6
Automated MAb Workflow: from Harvest Cell Culture to Intact Mass Analysis of Variants
Conclusion
§
Using Protein-A Affinity, MAbPac SEC, and MAbPac SCX-10 columns, HCC was
characterized by affinity purification, followed by SEC and charge variant analysis in
less than one hour.
§
The separation of the lysine variants demonstrated that the pH-based gradient method
is an effective approach, orthogonal to salt gradient separation.
§
The combination of off-line IEC separation and on-line LC MS detection provides an
efficient way to obtain structural information of MAb variants.
References
1.Decrop, W.; Swart, R. Development of an Automated Method for Antibody Purification
and Analysis.
J. Biomol. Tech
.
2011
, October; 22 (Supplement).
2.Rea, J., Moreno, T., Lou, Y., and Farnan, D. Validation of a pH Gradient-Based
Ion-exchange Chromatography Method for High-Resolution Monoclonal Antibody
Charge Variant Separations.
J. Pharm. Biomed. Anal
.
2011
54
(2), 317–23.
Acknowledgements
We would like to thank Terry Zhang at Thermo Fisher Scientific for assistance in acquiring
the MS data.
Poros is a registered trademark of Applied Biosystems. 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 manner that
might infringe the intellectual property rights of others.
LPN 3078-1
fraction collected
purified IgG fraction
11.0
12.0
13.0
14.0
15.0
UV_VIS_1
min
WVL:280 nm
14.0
15.0
16.0
17.0
18.0
19.0
20.0
UV_VIS_1
min
WVL:280 nm
30.0
100.0
10.0
ue 6.8
ue 10.8
26.0
27.0
28.0
29.0
30.0
UV_VIS_1
min
WVL:280 nm
94%B
ac SCX-10 column
FIGURE 6. Full scan MS spectra
FIGURE 7. Deconvoluted MS spectra
Peak 1
Peak 2
Peak 3
2700
2750
2800
2850
2900
2950
3000
3050
3100
3150
3200
m/z
0
20
40
60
80
100
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
2960.87
2902.83
2847.02
3021.26
2793.33
2741.63
3084.18
3149.78
2967.30
2798.52
2855.24
3028.05
2911.23
2747.74
3090.75
3156.70
2975.31
2863.91
2807.80
3041.60
2951.71
2733.32
3011.95
2838.32
3107.52
2894.38
3170.63
2784.74
3140.12
3074.80
2905.33
2963.41
2849.48
2795.74
3023.89
2744.00
3086.89
3152.54
2911.68
2749.89
3030.55
2969.97
2801.07
2855.50
3095.61
3159.43
2916.99
2704.71
2756.43
3041.95
2954.31
2813.00
2787.13
2985.08
2735.47
2868.58
3103.91
3016.73
3172.75
3079.46
3142.85
2907.88
2798.14
2851.90
3026.53
2966.08
2746.40
3089.51
3033.20
2858.12
3155.38
2916.29
2972.60
2804.18
2752.32
3101.45
3041.54
2924.86
2956.93
2898.96
2710.89
2983.51
2763.17
2817.44
3161.22
3021.17
2738.07
3080.34
3176.67
3117.24
-Lys
-Lys
Peak 1
Peak 2
Peak 3
