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©2013 Thermo Fisher Scientific Inc. All rights reserved. ReSpect is a trademark of Positive Probability, Ltd. ISO is a trademark of the International
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BioPharma_PN63944_E 11/13S
Conclusion
In this study, a workflow was developed that combines high resolution Orbitrap
MS, fast chromatography, high throughput msx HCD and accurate data analysis
to characterize intact mAb. The precise mass measurement and extensive, high
confident amino acid sequence obtained from this workflow provides the
following information for intact mAb and its substructure:
•
Accurate measurement of intact molecular mass
•
Reproducible quantification of glycoform relative abundance
•
Confident amino acid sequence information and structural information
CMS
b heavy chain
level mass error
(ppm)
6.4
0.7
0.7
partially reduced light chain
Figure 5: Top-down sequencing maps disulfide linkage on partially reduced light chain
Top-down HCD
ProSight PC
ions were then detected all together in a single Orbitrap detection event (Figure 4 top). More
than 30% of fragments from backbone cleavage were detected for both light chain and Fab
heavy chain (Figure 4 bottom) with excellent P score from ProSight PC software.
Top-down sequencing was also performed on a partially reduced light chain which is 4.02 Da
less in molecular mass than the fully reduced species. Analysis of the HCD spectrum in
ProSight PC software matched two disulfide linkages, which is typical of this type of IgG
molecule (Figure 5).
e 3 to Figure 5. Fab
hinge. Fab was then
top). LC/MS of intact
ies as shown above
in level were 6.4 ppm
40,000 for light chain
PPN_noCys_Red_140K
#
129-133
RT:
13.79-14.10
AV:
5
NL:
8.83E4
T:
FTMS+pESIFullms [1000.00-2700.00]
1151.5
1152.0
1152.5
1153.0
1153.5
1154.0
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
1152.73
z=20
1152.78
z=20
1152.63
z=20
1152.83
z=20
1152.58
z=20
1152.93
z=20
1152.98
z=20
1152.53
z=20
1153.03
z=20
1152.48
z=20
1153.13
z=20
1151.98
z=20
1151.48
z=?
1152.38
z=20
1151.77
z=20
1153.53
z=?
1153.18
z=20
1153.68
z=? 1153.98
z=?
1154.32
z=?
+20 charged
Spectrum of a partially reduced light chain
(+20 charged)
ReSpect is a trademark of Positive Probability, Ltd. All other 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.
Fab Heavy
24160
24170
24180
49.94
150.94
4151.94
24152.95
24153.93
24154.97
24156.00
24163.90
24166.90
24169.92
=140 K
Monoisotopic Mass
