The approach makes use of simple bench-top chemistry that introduces GEE tags on solvent-accessible side chains of carboxyl residues,32,35,36,40and requires no special equipment and can be done in any analytical lab. conformational effects brought on by conjugation of mAbs with drug-linkers. All 59 monitored carboxyl residues displayed similar solvent-accessibility between the ADC and mAb under native conditions, which suggests the ADC and mAb share similar side chain conformation. The findings are well correlated and complementary with results from other assays. This work illustrated that site-specific CGF is capable of pinpointing local conformational changes in mAbs or ADCs that might arise during development and manufacturing. The methodology can be readily implemented within the industry to provide comprehensive conformational assessment of these molecules. KEYWORDS:Antibody-drug conjugate, carboxyl group footprinting, covalent labeling, hydrogen-deuterium exchange, mass spectrometry, monoclonal antibody, Protein conformation, rate constant, side chains, solvent accessibility == Abbreviations == monoclonal antibody antibody-drug conjugate hydrogen/deuterium exchange mass spectrometry valine-citrulline-monomethyl auristatin E circular dichroism differential scanning calorimetry Immunoglobulin G fragment crystallizable fragment antigen-binding liquid chromatography mass spectrometry rate constant glycine ethyl ester 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride == Introduction == Monoclonal antibodies (mAbs) have played a substantial role in advancing options for treating many types of diseases, including cancer, infectious diseases and immune-mediated disorders, during the past two decades.1More recently, antibody-drug conjugates (ADCs) consisting of a mAb scaffold that is chemically linked to a cytotoxic small-molecule drug have emerged as effective cancer therapies and therapeutic candidates.,2,3Several properties of mAbs make them highly useful in a therapeutic setting: they bind specifically to a cognate epitope, have relatively long half lives in vivo and may elicit a clinically relevant immune response. In ADCs, the targeting specificity of the mAb component HSPA6 ensures that the cytotoxic payload is delivered only to cancer cells expressing the antigen, thereby avoiding some of the more debilitating side effects of traditional chemotherapy.4,5 Therapeutic mAbs and ADCs are complicated multimeric molecules, and chemical modifications to their primary amino acid sequence may be incurred during the manufacturing and storage. There is potential for the clinical efficacy of mAbs and ADCs to be compromised by post-translational modifications (PTMs) and chemical modifications due to their effects on antigen binding or biological clearance. Disruption of mAb/ADC conformation or higher-order structure also has the potential to impair the potency and stability of the therapeutic. While indirect evidence of conformational changes might be apparent in some analytical assays used for product release and characterization Alectinib Hydrochloride such as binding, potency assays and some chromatographic assays,6more traditional biophysical assays such as circular dichroism (CD), fluorescence spectroscopy, and differential scanning calorimetry (DSC) have typically been used for conformational characterization.7-11These assays can monitor the global state conformation of protein samples in a simple and rapid manner, but are not suitable for detecting small localized, but potentially significant, conformational differences that may exist between protein samples. As use of high-resolution mass spectrometry (MS) has become more commonplace, chemical labeling coupled with MS has become a viable approach for investigating protein conformation. This general approach has contributed to the understanding of conformational features of globular proteins, membrane proteins and large protein complexes at the peptide or residue level.12-19While MS-based technology has not been widely used for conformational elucidation Alectinib Hydrochloride of protein therapeutics yet, some applications demonstrating the utility of the approach can be found in the literature. Hydrogen/deuterium exchange (HDX) coupled with Alectinib Hydrochloride MS is the most common approach used in the studies of biotherapeutic proteins.20-30Classic or bottom-up HDX MS monitors isotope exchange kinetics of amide hydrogens on a protein to gain conformational insights into.