2
Overview
Purpose:
To develop a high resolution LC/MS-based workflow solution for robust,
accurate and comprehensive intact monoclonal antibody (mAb) characterization.
Methods:
Thermo
Scientific™
Q
Exactive™
hybrid quadrupole-Orbitrap mass
spectrometers were used for intact mass measurement and top-down sequencing. Full
MS spectra of intact and reduced mAbs were analyzed using Thermo
Scientific™
Protein
Deconvolution™
software version 2.0 that utilizes the ReSpect
TM
algorithm for molecular
mass determination. The top-down msx HCD spectra were analyzed using Thermo
Scientific™
ProSightPC
™
software version 2.0.
Results:
A mass error of less than 10 ppm was routinely achieved for intact mAb mass
measurement. Low mass modifications, such as oxidation, can be confidently identified on
substructure level such as intact Fab, or light chain. Using an on-line high resolution top-
down MS/MS approach, over 30% of the fragmentation site was covered for intact light
chain as well as for Fab heavy chain. Sequence coverage from top-down approach also
confirmed disulfide linkage on partially reduced samples.
Introduction
Monoclonal antibodies (mAbs) are increasingly being developed and utilized for
diagnosing and therapeutic treatment of diseases including cancer. Due to the
heterogeneity of mAb products, thorough characterization is necessary for their
reproducible and safe production. Among the analytical tools used for the analysis of
therapeutic mAbs, mass spectrometry has become more and more important in providing
valuable information on various protein properties. Such information includes intact mass,
amino acid sequence, post-translational modifications including glycosylation form
distribution, minor impurities due to sample processing and handling, and high order
structure. Characterization at the intact protein level is usually the first step. In this study, a
high resolution LC-MS based workflow solution was developed for robust, accurate and
comprehensive mAb characterization at the intact protein level.
The fast chromatography,
superior resolution and mass accuracy provided by the Q Exactive LC/MS system, and
accurate data analysis of this workflow, provides a high-confident screening tool to
accelerate biopharmaceutical product development.
Methods
Samples:
Four intact mAbs were used in this study. To reduce intact mAb, the sample was
incubated for one hour at 60 °C or 37 °C in 6 M guanidine-HCl containing 5 mM DTT for
complete or partial reduction, respectively. Fab was generated using papain in 1mM EDTA,
10 mM Cys, 50 mM sodium phosphate buffer, pH 7.0. Before digestion, the enzyme
suspension (10 mg/ml) was activated for 15 min at 37 °C in the same buffer at an enzyme:
buffer ratio of 1:9. The digestion was performed at 37 °C overnight using an enzyme:
antibody ratio of 1:99 w/w.
HPLC:
A Thermo Scientific
TM
ProSwift
™
RP-10R monolithic column (1 x 50mm) was used
for desalting and separation of light and Fab heavy chain. LC solvents were 0.1% formic
acid in H
2
O (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B). The column was
heated to 80 °C during analysis. The flow rate was 60 µL/min. After injection of 1 µg mAb,
a 15 min gradient was used to elute mAbs from the column (0.0 min, 20% B; 1.0 min, 35%
B; 3.0 min, 55% B; 4.0 min, 98% B; 7.0 min, 98% B; 7.1 min, 20% B; 15.0 min, 20% B).
Mass Spectrometry:
Q Exactive instruments were used for this study. Intact and reduced
mAbs were analyzed by ESI-MS for intact molecular mass. Top-down MS/MS was
performed using high energy collision dissociation with a unique spectrum multiplexing
feature (msx HCD). In this data acquisition mode, fragment ions produced from several
individual HCD events, each on a precursor of a different charge state of the reduced mAb,
were detected together in the Thermo
Scientific™
Orbitrap™
mass analyzer. The spray
voltage was 4 kV. Sheath gas flow rate was set at 10. Auxiliary gas flow rate was set at 5.
Capillary temperature was 275 °C. S-lens level was set at 55. In-source CID was set at 45
eV. For full MS, resolution was 17,500 for intact mAb and intact Fab average mass
measurement, or 140,000 for light chain and Fab heavy chain monoisotopic mass
Figure 1: Intact mAb a
One microgram of mAb
column using a 15 min gr
shown in Figure 1, the m
spectrum over the elution
the mAb (B). A zoom-in vi
that are baseline separate
After each of the mAb dat
the masses were compare
with the various combinati
is shown below in Figure 2
Results
Figure 2: Deconvoluted
errors of average molec
input m/z spectrum were
the masses were compar
commonly found glycofor
ProSightPC software in th
0
1
2
0
25
50
75
100
0.72
RelativeAbundance
1800
2000
0
25
50
75
100
2
2740
0
25
50
75
100
2745.7679
2742.8007
2748.771
2751.7
275
A
B
C
