Structural Characterization of a Monoclonal Antibody–Maytansinoid Immunoconjugate

Abstract

Structural 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 C(H)2 domain of the molecule, which is likely due to conjugation of DM1 on lysine residues in the C(H)2 domain. As a result, methionine at position 258 of the heavy chain, which is located in the C(H)2 domain of the antibody, is more susceptible to oxidation in thermally stressed ADC samples when compared to that of the naked antibody.

Quantitative collision‐induced unfolding differentiates model antibody–drug conjugates

“…Such conjugation allows for the high selectivity of mAbs to be combined with cytotoxic drugs, achieving discrimination between healthy and diseased tissue in contrast to traditional chemotherapies. While both cysteine and lysine‐targeted chemistries are widely used in ADC generation, the latter typically results in a more heterogeneous drug‐to‐antibody ratio (DAR) distribution, often creating therapeutics with increased structural complexity, owing to the large number of native lysine residues in mAb sequences …”

The impact of trisulfide modification of antibodies on the properties of antibody-drug conjugates manufactured using thiol chemistry

“…This fragment ion was generated by cleavage of the ester bond in DM4, as shown in the published results. 18,34 The other fragment ions, such as m/z 262.14 corresponding to the y2 fragment, originated from the peptide. This observation indicated that DM4 was directly conjugated to the side chain of cysteine.…”

Antibody drug conjugates

“…In addition to drug-antibody ratio, drug load, free drug, linker, and residual solvent levels are determined. Moreover, container closure, intravenous infusion bag (if applicable), transport, and storage stabilities are rigorously tested (Wakankar et al 2011;Luo et al 2016;Ross and Wolfe 2016).…”