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

Rituximab is the first FDA-approved N-glycosylated chimeric anti-CD20 monoclonal antibody (mAb) used to treat non-Hodgkin lymphomas and autoimmune diseases. Glycosylation plays aimportant role in the clinical efficacy and safety of therapeutic mAbs, making it a critical quality attribute. Specifically, glycosylation in the Fc region modulates essential biological functions, such as interactions with Fc gamma receptors (FcγRs) and complement activation. Peptide mapping combined with high-resolution mass spectrometry (MS) enables precise characterization and inference of glycosylation patterns, supporting structural-functional analyses and quality control in biopharmaceutical development. This study aimed to perform a detailed structural characterization of rituximab and to determine the glycosylation profile present in its Fc region using a high-resolution MS-based peptide mapping approach. Commercial rituximab (100 µg) was incubated with 0.2% RapiGest SF, reduced with dithiothreitol, and alkylated with iodoacetamide. Tryptic digestion was performed at 37 °C for 16 hours. Peptides were separated by reverse-phase liquid chromatography using an EASY-Spray™ PepMap RSLC analytical column (C18, 2 µm, 100 Å, 75 µm × 50 cm), with 1 μL sample injections and a constant flow rate of 400 nL/min. The elution was performed using a gradient from 5% to 98% solvent B (acetonitrile + 0.1% formic acid) in solvent A (0.1% formic acid). MS analysis was conducted on an Orbitrap instrument in positive mode. The nano-electrospray ionization (NSI) spray voltage was set to 1.9 kV. Precursor masses were measured at a resolution of 70,000 over a 375–1500 m/z range, with an automatic gain control (AGC) target of 3 × 10^6 and maximum injection time of 100 ms. Data-dependent acquisition (DDA) selected the five most abundant precursor ions for fragmentation using higher-energy collision-induced dissociation (HCD), with a 4.0 m/z isolation window and normalized collision energy of 30. Product ions were scanned at a resolution of 17,500, with AGC set to 1 × 10^5 and a 50 ms injection time. MS/MS spectra were searched against the Chinese Hamster Ovary (CHO) protein database from UniProt using PatternLab 5.0 and Biopharma Finder 5.1. Carbamidomethylation was set as a fixed modification, while oxidation of methionine and tryptophan was defined as a variable modification. Up to two missed cleavages were allowed, with a 20 ppm mass tolerance. Glycosylation profiling revealed N-linked glycosylation at the conserved asparagine residue (N301) of the heavy chain, with a predominance of complex, biantennary, fucosylated structures such as A2G0F, A2G1F, and A2G2F. These glycoforms influence antibody effector functions due to charge and steric effects. For example, afucosylated and hybrid glycans enhance ADCC by increasing affinity to FcγRIIIa, whereas sialylated forms may promote anti-inflammatory responses. Terminal galactose residues contribute to CDC activity, and lower galactosylation correlates with reduced CDC. Although rituximab is clinically effective, its lack of afucosylated glycoforms suggests potential for further optimization of ADCC. This structural characterization underscores the importance of glycosylation control in therapeutic mAb development and supports its use in biosimilarity assessments and in designing variants with enhanced clinical performance.

Agradecimentos: Fiocruz Ceará - SUS - Programa PEC Fiocruz