LC/MS Analysis of the Monoclonal Antibody
Rituximab Using the Q Exactive Benchtop
Orbitrap Mass Spectrometer
Martin Samonig
1,2
, Christian Huber
1,2
and Kai Scheffler
2,3
1
Division of Chemistry and Bioanalytics, University of Salzburg, Salzburg, Austria
2
Christian Doppler Laboratory for Innovative Tools for Biosimilar Characterization, University of Salzburg, Salzburg, Austria
3
Thermo Fisher Scientific, Dreieich, Germany
Application Note 591
Key Words
Monoclonal antibody, intact protein mass measurement, sequence
confirmation, protein deconvolution, top-down sequencing
Goal
Analysis and characterization of a monoclonal antibody using an optimized
LC/MS workflow based on monolithic columns coupled online with the
Thermo Scientific
™
Q Exactive
™
benchtop Orbitrap
™
mass spectrometer.
Introduction
Monoclonal antibodies (mAbs) are one of the fastest
growing classes of pharmaceutical products. They play a
major role in the treatment of a variety of conditions such
as cancer, infectious diseases, allergies, inflammation, and
auto-immune diseases. Because mAbs can exhibit
significant heterogeneity, extensive analytical
characterization is required to obtain approval for a new
mAb as a therapeutic product. Mass spectrometry has
become an essential tool in the characterization of mAbs,
providing molecular weight determinations of intact
proteins as well as separated light and heavy chains,
elucidation of glycosylation and glycan structures,
confirmation of correct amino acid sequences, and
identification of impurities such as host cell proteins
(HCP) inherent to the production process.
Rituximab, which is known under the trade names
Rituxan
®
(Biogen Idec/Genentech) in the United States
and MabThera
®
(Roche) in Europe, is a recombinantly
produced, monoclonal chimeric antibody against the
protein CD20. It was one of the first new generation
drugs in cancer immune therapy. Rituximab was approved
by the U.S. Food and Drug Administration in 1997 and
by the European Commission in 1998 for cancer therapy
of malignant lymphomas. The variable domain of the
antibody targets the cell surface molecule CD20, that can
be found in some non-Hodgkin lymphomas.
In this application note, the capabilities and performance
of the Q Exactive benchtop Orbitrap mass spectrometer
in analyzing the intact and reduced forms of rituximab are
demonstrated as well as sequence confirmation analyses
using a combined top-down and bottom-up approach.
Furthermore, the sensitivity of two chromatographic
setups using monolithic columns coupled online to the
mass spectrometer is evaluated. The data obtained
demonstrate superior resolution and mass accuracy of the
Q Exactive mass spectrometer and present it as a high-
confidence screening tool for accelerated and accurate
biopharmaceutical product development and
characterization.
