Maria Catarina Teles do Nascimento Gomes¹, Mariana Santos de Freitas¹, Matheus Ferreira de Aguiar¹, Donat Alexander de Chapeaurouge¹, Bárbara Cibelle Soares Farias Quintela¹, Anna Carolina Machado Marinho¹

Rituximab is a chimeric monoclonal antibody used to treat lymphomas, chronic lymphocytic leukemia, and rheumatoid arthritis. Its mechanism involves Fab domain binding to CD20 on B lymphocytes, inducing cell lysis. IgGs contain conserved N-linked glycans in the Fc region, typically composed of biantennary structures with varying degrees of core fucosylation, N-acetylglucosamine, galactose, and sialic acid. As glycan composition critically affects antibody function and immune response, this study aimed to perform a detailed structural characterization of rituximab, focusing on Fc glycosylation via high-resolution intact protein MS. Commercial rituximab was desalted and concentrated using 30 kDa MWCO centrifugal filters, exchanging the formulation buffer for 0.1% formic acid. For intact MS, samples were diluted 1:10 in 0.1% formic acid (~1 μg/μL protein). LC separation was performed on a Dionex Ultimate™ 3000 RSLCnano with a MabPac™ RP column (4 μm, 1500 Å, 2.1 × 100 mm) using a 20-min gradient from 25–80% solvent B (acetonitrile with 0.1% formic acid) at a flow rate of 250 μL/min. MS acquisition was performed on an Orbitrap instrument in Full MS mode (resolution 280,000), under HMR positive mode, with extended m/z range (2400–4000), AGC target 3 × 10⁶, CID energy 80 eV, and maximum IT 200 ms. Ionization parameters included a spray voltage of 4.2 kV, 75 °C source temp, and 100 eV S-lens RF. Mass deconvolution was performed with Biopharma Finder 5.1 (Intact Mass Analysis module), using the Xtract™ algorithm (20 ppm mass tolerance), accounting for PTMs such as CHO-specific glycosylation and C-terminal lysine clipping. Intact MS revealed a single, well-defined chromatographic peak with a retention time of ~6 min. The non-deconvoluted mass spectra exhibited clear ion envelope distributions with well-resolved charge states, demonstrating good reproducibility across injections. Deconvoluted spectra identified a major species at 147,124.70 Da, along with significant heterogeneity as evidenced by the detection of 13 distinct glycoforms. Glycosylation profiling confirmed N-linked glycosylation at the conserved asparagine residue (N301), with predominance of complex biantennary structures (A2G0F, A2G1F, A2G2F) and triantennary forms (A3G0), both fucosylated and afucosylated forms. High-mannose species (Man5) and sialylated glycans were also detected. Terminal mannose and galactose residues are linked to enhanced clearance and reduced half-life, while high-mannose glycans increase Antibody-Dependent Cell Cytotoxicity (ADCC) and immunogenicity. Galactose can modulate Complement-Dependent Cytotoxicity (CDC), and sialylated structures, especially those with N-glycolylneuraminic acid (produced in nonhuman cell lines), are associated with anti-inflammatory effects and reduced ADCC. Given the inherent macro- and microheterogeneity of glycosylation, which leads to variability even among batches of the same originator biologic, this study underscores the importance of rigorous glycosylation profiling in biosimilar development. Regulatory guidelines recommend assessing glycosylation similarity based on predefined quality ranges, ensuring consistent therapeutic efficacy and safety. Our high-resolution intact mass analysis provides a robust approach for characterizing rituximab’s glycosylation profile, offering valuable insights for biopharmaceutical quality control and biosimilarity assessments.

Agradecimentos: Fiocruz Ceará - SUS - Programa PEC Fiocruz