Search

Antibody-drug conjugates (ADCs) offer new therapeutic options for advanced cancer patients through precision killing with fewer side effects. The stability and efficacy of ADCs are closely related, emphasizing the urgency and importance of gaining a comprehensive understanding of ADC stability. In this work, a chemical de-conjugation approach was developed to investigate the in-situ stability of the small molecule drug while it is conjugated to the antibody. This method involves chemical-mediated release of the small molecule drug from the ADC and subsequent characterization of the released small molecule drug by HPLC. The feasibility of this technique was demonstrated utilizing a model ADC containing a disulfide linker that is sensitive to the reducing environment within cancer cells. Five reducing agents were screened for use in de-conjugation; tris(2-carboxyethyl) phosphine (TCEP) was selected for further optimization due to its high efficiency and clean impurity profile. The optimized de-conjugation assay was shown to have excellent specificity and precision. More importantly, it was shown to be stability indicating, enabling the identification and quantification of the small molecule drug and its degradation products under different formulation pHs and storage temperatures. In summary, the chemical de-conjugation strategy demonstrated here offers a powerful tool to assess the in-situ stability of small molecule drugs on ADCs and the resulting information will shed light on ADC formulation/process development and storage condition selection., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The development of an accurate HPLC method for a reactive linker drug intermediate for antibody drug conjugates (ADCs) is challenging as the linker drug is designed to be reactive. This reactivity can lead to the generation of artifact peaks in the chromatograms and deliver inaccurate impurity content results. In this study, the linker drug contains an amine reactive tetrafluorophenyl (TFP) ester that readily undergoes hydrolysis and internal succinimide cyclization in aqueous solution. While complete elimination of the on-column degradation was nearly impossible, optimization of the critical chromatography conditions guided by chemical kinetics minimizes the degree of on-column degradation enabling an accurate assay and purity method for this reactive linker drug intermediate. Kinetics of the linker drug reactions were studied in different solution pHs and temperatures while the reaction rates were used to guide the selection of the optimum diluent, mobile phase pH, and column temperature to minimize the on-column degradation. An UHPLC column was used to achieve fast analysis to further reduce the degree of on-column degradation. The actual amount of on-column degradation of the final HPLC method was determined to be <0.1%, lower than the ICH reporting limit of, therefore demonstrate the effectiveness of the strategy., (Copyright © 2014 Elsevier B.V. All rights reserved.)