Analytical methods for physicochemical characterization of antibody drug conjugates

Abstract

Antibody-drug conjugates (ADCs), formed through the chemical linkage of a potent small molecule cytotoxin (drug) to a monoclonal antibody, have more complex and heterogeneous structures than the corresponding antibodies. This review describes the analytical methods that have been used in their physicochemical characterization. The selection of the most appropriate methods for a specific ADC is heavily dependent on the properties of the linker, the drug, and the choice of attachment sites (lysines, inter-chain cysteines, Fc glycans). Improvements in analytical techniques such as protein mass spectrometry and capillary electrophoresis have significantly increased the quality of information that can be obtained for use in product and process characterization, and for routine lot release and stability testing.

Tissue Distribution Studies of Protein Therapeutics Using Molecular Probes: Molecular Imaging

“…The Lindmo assay (15) is very widely used for this purpose, but flattering results are easily obtained if the assay is not performed under strict conditions (16). These assays are critically dependent on knowing the true protein concentration, but the presence of a label may complicate the extinction coefficients (17) or (with chelating groups) interfere with copper-dependent Lowry and bicinchoninic acid protein assays.…”

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

“…Sites for conjugation within the complementarity-determining regions (CDRs) may be more likely to be modified, and the ability of critical reagents to detect ADA that target these regions (likely to be NAbs) could be compromised by high levels of labeling. Analytical techniques that probe the biophysical parameters of the therapeutic and variants thereof (size-exclusion chromatography to detect aggregation and capillary isoelectric focusing for charge) should be applied to identify issues early in assay development (35), allowing for optimization of labeling and assay buffers. Likewise, an SPR qualification step can demonstrate that immunologic reactivity of critical ADA reagents is similar to the parent molecule (within the scope of whatever surrogate controls are available).…”

Biophysical characterization of a model antibody drug conjugate

“…Thus, focus has been given to determine the ratio of the drug to the antibody and its heterogeneity using various chromatographic techniques (13). Here, we show below structure characterizations of ADCs using nonchromatographic techniques.…”

“…Differential scanning calorimetry (DSC) is used to characterize the thermal stability of proteins. It would be of great interest to see if these techniques can be successfully applied to characterize ADCs, as the conjugated drugs may interfere with the optical and hydrodynamic properties of ADCs (13). Here, we have initiated a study on the biophysical characterization of a model ADC.…”