Identification and Quantification of Oxidation Products in Full-Length Biotherapeutic Antibodies by NMR Spectroscopy
| Autor: | Mario Schubert, Vesna Stanojlovic, Christof Regl, Christian G. Huber, Arthur Hinterholzer, Chiara Cabrele |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Magnetic Resonance Spectroscopy
medicine.drug_class 010402 general chemistry Protein oxidation Monoclonal antibody 01 natural sciences Article Analytical Chemistry chemistry.chemical_compound medicine Immunologic Factors Amino Acids Methionine Methionine sulfoxide 010401 analytical chemistry Tryptophan Adalimumab Nuclear magnetic resonance spectroscopy Hydrogen Peroxide Oxidants 0104 chemical sciences Biochemistry chemistry Antirheumatic Agents Forced degradation Critical quality attributes Rituximab Oxidation-Reduction |
| Zdroj: | Analytical Chemistry |
| ISSN: | 1520-6882 0003-2700 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|