6 research outputs found
A Generic HPLC Method for Absolute Quantification of Oxidation in Monoclonal Antibodies and Fc-Fusion Proteins Using UV and MS Detection
Oxidation
of biopharmaceuticals may affect their bioactivity, serum
half-life, and (bio)Âchemical stability. The Fc domain of IgG monoclonal
antibodies (mAbs) contains two methionine residues which are susceptible
to oxidation. Here, we present 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. UV
spectroscopy showed the lowest limits of quantification (LOQ) (0.96
ng μL<sup>–1</sup>) and featured the lowest relative
process standard deviation (<i>V</i><sub>x0</sub>%) of 7.2%
compared to MS and AIF-MS with LOQs of 1.24–4.32 ng μL<sup>–1</sup> and relative process standard deviations of 9.0–14%,
respectively. Our 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
MoFi: A Software Tool for Annotating Glycoprotein Mass Spectra by Integrating Hybrid Data from the Intact Protein and Glycopeptide Level
Hybrid
mass spectrometry (MS) is an emerging technique for characterizing
glycoproteins, which typically display pronounced microheterogeneity.
Since hybrid MS combines information from different experimental levels,
it crucially depends on computational methods. Here, we describe a
novel software tool, MoFi, which integrates hybrid MS data to assign
glycans and other post-translational modifications (PTMs) in deconvoluted
mass spectra of intact proteins. Its two-stage search algorithm first
assigns monosaccharide/PTM compositions to each peak and then compiles
a hierarchical list of glycan combinations compatible with these compositions.
Importantly, the program only includes those combinations which are
supported by a glycan library as derived from glycopeptide or released
glycan analysis. By applying MoFi to mass spectra of rituximab, ado-trastuzumab
emtansine, and recombinant human erythropoietin, we demonstrate how
integration of bottom-up data may be used to refine information collected
at the intact protein level. Accordingly, our software reveals that
a single mass frequently can be explained by a considerable number
of glycoforms. Yet, it simultaneously ranks proteoforms according
to their probability, based on a score which is calculated from relative
glycan abundances. Notably, glycoforms that comprise identical glycans
may nevertheless differ in score if those glycans occupy different
sites. Hence, MoFi exposes different layers of complexity that are
present in the annotation of a glycoprotein mass spectrum
MoFi: A Software Tool for Annotating Glycoprotein Mass Spectra by Integrating Hybrid Data from the Intact Protein and Glycopeptide Level
Hybrid
mass spectrometry (MS) is an emerging technique for characterizing
glycoproteins, which typically display pronounced microheterogeneity.
Since hybrid MS combines information from different experimental levels,
it crucially depends on computational methods. Here, we describe a
novel software tool, MoFi, which integrates hybrid MS data to assign
glycans and other post-translational modifications (PTMs) in deconvoluted
mass spectra of intact proteins. Its two-stage search algorithm first
assigns monosaccharide/PTM compositions to each peak and then compiles
a hierarchical list of glycan combinations compatible with these compositions.
Importantly, the program only includes those combinations which are
supported by a glycan library as derived from glycopeptide or released
glycan analysis. By applying MoFi to mass spectra of rituximab, ado-trastuzumab
emtansine, and recombinant human erythropoietin, we demonstrate how
integration of bottom-up data may be used to refine information collected
at the intact protein level. Accordingly, our software reveals that
a single mass frequently can be explained by a considerable number
of glycoforms. Yet, it simultaneously ranks proteoforms according
to their probability, based on a score which is calculated from relative
glycan abundances. Notably, glycoforms that comprise identical glycans
may nevertheless differ in score if those glycans occupy different
sites. Hence, MoFi exposes different layers of complexity that are
present in the annotation of a glycoprotein mass spectrum
MoFi: A Software Tool for Annotating Glycoprotein Mass Spectra by Integrating Hybrid Data from the Intact Protein and Glycopeptide Level
Hybrid
mass spectrometry (MS) is an emerging technique for characterizing
glycoproteins, which typically display pronounced microheterogeneity.
Since hybrid MS combines information from different experimental levels,
it crucially depends on computational methods. Here, we describe a
novel software tool, MoFi, which integrates hybrid MS data to assign
glycans and other post-translational modifications (PTMs) in deconvoluted
mass spectra of intact proteins. Its two-stage search algorithm first
assigns monosaccharide/PTM compositions to each peak and then compiles
a hierarchical list of glycan combinations compatible with these compositions.
Importantly, the program only includes those combinations which are
supported by a glycan library as derived from glycopeptide or released
glycan analysis. By applying MoFi to mass spectra of rituximab, ado-trastuzumab
emtansine, and recombinant human erythropoietin, we demonstrate how
integration of bottom-up data may be used to refine information collected
at the intact protein level. Accordingly, our software reveals that
a single mass frequently can be explained by a considerable number
of glycoforms. Yet, it simultaneously ranks proteoforms according
to their probability, based on a score which is calculated from relative
glycan abundances. Notably, glycoforms that comprise identical glycans
may nevertheless differ in score if those glycans occupy different
sites. Hence, MoFi exposes different layers of complexity that are
present in the annotation of a glycoprotein mass spectrum
Toxicity and induction data
Includes raw data of toxicity and induction experiments mentioned in the manuscript. For more information refer to the ReadMe file
Glycan array TAP1 and XCL
Contains raw data for the Glycan array analysis of the lectins TAP1 and XCL.
Files are also available at the Consortium for Functional Glycomics (CFG) http://www.functionalglycomics.org/static/consortium/resources/resourcecoreh.shtm