2
Analysis of Monoclonal Antibodies, Aggregates, and Their Fragments by Size Exclusion Chromatography Coupled with an Orbitrap Mass Spectrometer
Overview
Purpose:
Demonstrate SEC-MS as a characterization platform for a monoclonal
antibody (mAbs) and its fragments.
Methods:
Coupling size-exclusion chromatography with high resolution Orbitrap mass
spectrometer (SEC-MS) enables accurate mass measurement of mAb, its aggregates,
and its fragments.
Results:
1. mAb monomer and dimer aggregate intact masses can be measured by SEC-MS
under non-denaturing condition using near neutral pH eluent
2.
Thermo Scientific™ Exactive Plus™ EMR
Orbitrap mass spectrometer enables the
accurate detection of mAb at
m/z
350- 20,000
3.
Thermo Scientific™
MAbPac
™ SEC
-1 column successfully separates the HC and
LC, and partially separates Fab and Fc fragments using denaturing eluent.
Introduction
The biopharmaceutical industry has continued its focus on the development of
biotherapeutic monoclonal antibody (mAbs) drugs
1
. mAbs produced from mammalian
cell culture may contain significant amounts of dimers and higher-order aggregates.
Size exclusion chromatography (SEC) is a well-accepted technique for the detection
and accurate quantification of protein aggregates in biological drug products. It is
routinely used for the characterization and quality control of mAb products.
There is a growing trend to obtain intact mass information as well as the glycan profile
in the QC of monoclonal antibodies using high resolution mass spectrometry. The most
commonly employed LC/MS method is to desalt the mAb via reversed phase
chromatography followed by the MS analysis. However, the extreme low pH and
organic solvent used in the reverse phase chromatography often denatures the mAb. In
the case of antibody-drug conjugate (ADC) with interchain cysteine linked drugs, the
harsh solvent condition will dissociate the heavy and light chains of the ADC and
prevent the measurement of intact mass. A non-denaturing SEC-based desalting mass
spectrometry method enables mass measurement of the mAb in its native state. The
volatile ammonium formate buffer is compatible with MS and preserves intact protein
structure.
2
Full characterization of mAb includes determination of mass of the mAb fragments,
such as heavy chain (HC) and light chain (LC) generated by reduction of interchain
disulfide bonds, as well as Fab and Fc generated by papain digestion. Using
denaturing eluent containing 20% acetonitrile, 0.1% TFA, and 0.05% formic acid, SEC
can baseline separate HC and LC, as well as partially separate Fab and Fc. It serves
as a platform method for mAb fragment analysis.
The Exactive Plus EMR mass spectrometer combines high-resolution, accurate mass
data with an extended mass range (EMR). It has an
m/z
range up to 20,000 and
improved transmission of higher mass ions for stronger signals. All these features
make the Exactive Plus EMR mass spectrometer a superb tool for accurate intact mass
measurement of mAb and high performance screening of mAb glycosylation profile.
The mAbPac SEC-1 is a size exclusion chromatography (SEC) column designed for
monoclonal antibody (mAb) analysis, including monomers, aggregates, and fragments.
Its stable surface bonding leads to low column bleed and compatibility with MS
detection. In this study, we demonstrate the compatibility of mAbPac SEC-1 with
Exactive Plus EMR Mass Spectrometer. SEC-MS enables intact mass detection of
mAb monomer, dimer aggregate under non-denaturing condition and fragments
(including heavy chain, light chain, Fab, and Fc) under denaturing conditions.
Liquid Chromatography
HPLC experiments were carri
3000 RSLCnano System equ
3500RS dual-gradient pump
Separation Thermostatted Au
mode. For the 4.0 mm ID col
column, flow rate was set at
Reduction of mAb to heavy
Reduction of inter-chain disul
mAb with 20 mM DTT at 50
formic acid to final concentrat
Papain digestion of mAb to
The digestion was carried out
in 100 mM Tris-HCl, pH 7.6,
4 hours, the digestion was st
0.1%.
Non-denaturing SEC mobil
20 mM ammonium formate (
Denaturing SEC mobile ph
20% acetonitrile, 0.1% formic
MS Conditions
The Exactive Plus EMR mas
mAb fragments were analyze
details.
Data processing
Full MS spectra of intact mAb
Thermo Scientific™ Protein
algorithm for molecular mass
Methods
Chemicals and reagents
High purity ammonium formate
(≥99.995%) was purchased from Sigma
®
. Other reagents
were purchased from reputable suppliers. Monoclonal antibodies mAb1 and mAb2 were
gifts from a biotech company.
Columns
MAbPac SEC-1, 5 µm, 4
㽢
300 mm (P/N 074696)
MAbPac SEC-1, 5 µm, 2.1
㽢
150 mm (P/N 088462)
Results
Analysis of mAb by non-de
The analysis of mAbs by SE
at near-physiological pH rang
with 300 mM NaCl. However
content makes this buffer non
Therefore, we explored using
SEC separation and directly
instrument. Figure 1 shows t
the extracted ion chromatogr
charge envelope of +24 to +2
condition, the charge envelop
20 mM ammonium formate el
mAb shifts to higher mass ra
(
m/z
above 6000) is made po
instrument. Figure 1c shows
peak at mass 148,033 u and
corresponding to different gly
peak at mass 148,163 u, is 1
variant.
TABLE 1. MS conditions.
used for this study. The follo
Source DC Offset: -25V; Inje
Flatapole DC: -6V. Other ion t
Instrument Conditions Non-dena
EMR mode
On
Mass range
m/z 400–
Spray voltage
4.3 kV
Sheath gas
30 arb. u
Auxiliary gas
10 arb. u
Capillary temperature 275 °C
S-lens level
200
In-source CID
100 eV
HCD CE
10
Microscans
5
AGC target
1 × 10
6
Maximum IT
300 ms
Resolving power
35,000
Probe temperature
400 °C
