Cutting-edge multi-level analytical and structural characterization of antibody-drug conjugates: present and future

Abstract

Introduction: The development and optimization of antibody drug conjugates (ADCs) rely on improving their analytical and bioanalytical characterization, by assessing critical quality attributes (CQAs). Among the CQAs, the glycoprofile, drug load distribution (DLD), the amount of unconjugated antibody (D0), the average drug-to-antibody ratio (DAR), the drug conjugation sites and the residual drug-linker and related product proportions (SMDs) in addition to high and low molecular weight species (H/LMWS) are the most important ones. Areas covered: The analytical and structural toolbox for the characterization of 1 st , 2 d and 3 d generation ADCs was significantly extended in the last 3 years. Here, we reviewed state-ofthe-art techniques, such as liquid chromatography, high resolution native and ion mobility mass spectrometry, multidimensional LC and capillary electrophoresis hyphenated to mass spectrometry, reported mainly since 2016. Expert commentary: These emerging techniques allow a deep insight into important CQAs that are related to ADC Chemistry Manufacturing and Control (CMC) as well as an improved understanding of in vitro and in vivo ADC biotransformations. This knowledge and the development of quantitative bioanalytical assays will continue to contribute to earlydevelopability assessment for the optimization of all the ADC components (i.e., antibody, drug, and linker) and help to bring next-generation candidate ADC into the clinic and hopefully to the market.

Analytical Comparison of Antibody-drug Conjugates Based on Good Manufacturing Practice Strategies

“…[1][2][3] In accordance with the growth of the ADC field, analytical characterization continues to play a crucial role in the discovery, development, and manufacturing of ADCs. 4,5 The FDA in particular, requires the pharmaceutical industry to carefully analyze antibody-based biopharmaceuticals, 6,7 which remains a challenge due to the heterogeneity and structural complexity of the compounds. Several analytical methods have been established in an effort to address this issue.…”

“…Several analytical methods have been established in an effort to address this issue. 4,5 High performance liquid chromatography (HPLC) analysis is the most popular analytical approach for drug antibody ratio (DAR) characterization, but it is important to develop multiple methods for application at different project stages to ensure accuracy of DAR determination. Previously, we reported analytical comparison for site-specific ADCs using six analytical techniques to establish the appropriate analytical method for each phase in the project cycle.…”

Native size-exclusion chromatography-mass spectrometry: suitability for antibody–drug conjugate drug-to-antibody ratio quantitation across a range of chemotypes and drug-loading levels

“…There are several examples in the literature demonstrating that MS can be used for quantitative or qualitative assessments of DAR and drug-distribution for the types of molecules described in Table 1. [21][22][23][24] All of these MS-based approaches have commonalities: the multiply charged mass spectrum of the ADC is deconvolved into a zero charge mass spectrum, the species observed in the mass spectrum are identified on the basis of agreement between theoretical and observed mass and, finally, the relative levels of each of the individual drug-loaded species are inferred based on the apparent-relative height or area of that species. Compared to the conventional methods for quantitating ADC DAR, MS-based approaches have inherent advantages because DAR quantitation does not necessarily depend on liquid chromatography (LC)-based separation of the individual drugloaded species.…”

Insights from capillary electrophoresis approaches for characterization of monoclonal antibodies and antibody drug conjugates in the period 2016–2018

“…Nevertheless, this method is more limited for the analysis of ADCs depending of the conjugation chemistry and the attachment sites as lysines, cysteines or glycans [41][42][43][44]. Concerning lysine-conjugated ADCs, in 2016, Chen and co-workers published non-reducing CE-SDS reports of trastuzumab emtansine (T-DM1) and the unconjugated trastuzumab.…”