Structural Characterization of the Aggregates of Gemtuzumab Ozogamicin

Abstract

The preparation of antibody drug conjugates (ADCs), particularly those containing hydrophobic payloads, may promote aggregate formation. The aggregate (high molecular mass species, HMMS) is an impurity that must be controlled in the final drug substance and drug product formulation to meet product safety and efficacy. Although there are numerous methods designed to mitigate protein aggregation, some of the reasons for the formation of aggregates in ADCs are unique and specific to the synthetic preparation and structure of each conjugate. Proper structural characterization and identification of causes of aggregation are key to improving the process for preparation of ADCs for minimizing aggregate formation. In this article, we have characterized aggregates generated during the preparation of a lysine conjugate, gemtuzumab ozogamicin, prepared from an IgG4 antibody (hP67.6) and a calicheamicin-based linker-payload. Using analytical and biophysical techniques, structural details of the aggregates formed in the conjugation process are elucidated. The aggregates are predominantly composed of a mixture of dimers and multimers with high drug loaded species. The level of conjugation to the N-termini of the mAb was higher in aggregates than in the monomeric ADC. Studies performed on the biological activity revealed differences in binding affinity and cytotoxicity between aggregates and monomeric ADC species.

Alteration of Physicochemical Properties for Antibody-Drug Conjugates and Their Impact on Stability

“…24 Further was shown that greater labeling of the mAb can result in higher tendencies to form aggregates. 36,37 Structural changes upon bioconjugation of polyethylene glycol (PEG), which is a commonly used linker system for ADCs, have been reported to reduce susceptibility by proteases by reduced conformational accessibility, when conjugated to lysine residues. A labeling of 6.8% of all lysine residues was sufficient to reduce trypsin digestion and even further mediated a higher resistance against pepsin and chymotrypsin digestion at a labeling degree of 14% compared to the mAb.…”

Recent developments in chemical conjugation strategies targeting native amino acids in proteins and their applications in antibody–drug conjugates

“…2 Such heterogenicity was further studied by its developer with size-exclusion chromatography – the species with higher average drug loading were found to be responsible for forming aggregates. 55 Physical instability such as aggregation is a stability limiting parameter for ADC; the product was voluntarily withdrawn from the US market in 2010 due to safety concerns. 56 InO has an average DAR of 6, and the drug load distribution varies from 0 to 9.…”

“…While it is unclear whether the conjugation of the drug to the N-termini of the mAb directly leads to the formation of aggregates, the structural characterization studies have shown that the level of conjugation to the N-termini of the mAb was higher in aggregates than in the monomeric ADC. 55 …”

“…While it is unclear whether the conjugation of the drug to the N-termini of the mAb directly leads to the formation of aggregates, the structural characterization studies have shown that the level of conjugation to the N-termini of the mAb was higher in aggregates than in the monomeric ADC. 55 However, it is debatable whether they directly lead to inferior therapeutic proles. It is worth introducing a preclinical study on cervical cancer, where a lysine conjugated ADC afforded a greater efficacy on a molar payload basis than a site-specic engineered cysteine conjugated ADC.…”

Advances and Limitations of Antibody Drug Conjugates for Cancer

“…It is conjugated via an acid cleavable hydrazone linker, 4-(4-acetylphenoxy) butanoic acid (AcBut) to a cytotoxic agent (N-acetyl gamma calicheamicin) [ 21 , 22 ]. It has an average DAR of 2 to 3 [ 23 ]. The linker moiety affixes to exposed lysines (lys) on the antibody.…”