A Generic HPLC Method for Absolute Quantification of Oxidation in Monoclonal Antibodies and Fc-Fusion Proteins Using UV and MS Detection

ABSTRACT: Absolute quantification of methionine oxidation in the Fc domain of biopharmaceuticals. Wedeveloped a middle-down approach employing the cysteine protease IdeS under reducing conditions to obtain three mAb subunits of approximately 25 kDa: Fc/2, Fd’ and LC. These subunits were separated by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) and detected by UV-spectroscopy as well as Orbitrap mass spectrometry (MS), as well as MS upon all-ion fragmentation (AIF-MS). We evaluated the feasibility of three strategies for absolute quantification of oxidation in the Fc region of hydrogen peroxide-stressed Rituximab, using a single, commercially available software platform both for data acquisition and evaluation: UV-spectroscopy, full scan MS, and monitoring of product ions obtained by AIF-MS. The approach is generic in that it allows monitoring and quantification of oxidation in the Fc regions of fully human and humanized IgG1 mAbs as well as of Fc-fusion proteins. This is exemplified by limits of detection of 1.2%, 1.0%, and 1.2% of oxidation in drug products containing the biopharmaceuticals Rituximab, Adalimumab, and Etanercept, respectively. The presented method is an attractive alternative to conventional time-intensive peptide mapping which is prone to artificial oxidation due to extensive sample preparation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Christof Regl  

LAB HEAD: Christian G. Huber

PROVIDER: PXD006873 | Pride | 2018-04-05

REPOSITORIES: Pride