Selective Tryptophan Oxidation of Monoclonal Antibodies: Oxidative Stress and Modeling Prediction.

Oxidation of tryptophan not only generates heterogeneity of a therapeutic monoclonal antibody (mAb) but also can be a potential critical quality attribute (CQA) of the product. In this study, mAbs A-C of IgG1 and IgG4 (immunoglobulin G, IgG) isotypes with oxidized tryptophan (Trp) residues were selectively generated by incubating the mAbs with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) in formulations containing l-methionine. The site-specific oxidation of tryptophan residues were confirmed by liquid chromatography coupled with mass spectrometry (LC-MS) studies. The site of oxidation was identified to be a conserved tryptophan residue in the heavy chain complementarity determining region 3 (CDR3) of mAbs A and B with no significant oxidation found on other tryptophan residues including those in close proximity to CDR3. For mAb C, all tryptophan residues including one in the heavy chain CDR1 and a tryptophan in close proximity to heavy chain CDR3 were not susceptible to oxidation. For all three mAbs, the structure and tryptophan oxidation relationship was further studied by computational modeling of the variable domain of the antibodies (variable fragment, Fv). The computational modeling provided a structural understanding at the molecular level to the tryptophan oxidation, where high solvent accessibility is a prerequisite for heavy chain CDR3 tryptophan oxidation. However, higher oxidation susceptibility of tryptophan in heavy chain CDR3 did not linearly correlate to higher solvent accessibility, suggesting that other factors including side-chain orientation and/or surrounding structure elements around the heavy chain CDR3 may also be involved. Through this study, we demonstrate that a selective oxidation system, together with computational modeling, can be an important tool to identify potential CQAs of a therapeutic mAb such as tryptophan oxidation liabilities during the mAb's development.

Decomposition of L-valine under nonthermal dielectric barrier discharge plasma.

L-Valine solutions in water and phosphate buffer were treated with nonthermal plasma generated by using a dielectric barrier discharge (DBD) device and the products generated after plasma treatments were characterized by (1)H NMR and GC-MS. Our results demonstrate that L-valine is decomposed to acetone, formic acid, acetic acid, threo-methylaspartic acid, erythro-methlyaspartic acid, and pyruvic acid after direct exposure to DBD plasma. The concentrations of these compounds are time-dependent with plasma treatment. The mechanisms of L-valine under the DBD plasma are also proposed in this study. Acetone, pyruvic acid, and organic radicals (•)CHO, CH3COCH2OO(•) (acetonylperoxy), and CH3COC(OH)2OO(•) (1,1-dihydroxypropan-2-one peroxy) may be the determining chemicals in DNA damage.