Abstract
Therapeutic proteins are an indispensable class of drugs and often therapeutics of last resort. They are sensitive to oxidation, which is of critical concern, because it can affect drug safety and efficacy. Protein oxidation, with methionine and tryptophan as the most susceptible moieties, is mainly monitored by HPLC–MS techniques. However, since several oxidation products display the same mass difference, their identification by MS is often ambiguous. Therefore, an alternative analytical method able to unambiguously identify and, ideally, also quantify oxidation species in proteins is highly desired. Here, we present an NMR-based approach to monitor oxidation in full-length proteins under denaturing conditions, as demonstrated on two biotherapeutic monoclonal antibodies (mAbs). We show that methionine sulfoxide, methionine sulfone, N-formylkynurenine, kynurenine, oxindolylalanine, hydroxypyrroloindole, and 5-hydroxytryptophan result in characteristic chemical shift correlations suited for their identification and quantification. We identified the five most abundant oxidation products in forced degradation studies of two full-length therapeutic mAbs and can also unambiguously distinguish oxindolylalanine from 5-hydroxytryptophan, which are undistinguishable by MS due to the same mass shift. Quantification of the abundant methionine sulfoxide by NMR and MS gave highly comparable values. These results underline the suitability of NMR spectroscopy for the identification and quantification of critical quality attributes of biotherapeutics.
Highlights
Therapeutic proteins are an indispensable class of drugs and often therapeutics of last resort
We show that methionine sulfoxide, methionine sulfone, N-formylkynurenine, kynurenine, oxindolylalanine, hydroxypyrroloindole, and 5-hydroxytryptophan result in characteristic chemical shift correlations suited for their identification and quantification
We identified the five most abundant oxidation products in forced degradation studies of two fulllength therapeutic monoclonal antibodies (mAbs) and can unambiguously distinguish oxindolylalanine from 5-hydroxytryptophan, which are undistinguishable by MS due to the same mass shift
Summary
By performing extended oxidation under denaturing conditions (1% H2O2 for 25 h in the formulation buffer, measured in 7 M urea and 11 mM TCEP), it was possible to detect Met(O2) (Figures S7 and S8) and the Trp oxidation products Oia, and Kyn. In particular, Cε3−Hε3 and Cζ3−Hζ3 cross-peaks of Oia and weak signals of Cε2−Hε2 and Cη2−Hη2 of Kyn were detected in a 2D 1H−13C HSQC spectrum For the quantification of the most abundant oxidation product Met(O) using 2D 1H−13C HSQC spectra of rituximab and adalimumab treated with H2O2, we evaluated several approaches that were based on the following considerations: (i) The wellisolated CH3 group of nonoxidized Met is sharp and well-suited for quantification, whereas the CH3 group of Met(O) is located close to other random-coil correlations, mainly of Asn. due to the difficulty of integrating overlapping signals, the Cε−Hε integral of Met(O) might have a larger error. It should be noted that consistent quantification data were only obtained when the sample was kept under argon atmosphere during the sample preparation steps in order to exclude oxidation by atmospheric oxygen
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
