ac5b03709_si_001.pdf (175.44 kB)
Structural Characterization of a Monoclonal Antibody–Maytansinoid Immunoconjugate
journal contribution
posted on 2016-01-05, 00:00 authored by Quanzhou Luo, Hyo Helen Chung, Christopher Borths, Matthew Janson, Jie Wen, Marisa K. Joubert, Jette WypychStructural characterization was performed
on an antibody–drug
conjugate (ADC), composed of an IgG1 monoclonal antibody (mAb), mertansine
drug (DM1), and a noncleavable linker. The DM1 molecules were conjugated
through nonspecific modification of the mAb at solvent-exposed lysine
residues. Due to the nature of the lysine conjugation process, the
ADC molecules are heterogeneous, containing a range of species that
differ with respect to the number of DM1 per antibody molecule. The
DM1 distribution profile of the ADC was characterized by electrospray
ionization mass spectrometry (ESI-MS) and capillary isoelectric focusing
(cIEF), which showed that 0–8 DM1s were conjugated to an antibody
molecule. By taking advantage of the high-quality MS/MS spectra and
the accurate mass detection of diagnostic DM1 fragment ions generated
from the higher-energy collisional dissociation (HCD) approach, we
were able to identify 76 conjugation sites in the ADC, which covered
approximately 83% of all the putative conjugation sites. The diagnostic
DM1 fragment ions discovered in this study can be readily used for
the characterization of other ADCs with maytansinoid derivatives as
payload. Differential scanning calorimetric (DSC) analysis of the
ADC indicated that the conjugation of DM1 destabilized the CH2 domain of the molecule, which is likely due to conjugation of DM1
on lysine residues in the CH2 domain. As a result, methionine
at position 258 of the heavy chain, which is located in the CH2 domain of the antibody, is more susceptible to oxidation
in thermally stressed ADC samples when compared to that of the naked
antibody.