SIMAO PEDRO JACINTO NOGUEIRA NETO^1,2, MARIA BEATRIZ DA SILVA SANTOS^1,2, SUELEN CARNEIRO DE MEDEIROS¹, BARBARA CIBELLE SOARES FARIAS QUINTELA¹, ANNA CAROLINA MACHADO MARINHO¹

Introduction: Tryptic hydrolysis is critical for peptide mapping by mass spectrometry, enabling controlled peptide generation and improved detection of post-translational modifications (PTMs). The Quality by Design (QbD) approach facilitates risk analysis and method optimization, ensuring reproducibility, reliability, and cost-effectiveness. Optimizing hydrolysis time is particularly important for monoclonal antibody characterization, as incomplete digestion can lead to missed PTM identification, while overdigestion may generate excessive short peptides, complicating data analysis. Objective: Develop an experimental design to assess the impact of hydrolysis time by evaluating different trypsin incubation periods with the commercial monoclonal antibody Rituximab. Materials and Methods: A standardized protocol was applied, testing trypsin incubation times of 3, 6, 16, and 24 hours for 100 μg of Rituximab. Samples (1 μL) were injected at 400 nL·min⁻¹ and separated using an EASY-Spray™ PepMap RSLC column (C18, 2 μm, 100 Å, 75 μm × 50 cm) with a 5–98% gradient of solvent B (ACN + 0.1% formic acid) in solvent A (0.1% formic acid). MS analysis was performed with a spray voltage of 1.9 kV, precursor resolution of 70,000 (m/z 375–1500), AGC target of 3 × 10⁶ (positive mode) and Top 5 precursor selection for HCD. Fragment ions were analyzed at 17,500 resolution (AGC 1 × 10⁵). Data were processed using PatternLab and Biopharma Finder with fixed carbamidomethylation, variable methionine oxidation, up to two missed cleavages, and 20 ppm mass tolerance. Results and Discussion: Enzymatic efficiency was highest after 16 hours of incubation, yielding the most peptides (366) with 88.5% fully cleaved peptides and only six missed cleavages. Fully specific peptides peaked at 16h (143 peptides), outperforming 3h, 6h, and 24h (94, 68, and 89 peptides, respectively). Semi-specific peptides were also highest at 16h (223 peptides). Conclusion: For this experimental setup, 16-hour tryptic digestion optimized peptide yield, specificity, and cleavage efficiency, demonstrating the utility of QbD in method development.

Agradecimentos: Fiocruz Ceará; SUS; Programa de Vocação Científica (PROVOC); Escola Eusébio de Queiroz; Escola Politécnica Joaquim Jose Venâncio (Fiocruz)