clipping, and/or N-terminal pyroglutamination. The light and heavy chains showed expected monoisotopic masses of 23,113.32 Da and 51,773.71 Da, respectively. The Fc and Fd fragments - comprising part of the Fab domain - showed average monoisotopic masses of 25,398.06 Da and 25,685.80 Da, respectively. These results confirm the antibody´s structural integrity and the efficiency of subunit generation. This study demonstrates the applicability of middle-up LC-MS as a robust tool for detailed mAb characterization, contributing to quality control and comparability assessments in biopharmaceutical development. Future research may explore the middle-down strategy, which involves the fragmentation of subunits in MS/MS and enhance this analysis by enabling the identification of PTMs with increased resolution.

Agradecimentos: Os autores agradecem à Fundação Oswaldo Cruz Ceará (Fiocruz Ceará), ao Sistema Único de Saúde (SUS) e ao Programa de Estágio Curricular (PEC Fiocruz) pelo apoio institucional e pela contribuição para a realização deste trabalho.