Physicochemical Stability of the Antibody−Drug Conjugate Trastuzumab-DM1: Changes due to Modification and Conjugation Processes

Abstract

In the manufacture of the antibody-drug conjugate Trastuzumab-DM1 (T-DM1), the lysine residues on the antibody trastuzumab (Tmab) are modified to form the intermediate Tmab-MCC (T-MCC) and then conjugated with the drug DM1. Our goal is to understand the effects of modification and conjugation steps on the physicochemical stability of the antibody. The structural stability of Tmab relative to its modified and conjugated forms was assessed, employing thermally induced stress conditions to formulations containing Tmab, T-MCC, and T-DM1. DSC, SEC, CE-SDS, and LC-MS were used to study the stability of Tmab, T-MCC, and T-DM1 to thermal stress. The DSC thermograms show a decrease in melting temperature for the CH2 transition, in the order Tmab > T-MCC > T-DM1. As per SEC analysis, a significant increase in level of aggregation was detected in T-MCC (∼32%) and T-DM1 (∼5%) after 14 days at 40 °C. Tmab did not show significant aggregate formation. CE-SDS and LC-MS data demonstrate that the aggregation in the case of T-MCC is largely covalent and involves mechanisms other than formation of intermolecular disulfide cross-links. The aggregation observed for T-MCC was significantly inhibited upon addition of amino acids with nucleophilic side chains containing thiol (Cys) and hydroxyl moieties (Ser, Tyr). The covalent aggregation observed for T-MCC and the ability of nucleophilic amino acids, particularly Cys, to inhibit it indicate that the maleimide moiety in the MCC linker may react to form intermolecular covalent cross-links between T-MCC molecules, possibly through a Michael addition mechanism. In addition, DSC results demonstrate that the conjugation of the drug moiety DM1 to Tmab results in destabilization of the CH2 domain of the antibody.

Antibody Conjugate Therapeutics: Challenges and Potential

“…Inside of the cell, most linkers are labile; however, some are stable, requiring degradation of the antibody and linker to release the cytotoxic agent. Thus, linkers are a key component of antibody-conjugate structures (42)(43)(44)(45). Currently used linkers most frequently react with lysine side chains or sulfhydryls in the hinge regions of the antibody.…”

Immunogenicity of Antibody Drug Conjugates: Bioanalytical Methods and Monitoring Strategy for a Novel Therapeutic Modality

“…As with any ligand binding assay, the biophysical properties (including charge, hydrophobicity, and solubility) and conformation of critical target reagents have profound effects on assay characteristics. Labeling and choice of assay buffers have been proven relevant for the use of mAbs as reagents in routine ADA immunoassays and are more critical now that the protein therapeutic in question has already been through one round (or more) of conjugation to a small-molecule drug (see (33,34) for examples of the consequences of conjugation on mAb biophysical properties). For example, if both conjugation and labeling chemistry utilize amine groups of lysine residues, the net positive charge of the doubly labeled ADC reagent may be reduced.…”

Improving biophysical properties of a bispecific antibody scaffold to aid developability

“…In addition, proper folding and stability of the CH3 domain could also contribute to the monomer-dimer equilibrium, 45 thus a stable CH3 heterodimer improves the homogeneity of the engineered antibody. From an application perspective, the next generation of antibody designs could very well involve bispecific antibodies that are themselves antibody drug conjugates, 46 and given that drug conjugation reactions tend to affect the stability and observed Tm of the parent antibody, 47 a more stable heterodimeric Fc can improve the manufacturability of these classes of therapeutics.…”