A novel method for in silico assessment of Methionine oxidation risk in monoclonal antibodies: Improvement over the 2-shell model

Abstract

Over the past decade, therapeutic monoclonal antibodies (mAbs) have established their role as valuable agents in the treatment of various diseases ranging from cancers to infectious, cardiovascular and autoimmune diseases. Reactive groups of the amino acids within these proteins make them susceptible to many kinds of chemical modifications during manufacturing, storage and in vivo circulation. Among these reactions, the oxidation of methionine residues to their sulfoxide form is a commonly observed chemical modification in mAbs. When the oxidized methionine is in the complementarity-determining region (CDR), this modification can affect antigen binding and thus abrogate biological activity. For these reasons, it is essential to identify oxidation liabilities during the antibody discovery and development phases. Here, we present an in silico method, based on protein modeling and molecular dynamics simulations, to predict the oxidation-liable residues in the variable region of therapeutic antibodies. Previous studies have used the 2-shell water coordination number descriptor (WCN) to identify methionine residues susceptible to oxidation. Although the WCN descriptor successfully predicted oxidation liabilities when the residue was solvent exposed, the method was much less accurate for partially buried methionine residues. Consequently, we introduce a new descriptor, WCN-OH, that improves the accuracy of prediction of methionine oxidation susceptibility by extending the theoretical framework of the water coordination number to incorporate the effects of polar amino acids side chains in close proximity to the methionine of interest.

Combined Presence of Ferrous Ions and Hydrogen Peroxide in Normal Saline and In Vitro Models Induces Enhanced Aggregation of Therapeutic IgG due to Hydroxyl Radicals

“…It has been reported in different studies that the amino acids such as methionine, histidine, tryptophan, tyrosine, and phenylalanine were found in the oxidized state due to the presence of various reagents. ,,− For example, in a previously published report by Glover et al, the oxidation in the same mAb (mAb1) used in this study, but produced by a different manufacturer, was analyzed upon exposure to Fe 2+ and H 2 O 2 in formulation buffer , and it was found that methionine at 255 and 431 positions in the heavy chain of the mAb was oxidized. Furthermore, various studies have reported different metal and various oxidative agent mediated degradation of proteins including mAb. ,,− In this work, fragments were also observed in the SEC chromatogram of mAb samples in saline containing both Fe 2+ and H 2 O 2 . The fragments in SEC chromatogram obtained beyond 37 min and the ones that possibly interacted with metal/H 2 O 2 were not characterized.…”

“…The generation of excessive ROS due to the reaction between metal ions and H 2 O 2 is responsible for the rapid degradation of proteins. Fenton reaction, which leads to the formation of hydroxyl free radicals, has been studied in many chemical and biochemical systems. ,, The free radicals perturb the protein structure by oxidatively damaging the amino acids, eventually leading to the formation of aggregates and breakage of polypeptide chains. ,, There are various published studies where the oxidation of amino acids and oxidation mediated damage of proteins in terms of aggregation and fragmentation have been explored. ,,− The detailed mechanism of Fenton-reaction-mediated aggregation is shown in Figure . Furthermore, interactions of metal ions to the protein backbone and amino acids, and the presence of nonprotein components in protein aggregates have also been published in various studies which explored metal-catalyzed degradation of proteins. ,, Hence, non-protein components in aggregates of mAb in the current study can be characterized further in future studies.…”

Assessments of Alpha-Amylase Inhibitory Potential of Tagetes Flavonoids through In Vitro, Molecular Docking, and Molecular Dynamics Simulation Studies

“…The crystal structure of human pancreatic alpha-amylase in complex with myricetin (MYC) was obtained from the Protein Data Bank (PDB ID: 4GQR) [ 50 ]. The protein was prepared for further analysis using the Protein Preparation Wizard in Maestro Schrödinger [ 51 ]. This included the addition of missing hydrogen atoms to the residues, the correction of metal ionization states, and the removal of water molecules beyond 5 Å.…”

Investigating the mechanisms of antibody binding to alpha-synuclein for the treatment of Parkinson’s Disease

“…The sequence data for PRX002 was obtained from the Kyoto Encyclopedia of Genes and Genomes (KEGG) [31], and Schrödinger software BioLuminate [32, 33, 34, 35, 36, 37] and PyMol [38] were used to predict the protein structure of the Fab region of the mAb, with Chothia numbering used to annotate the final models. Thirty-four different variations of α-syn were obtained from the Protein Data Bank (PDB ID 2KKW [34]), which all had different structures gathered by nuclear magnetic resonance imaging.…”