4 research outputs found
Fast and Automated Characterization of Antibody Variants with 4D HPLC/MS
Characterization
of unknown monoclonal antibody (mAb) variants
is important in order to identify their potential impact on safety,
potency, and stability. Ion exchange chromatography (IEC) coupled
with UV detection is frequently used to separate and quantify mAb
variants in routine quality control (QC). However, characterization
of the chromatographic peaks resulting from an IEC separation is an
extremely time-consuming process, involving many cumbersome steps.
Presented here is an online four-dimensional high performance liquid
chromatographyâmass spectrometry (4D HPLC/MS) approach, developed
to circumvent these limitations. To achieve this, a 2D HPLC system
was extended through the introduction of additional modules, hence
enabling fully automated bioseparation of mAbs, fractionation of peaks,
reduction, tryptic digestion, and reversed-phase (RP) separation of
resulting peptides followed by MS detection. The entire separation
and analytical process for an unknown peak is performed in less than
1.5 h, leading to a significant time savings, with comparable sequence
coverage. To show the comparability with the traditional offline process,
a proof of concept study with a previously characterized mAb is presented in this paper
Detailed Characterization of Monoclonal Antibody Receptor Interaction Using Affinity Liquid Chromatography Hyphenated to Native Mass Spectrometry
We report on the
online coupling of FcRn affinity liquid chromatography
(LC) with electrospray ionization mass spectrometry (ESI-MS) in native
conditions to study the influence of modifications on the interaction
of recombinant mAbs with the immobilized FcRn receptor domain. The
analysis conditions were designed to fit the requirements of both
affinity LC and ESI-MS. The mobile phase composition was optimized
to maintain the proteins studied in native conditions and enable sharp
pH changes in order to mimic properly IgGs Fc domain/FcRn receptor
interaction. Mobile phase components needed to be sufficiently volatile
to achieve native MS analysis. MS data demonstrated the conservation
of the pseudonative form of IgGs and allowed identification of the
separated variants. Native FcRn affinity LCâESI-MS was performed
on a therapeutic mAb undergoing various oxidation stress. Native MS
detection was used to determine the sample oxidation level. Lower
retention was observed for mAbs oxidized variants compared to their
intact counterparts indicating decreased affinities for the receptor.
This methodology proved to be suitable to identify and quantify post-translational
modifications at native protein level in order to correlate their
influence on the binding to the FcRn receptor. Native FcRn affinity
LCâESI-MS can tremendously reduce the time required to assess
the biological relevance of the IgG microheterogeneities thus providing
valuable information for biopharmaceutical research and development
Chemo-Enzymatic Synthesis of <sup>13</sup>C Labeled Complex NâGlycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry
Methods for the absolute quantification
of glycans are needed in
glycoproteomics, during development and production of biopharmaceuticals
and for the clinical analysis of glycan disease markers. Here we present
a strategy for the chemo-enzymatic synthesis of <sup>13</sup>C labeled
N-glycan libraries and provide an example for their use as internal
standards in the profiling and absolute quantification of mAb glycans
by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF)
mass spectrometry. A synthetic biantennary glycan precursor was <sup>13</sup>C-labeled on all four amino sugar residues and enzymatically
derivatized to produce a library of 15 glycan isotopologues with a
mass increment of 8 Da over the natural products. Asymmetrically elongated
glycans were accessible by performing enzymatic reactions on partially
protected UV-absorbing intermediates, subsequent fractionation by
preparative HPLC, and final hydrogenation. Using a preformulated mixture
of eight internal standards, we quantified the glycans in a monoclonal
therapeutic antibody with excellent precision and speed
Chemo-Enzymatic Synthesis of <sup>13</sup>C Labeled Complex NâGlycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry
Methods for the absolute quantification
of glycans are needed in
glycoproteomics, during development and production of biopharmaceuticals
and for the clinical analysis of glycan disease markers. Here we present
a strategy for the chemo-enzymatic synthesis of <sup>13</sup>C labeled
N-glycan libraries and provide an example for their use as internal
standards in the profiling and absolute quantification of mAb glycans
by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF)
mass spectrometry. A synthetic biantennary glycan precursor was <sup>13</sup>C-labeled on all four amino sugar residues and enzymatically
derivatized to produce a library of 15 glycan isotopologues with a
mass increment of 8 Da over the natural products. Asymmetrically elongated
glycans were accessible by performing enzymatic reactions on partially
protected UV-absorbing intermediates, subsequent fractionation by
preparative HPLC, and final hydrogenation. Using a preformulated mixture
of eight internal standards, we quantified the glycans in a monoclonal
therapeutic antibody with excellent precision and speed