Development of trypsin peptide mapping for hydroxylysine modification quantification in an IgG4 bispecific antibody and investigation of influencing factors.

We report the first identification of a rare hydroxylysine modification in the heavy chain 2 (HC2) region of an IgG4 bispecific antibody using peptide mapping coupled with liquid chromatography-mass spectrometry (LC-MS). The modified peptide matches the XKG consensus sequence recognized by lysyl hydroxylases. This modification severely impaired trypsin digestion efficiency: approximately 81% of missed cleavage peptides at this site contained hydroxylysine, indicating a strong association between the modification and missed cleavage. To reduce missed cleavage and enable accurate quantification of hydroxylysine, we systematically optimized the digestion conditions (buffer composition, pH, incubation temperature, digestion time, enzyme to antibody ratio) to enhance trypsin efficiency. This optimized method was subsequently applied to investigate the factors contributing to hydroxylation variability during antibody manufacturing. Our results showed that the downstream purification processes and storage stability conditions did not influence the hydroxylation levels. In contrast, key upstream cell culture parameters distinctly modulated hydroxylation: both a lower post-shift temperature and an earlier shift time resulted in a greater reduction in the modification level, whereas prolonged culture duration caused only a mild decrease. This work presents an optimized trypsin peptide mapping method that overcomes hydroxylysine-induced missed cleavage, enabling reliable quantification of this rare modification. Furthermore, it provides critical insights into the process dependence of hydroxylysine formation, facilitating the development of improved quality control strategies for therapeutic antibody production.