PXD006873

PXD006873 is an original dataset announced via ProteomeXchange.

Title	A Generic HPLC Method for Absolute Quantification of Oxidation in Monoclonal Antibodies and Fc-Fusion Proteins Using UV and MS Detection
Description	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.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_04:39:19.508.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Christof Regl
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2017-07-06 01:10:13	ID requested
1	2018-04-05 03:50:43	announced
⏵ 2	2024-10-22 04:39:20	announced	2024-10-22: Updated project metadata.

Regl C, Wohlschlager T, Holzmann J, Huber CG, A Generic HPLC Method for Absolute Quantification of Oxidation in Monoclonal Antibodies and Fc-Fusion Proteins Using UV and MS Detection. Anal Chem, 89(16):8391-8398(2017) [pubmed]

10.1021/acs.analchem.7b01755;

ProteomeXchange project tag: Human Proteome Project

curator keyword: Technical

submitter keyword: ion-pair reversed-phase, Q Exactive,IdeS

Christian G. Huber
contact affiliation	1: Department of Molecular Biology, Division of Chemistry andBioanalytics, University of Salzburg, Hellbrunner Strasse 34, 5020 Salzburg, Austria 2: Christian Doppler Laboratory for Innovative Tools for Biosimilar Characterization, University of Salzburg, Hellbrunner Strasse 34, 5020 Salzburg, Austria
contact email	c.huber@sbg.ac.at
lab head
Christof Regl
contact affiliation	University of Salzburg
contact email	christof.regl@sbg.ac.at
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD006873
     1. Label: PRIDE project
     2. Name: A Generic HPLC Method for Absolute Quantification of Oxidation in Monoclonal Antibodies and Fc-Fusion Proteins Using UV and MS Detection