Understanding the Conformational Impact of Chemical Modifications on Monoclonal Antibodies with Diverse Sequence Variation Using Hydrogen/Deuterium Exchange Mass Spectrometry and Structural Modeling

Abstract

Chemical modifications can potentially induce conformational changes near the modification site and thereby impact the safety and efficacy of protein therapeutics. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a powerful analytical technique with high spatial resolution and sensitivity in detecting such local conformational changes. In this study, we utilized HDX-MS combined with structural modeling to examine the conformational impact on monoclonal antibodies (mAbs) caused by common chemical modifications including methionine (Met) oxidation, aspartic acid (Asp) isomerization, and asparagine (Asn) deamidation. Four mAbs with diverse sequences and glycosylation states were selected. The data suggested that the impact of Met oxidation was highly dependent on its location and glycosylation state. For mAbs with normal glycosylation in the Fc region, oxidation of the two conserved Met252 and Met428 (Kabat numbering) disrupted the interface interactions between the CH2 and CH3 domains, thus leading to a significant decrease in CH2 domain thermal stability as well as a slight increase in aggregation propensity. In contrast, Met oxidation in the variable region and CH3 domain had no detectable impact on mAb conformation. For aglycosylated mAb, Met oxidation could cause a more global conformational change to the whole CH2 domain, coincident with the larger decrease in thermal stability and significant increase in aggregation rate. Unlike Met oxidation, Asn deamidation and Asp isomerization mostly had very limited effects on mAb conformation, with the exception of succiminide intermediate formation which induced a measurable local conformational change to be more solvent protected. Structural modeling suggested that the succinimide intermediate was stabilized by adjacent aromatic amino acids through ring-ring stacking interactions.

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

“…Significant increases in flexibility of this same segment have been correlated previously in our labs in another IgG1 mAb with arginine and sodium thiocyanate-induced decreases in conformational stability, and concomitant increases in the aggregation propensity. 33,34 Furthermore, several reports by others have correlated increased flexibility of this same primary sequence segment in the C H 2 domain with methionine oxidation, 38,39,54 destabilization by deglycosylation, 35 changes in glycosylation 38 and even decreased stability due to drug conjugation to free cysteine residues. 41 The 244-255 segment in the C H 2 domain is relatively apolar in nature, with 2 valine residues and 2 phenylalanine residues, and its phenyl rings pack closely with the glycans in crystal structures.…”

“…33,34 Hydrogen/deuterium exchange coupled to mass spectrometry (H/D-MS) is now a well-established technique to explore the local dynamics of the amide backbone of mAbs with peptidelevel resolution. 35,36 H/D-MS has been used to study changes in mAb conformational dynamics imparted by stabilizing and destabilizing excipients 34 and salts, 33 mutation in the IgG1 C H 3 domain, 37 deglycosylation, 35 chemical and post-translational modifications, 38,39 thermal and freeze-thaw stress, 40 and cytotoxic drug conjugation 41 in IgG1 mAbs. The work reported here investigated the nature of the relative differences in physical stability between an IgG1 mAb (mAb-A) and its corresponding YTE-mutant (mAb-E) by examining effects of the YTE-mutations on amide backbone local dynamics measured by amide H/D exchange, conformational stability measured by differential scanning calorimetry (DSC), and aggregation propensity measured by size exclusion chromatography (SEC).…”

Conformational processing of oncogenic v-Src kinase by the molecular chaperone Hsp90

“…Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) experiments were performed on a fully automated system equipped with a Leap robot (HTS PAL; Leap Technologies), a Waters ACQUITY UPLC, a HDX manager, and the Synapt G2-S mass spectrometer as described elsewhere (Waters) (65). Src kinases were vacuum concentrated in the presence of 100 mM trehalose (Sigma Trapping and chromatographic separation were carried out at 0°C to minimize back-exchange.…”

Comparative higher-order structure analysis of antibody biosimilars using combined bottom-up and top-down hydrogen-deuterium exchange mass spectrometry

“…To the best of our knowledge, this kind of study has not yet been done. The HDX-MS work done so far on antibodies used either the bottom-up [39][40][41][42][43][44] or the top-down approach [27,45]. Our current work fills this gap by characterizing the higher-order structure of an originator antibody drug BEV (BEV) and two batches of its biosimilars side by side, using the two methods.…”