In-Depth Characterization of a Pro-Antibody–Drug Conjugate by LC–MS

Abstract

Pro-antibody-drug conjugate (PDC) is a hybrid structural format of immunoconjugate, where the structural complexity of pro-antibody and intrinsic heterogeneity of ADCs impose a prominent analytical challenge to the in-depth characterization of PDCs. In the present study, we successfully prepared and characterized PanP-DM1 as a model of PDCs, which is an anti-EGFR pro-antibody following conjugation with DM1 at lysine residues. The drug-to-antibody ratio (DAR) of PanP-DM1 was determined by LC-MS after deglycosylation, and verified by UV/vis spectroscopy. Following reduction or IdeS digestion, the pro-antibody fragments linked with DM1 were investigated by middle-down mass spectrometry. Furthermore, more than 20 modified lysine conjugation sites were determined by peptide mapping after trypsin digestion. Additionally, more than ten glycoforms of PanP-DM1 were also identified and quantified. In summary, critical quality attributes (CQAs) of PDCs including DAR, drug load distribution, and conjugation sites were fully characterized, which would contribute to the development of other PDCs for cancer treatment.

Elimination of tumor by CD47/PD-L1 dual-targeting fusion protein that engages innate and adaptive immune responses

“…23,46 Characterization of IAB by SEC-UPLC, LC-MS, and competitive ELISA The homogeneity of IAB were verified by size-exclusion chromatography-ultra performance liquid chromatography (SEC-UPLC) and molecular weight was accurately measured by LC-MS, as previously described. 47,48 The CD47/PD-L1 binding activity of IAB was evaluated by competitive ELISA. Briefly, ELISA plates (Costar) were coated overnight at 4 Cwith hPD-L1 Fc (Shanghai Sinomab Biotech Co., ZJ-01-041) or hCD47 Fc (Shanghai Sinomab Biotech Co., ZJ-01-053) in phosphate-buffered saline (PBS) buffer.…”

Ab locks for improving the selectivity and safety of antibody drugs

“…• Cetuximab [35] • anti-VCAM-1 Ab [43] • Panitumumab (anti-EGFR mAb) [193] • Panitumumab-DM1 [93,192] • anti-HIV p17 Ab [70] of immunogenicity of anti-Hinge Ab from the host; (3) Ab lock can significantly inhibit the binding and neutralizing ability of anti-idiotypic Ab to original Ab drugs, at least in the case of Infliximab; (4) The changeable design of protease substrate between Ab lock and Infliximab can be applied to any alternative Ab drugs that are used for RA or even other disease treatments. It is expected that a protease cleavable and efficient Ab lock can significantly increase the selective activation of Ab drugs at the disease site and reduce the on-target toxicities of Ab drugs during systemic circulation, thus potentially improving the clinical benefit and quality of life of patients.…”

“…Similar strategies were also applied to other Abs such as anti-vascular cell adhesion molecule 1 (VCAM-1) mAb [43] and anti-EGFR mAb (Panitumumab, Vectibix®) [193]. Furthermore, Liu et al [93] and Yang et al [192] applied the Probody approach to the maytansine (DM1)conjugated anti-EGFR mAb (Panitumumab) and generated a pro-antibody-drug conjugate (PDC) named PanP-DM1. Regardless of DM1 conjugation, the PanP-DM1 still displayed 12-fold weaker binding ability to immobilized EGFR antigen than parental Panitumumab and significantly improved the cancer-selective activity, therapeutic and safety index over traditional ADC.…”

Analytical techniques for the characterization of Antibody Drug Conjugates: Challenges and prospects

“…Average DAR of ADCs pool containing different drug load can be determined using UV-Vis spectroscopy. Average DAR for ADCs with drugs like vinca alkaloid 4-desacetylvinblastine-3-carbohydrazide (DAVLB), 13 methotrexate, 14 adriamycin, 15 and monomethyl auristatine E (MMAE), 16 maytansinoid, 17 emtansine 18 have been successfully determined. Hamblett et al have demonstrated the validity of DAR determined by UV-Vis method using hydrophobic chromatography.…”