6
Structure Characterization of Intact Monoclonal Antibody Using an Orbitrap Tribrid Mass Spectrometer
Conclusion
The monoclonal antibody could be successfully analyzed by both Orbitrap Elite
and Orbitrap Fusion mass spectrometers.
With optimized experiment conditions, a molecular mass for several glycoforms
of the example monoclonal antibody was accurately measured with low ppm
mass error using Orbitrap Fusion MS.
67% fragment coverage was obtained for NIST mAb light chain using Orbitrap
Elite with LC-MS analysis.
22% fragment coverage was obtained for example mAb heavy chain using
Orbitrap Fusion MS with infusion.
Acknowledgements
We would like to thank NIST for providing us with the monoclonal antibody.
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the
intellectual property rights of others.
PO64092-EN 0614S
FIGURE 6. Example mAb heavy chain sequence coverage of ETD (blue, 6ms)
and HCD (red, 15%) using Orbitrap Fusion MS.
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
5.76 6.04 6.32
7.69
7.02 7.59
7.76 7.95
9.99
8.22
9.81
8.75 9.20
NL:
1.34E9
TIC MS
Denatured_
DTT_reduc
ed_waters
_5
1800 1900 2000 2100 2200 2300 2400
2017.4332
z=?
1862.4284
z=?
2200.7980
z=?
6
2276.3028
z=?
789.7590
z=?
1937.6242
z=?
2134.7608
z=?
2315.2185
z=?
NL:1.57E6
Denatured_DTT_reduced_wa
ters_5#1050-1185 RT:
3.57-3.83 AV:136T:FTMS
+pESIFullms
[400.00-4000.00]
NL:3.00E5
Denatured_DTT_reduced_wa
ters_5#1377-1708 RT:
4.25-4.89 AV:332T:FTMS
+pESIFullms
[400.00-4000.00]
own ETD MS/MS
Top-down HCD MS/MS
hain
MS of light chain
ull MS of heavy chain
analysis was performed for
CD. Figure 4 a) is the
S spectra were acquired in
the light and heavy chains
hown). ETD with a reaction
e with 52% of light chain
18% yielded the most
e combination of the ETD
erage (Figure 5).
y chain
heavy chain analysis with
ainusing Orbitrap Elite.
sing Orbitrap Elite.
Deglycosylated and reduced mAb was infused and analyzed by Orbitrap Fusion. Six
ms ETD reaction time and 14% HCD collision energy were used for the separate ETD
and HCD fragmentation spectra. Around 22% backbone fragmentation coverage was
achieved combining both ETD and HCD (Figure 6).
