Abstract

Deamidation of asparagine (Asn) residues of monoclonal antibodies (mAbs) plays a pivotal role in the in vivo/vitro degradation or efficacy loss of biopharmaceuticals. However, a major challenge for MS analysis of deamidation of Asn-containing peptides in mAbs, is due to the fact that there is only a 1 Da mass shift between the native form (Asn residues) and deamidated forms (n-aspartyl (n-Asp) and isoaspartyl (isoAsp) residues with identical mass). Therefore, a chromatographic separation of the deamidated proteins and/or the peptides derived therefrom is needed prior to MS analysis. In this study, the monolithic column with various stationary phases, including reverse phase (RP), single phospholipid-functionalized and mixed phospholipid-functionalized monoliths, were prepared for the separation of the deamidation-sensitive signature peptide (IYPTNGYTR) of trastuzumab and its two deamidated products, n-Asp55 residue IYPTDGYTR and isoAsp55 residue IYPTisoDGYTR. Compared to the RP monolith, the phospholipid-functionalized monoliths provided mixed-mode interactions and exhibited better peak shape and separation selectivity. The effect of the parameters, including the type and concentration of buffer, temperature and pH value on the separation performance were investigated. Under the optimal conditions, the three peptides were fully separated on a mixed phosphocholine (PC) / phosphatidic acid (PA) functionalized monolith (poly (MDPC60PA40-co-EDMA)) due to the joint contribution of hydrophobic and electrostatic interactions. Therefore, the novel method based on the mixed phospholipids-functionalized monolithic column exhibited good potential for the analysis of deamidated peptides, which will be useful for the in-depth study of post-translational modifications of mAbs.

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call

Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.