![Page 1: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/1.jpg)
Tim Blanc, Gordon Freckleton, Babita Saxena, Ming-Ching Hsieh, Tun Liu, Rick Crowley, Qinwei Zhou.
ImClone Systems, a wholly-owned subsidiary of Eli Lilly and Company. Bioanalytical Science. 22 ImCLone Drive Branchburg, NJ 08876
Identification of antibody fragments in SDS-Capillary Gel Electrophoresis (cSDS) for
Comparability and Product related Impurities Studies of Monoclonal Antibody.
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Part 1 - Model for Challenging Resolution Reproducibility
Part 2 - Resolution of Antibody Fragments of Similar Molecular Weights
Identification of antibody fragments in SDS-Capillary Gel Electrophoresis (cSDS) for Comparability and Product related
Impurities Studies of Monoclonal Antibody.
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•cSDS as a Release and Stability Test - %Purity
•cSDS in a Protein Characterization, Comp. Study Testing and Clone Selection
(is there more to be revealed than just % Purity?)
For Comp. Studies, Product Related Impurities investigations and Clone Selection Studies we have found cSDS to be revealing beyond just % Purity
While seeing differences is useful, knowing what structures represent is a whole other ballgame. How do you move cSDS towards an assay where you have confidence that a peak represents a particular structure?
How does SDS-Capillary Gel Electrophoresis (cSDS) used in Studies of Monoclonal Antibody?
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Non-Reduced cSDS Electropherograms Regions
Minutes12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
Minutes12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
AU
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
0.0012
0.0014
0.0016
0.0018
0.0020
AU
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
0.0012
0.0014
0.0016
0.0018
0.0020
10Kd 50Kd25Kd 100Kd75Kd 125Kd 150Kd >150Kd
10Kd 50Kd25Kd 100Kd75Kd 125Kd 150Kd >150Kd
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Image Ref.: http://www.cartage.org.lb/en/themes/Sciences/LifeScience/GeneralBiology/Immunology/Recognition/AntigenRecognition/Antibodystructure/ab3.gif
Antibody Structure
(CDKTHTC)IgG1
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150 kD 100 kD 50 kD
+ +
50 kD50 kD
Hinge Region Breakdown
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150 kD 125 kD 100 kD 25 kD25 kD
+ +
Light Chain Loss
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Forced Degradation For Product Related Impurities(Chaos)
149.2kD
126.5kD
25.6 kD
103.8 kD
101.1 kD
48.1 kD
53.0 kD
27.4 kD
51.8 kD
48.1 kD
25.4 kD
22.7 kD
123.6 kD75.5 kD
49.9 kD
27.2 kD
24.5 kD
74.5 kD
1.8 kD26.4 kD 27.2 kD
78.4 kD
45.4 kD 52.6 kD
78.2 kD
98.0 kD
49.9 kD
22.7 kD
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150 kD 125 kD 75 kD100 kD 50kD 25 kD
Nominal Molecular Weights of Antibody Fragments
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150 kD 125 kD 75 kD100 kD 50kD 25 kD
Nominal Molecular weight of Antibody Fragments
53.0 kD48.1 kD
51.8 kD49.9 kD
75.5 kD
78.4 kD
74.5 kD
98.0 kD
101.1 kD
103.8 kD
123.6 kD
126.5 kD149.2 kD
78.2 kD
26.4 kD 22.7 kD
25.5 kD25.9 kD
27.2 kD
27.4 kD
45.4 kD
52.6 kD 25.6 kD
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Determine if cSDS distinguish these fragments of similar mass via adequate resolution and a reproducible migration/mobility measurements?
Goal
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M i n u t e s1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0
AU
- 0 . 0 0 5 0
- 0 . 0 0 2 5
0 . 0 0 0 0
0 . 0 0 2 5
0 . 0 0 5 0
0 . 0 0 7 5
0 . 0 1 0 0
0 . 0 1 2 5
0 . 0 1 5 0
0 . 0 1 7 5
0 . 0 2 0 0
AU
- 0 . 0 0 5 0
- 0 . 0 0 2 5
0 . 0 0 0 0
0 . 0 0 2 5
0 . 0 0 5 0
0 . 0 0 7 5
0 . 0 1 0 0
0 . 0 1 2 5
0 . 0 1 5 0
0 . 0 1 7 5
0 . 0 2 0 0P D A - 2 2 0 n mS . S . C o n t r o l R e d u c e d p H 9 . 0
Resolution Control Sample (3 IgG1s)Non-Reduced and Reduced
M i n u t e s
1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 2 8 3 0 3 2 3 4 3 6 3 8 4 0 4 2 4 4
AU
- 0 . 0 0 4
- 0 . 0 0 2
0 . 0 0 0
0 . 0 0 2
0 . 0 0 4
0 . 0 0 6
0 . 0 0 8
0 . 0 1 0
0 . 0 1 2
0 . 0 1 4
0 . 0 1 6
AU
- 0 . 0 0 4
- 0 . 0 0 2
0 . 0 0 0
0 . 0 0 2
0 . 0 0 4
0 . 0 0 6
0 . 0 0 8
0 . 0 1 0
0 . 0 1 2
0 . 0 1 4
0 . 0 1 6P D A - 2 2 0 n mS . S . C o n t r o l N o n - R e d u c e d p H 9 . 0
Antibody-B LC
Antibody-B
Antibody-C
Antibody-A
Antibody-C LCAntibody-A LC
Antibody-B HC
Antibody-A HC
Antibody-C HC
REDUCED
NON-REDUCED
10Kd Marker
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Reproducibility of Control Sample
10 Runs where performed under both Non-Reducing and Reducing Conditions. Samples were bracketed with the Control sample at the beginning and end of the run.
•Gel Lot •Capillary Lot•Capillary Vendor•Capillary Build
Ld= 20.0 cm, 19.4 cm and 20.6 cm
Note- with different capillary lengths you could not expect perfectly reproducible migration times. Capillary length variability, and the effect thereof, seemed like an important potential source of variability and therefore emphasized.
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Minutes
12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
AU
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
AU
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
Resolution Control Sample (3 IgG1s)Non-Reduced
Antibody-B
Antibody-CAntibody-A NON-REDUCED10Kd
Marker
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Minutes
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
AU
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
AU
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
Resolution Control Sample (3 IgG1s)Reduced
LC-B LC-C LC-A
REDUCED
10Kd Marker HC-B HC-CHC-A
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Non-Reduced Migration Time Data
Beginning
End
10kD SS‐NR SS‐NR SS‐NRMarker Ab‐B Ab‐A Ab‐CMT MT MT MT
Average 11.898 26.249 26.848 27.961Std.Dev. 0.283 0.711 0.768 0.755%RSD 2.377 2.707 2.859 2.700Min. 11.567 25.442 26.000 27.067Max. 12.342 27.250 27.908 29.050
10kD SS‐NR SS‐NR SS‐NRMarker Ab‐B Ab‐A Ab‐CMT MT MT MT
Average 12.162 26.847 27.476 28.575Std.Dev. 0.249 0.654 0.673 0.688%RSD 2.051 2.437 2.449 2.408Min. 11.800 26.067 26.675 27.742Max. 12.558 27.758 28.433 29.525
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Non-Reduced Migration Time DataExample with no Migration Time Overlap
Migration Time (min.)
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Non-Reduced Migration Time DataExample with no Migration Time Overlap
Migration Time (min.)
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Non-Reduced Migration Time Data
Migration Time (min.)
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Reduced Migration Time Data
Beginning
End
Marker Ab‐B LC Ab‐C LC Ab‐A LC Ab‐B HC Ab‐A HC Ab‐C HCMT MT MT MT MT MT MT
Average 12.216 14.980 15.123 15.641 18.649 18.873 19.781Std.Dev. 0.241 0.306 0.306 0.321 0.398 0.367 0.423%RSD 1.974 2.044 2.024 2.049 2.134 1.942 2.140Min. 11.850 14.517 14.667 15.167 18.083 18.325 19.183Max. 12.500 15.350 15.492 16.033 19.150 19.392 20.317
Marker Ab‐B LC Ab‐C LC Ab‐A LC Ab‐B HC Ab‐A HC Ab‐C HCMT MT MT MT MT MT MT
Average 12.456 15.301 15.433 15.967 19.022 19.294 20.175Std.Dev. 0.239 0.303 0.300 0.311 0.379 0.383 0.398%RSD 1.915 1.979 1.943 1.947 1.991 1.987 1.975Min. 12.075 14.817 14.958 15.475 18.458 18.717 19.567Max. 12.842 15.792 15.917 16.475 19.617 19.900 20.792
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Reduced Migration Time Data
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Non-Reduced Mobility Data
Beginning
End
Ab‐B Ab‐A Ab‐CMobility Mobility Mobility
Average 2.4181E‐05 2.3569E‐05 2.2586E‐05Std.Dev. 1.4944E‐06 1.5257E‐06 1.5665E‐06%RSD 6.180 6.473 6.936Min. 2.2227E‐05 2.1593E‐05 2.0581E‐05Max. 2.6429E‐05 2.5842E‐05 2.4965E‐05
Ab‐B Ab‐A Ab‐CMobility Mobility Mobility
Average 2.4845E‐05 2.4215E‐05 2.3270E‐05Std.Dev. 1.5784E‐06 1.6699E‐06 1.6831E‐06%RSD 6.353 6.896 7.233Min. 2.2983E‐05 2.2232E‐05 2.1234E‐05Max. 2.7016E‐05 2.6452E‐05 2.5595E‐05
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Non-Reduced Mobility Data
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Reduced Mobility Data
Beginning
End
Ab‐B LC Ab‐C LC Ab‐A LC Ab‐B HC Ab‐A HC Ab‐C HCMobility Mobility Mobility Mobility Mobility Mobility
Average 4.5983E‐05 4.5545E‐05 4.4036E‐05 3.6917E‐05 3.6274E‐05 3.4609E‐05Std.Dev. 4.1046E‐07 4.3859E‐07 4.8096E‐07 8.0356E‐07 1.0464E‐06 1.2914E‐06%RSD 0.893 0.963 1.092 2.177 2.885 3.731Min. 4.5023E‐05 4.4528E‐05 4.2922E‐05 3.5209E‐05 3.4440E‐05 3.1944E‐05Max. 4.6397E‐05 4.5987E‐05 4.4515E‐05 3.7663E‐05 3.7177E‐05 3.5672E‐05
Ab‐B LC Ab‐C LC Ab‐A LC Ab‐B HC Ab‐A HC Ab‐C HCMobility Mobility Mobility Mobility Mobility Mobility
Average 4.6129E‐05 4.5744E‐05 4.4251E‐05 3.7370E‐05 3.6699E‐05 3.5132E‐05
Std.Dev. 3.9352E‐07 4.2805E‐07 4.7873E‐07 7.6632E‐07 8.4663E‐07 9.3832E‐07%RSD 0.853 0.936 1.082 2.051 2.307 2.671Min. 4.5203E‐05 4.4752E‐05 4.3153E‐05 3.5591E‐05 3.5037E‐05 3.3363E‐05Max. 4.6535E‐05 4.6197E‐05 4.4751E‐05 3.8194E‐05 3.7707E‐05 3.6264E‐05
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Reduced Mobility Data
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Non-Reduced Relative MT Data
Beginning
End
(Start) (Start) (Start)Ab‐B Ab‐A Ab‐CRMT RMT RMT
Average 2.205 2.258 2.349Std.Dev. 0.015 0.017 0.017%RSD 0.682 0.735 0.713Min. 2.184 2.235 2.323Max. 2.230 2.283 2.373
(End) (End) (End)Ab‐B Ab‐A Ab‐CRMT RMT RMT
Average 2.207 2.259 2.349Std.Dev. 0.017 0.019 0.019%RSD 0.779 0.833 0.795Min. 2.183 2.234 2.324Max. 2.238 2.291 2.383
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Non-Reduced Relative MT Data
2.238
2.235
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Reduced Relative MT Data
Beginning
End
Ab‐B Ab‐A Ab‐CRMT RMT RMT
Average 2.205 2.258 2.349Std.Dev. 0.015 0.017 0.017%RSD 0.682 0.735 0.713Min. 2.184 2.235 2.323Max. 2.230 2.283 2.373
Ab‐B Ab‐A Ab‐CRMT RMT RMT
Average 2.207 2.259 2.349Std.Dev. 0.017 0.019 0.019%RSD 0.779 0.833 0.795Min. 2.183 2.234 2.324Max. 2.238 2.291 2.383
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Reduced Relative MT Data
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Part 1 Summary
Resolution was achieved under every condition
Relative migration times most robust for identifying peak
Relative Migration Times show the least over lap of these extremely challenging resolution pairs.
This model shows resolution at the cusp of the method ability
MT(RSD)
OverlapMobility(RSD)
OverlapRMT(RSD)
Overlap
Non‐Reduced 2.6% Severe* 6.5% Substantial 0.75% Minimal
Reduced 2.0% Severe* 2.0% Substantial 0.75% Minimal
![Page 31: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/31.jpg)
Generate antibody fragments to help identify peaks in electropherograms and investigate migration differences of fragments of similar mass
Directed Degradation to Generated Fragments of Similar Mass
150 kD 125 kD 75 kD100 kD 50kD 25 kD
![Page 32: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/32.jpg)
Resolution of Fragments Within the Same Nominal MW
100 Kd vs
vsvs50 Kd
Examples at 100 Kd and 50 Kd
![Page 33: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/33.jpg)
Tris pH 9 with 2M Urea
48hrs., 25C in the dark +
PartialPapain Digestion Protein
AEluate
Flow-Thru
Tris pH 8, 8M Urea, 20mM DTT +
149.2 kD
149.2 kD
126.5 kD
149.2 kD
103.8
101.1 kD
48.1 kD
53.0 kD 25.6 kD
51.8 kD22.7 kD
22.7 kD
Directed Degradation to Produce Fragments
![Page 34: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/34.jpg)
Minutes
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
AU
-0.00025
0.00000
0.00025
0.00050
0.00075
0.00100
0.00125
0.00150
0.00175
0.00200
AU
-0.00025
0.00000
0.00025
0.00050
0.00075
0.00100
0.00125
0.00150
0.00175
0.00200
Two samples, one further along in shelf life, showing fragments at approx. 100Kd
Directed Degradation to Generated Fragments of Similar Mass100Kd
100 Kd Region
Aged Sample
Recent Sample
![Page 35: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/35.jpg)
Directed Degradation to Generated Fragments of Similar Mass100Kd
17996.0 50398.2 82800.4 115202.6 147604.8 180007.0Mass (m/z)
0
1027.8
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
Voyager Spec #1=>RSM2000[BP = 101030.7, 1028]
101023.75
50529.7522785.8150699.94
101.1 kD
22.7 kD
Fc+Fab [1+]
Fc+Fab [2+]
MALDI Mass Spectrum of Fraction
![Page 36: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/36.jpg)
Minutes
12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
Full scale view
Native IgG1 sample
Fraction
101.1 kD
Fc+Fab
Directed Degradation to Generated Fragments of Similar Mass100Kd
![Page 37: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/37.jpg)
Minutes
12 14 16 18 20 22 24 26 28 30 32 34 36
AU
-0.0010
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
AU
-0.0010
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
Native IgG1 Sample
Fragment
Fc+FabZoomed-in view
Directed Degradation to Generated Fragments of Similar Mass100Kd
![Page 38: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/38.jpg)
Minutes
12 14 16 18 20 22 24 26 28 30 32 34 36
AU
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
0.0040
AU
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
0.0040Zoomed-in view
Native IgG1 Sample
Spiked Fraction
Fc+Fab
Directed Degradation to Generated Fragments of Similar Mass100Kd
![Page 39: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/39.jpg)
Minutes
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
AU
-0.00025
0.00000
0.00025
0.00050
0.00075
0.00100
0.00125
0.00150
0.00175
0.00200
AU
-0.00025
0.00000
0.00025
0.00050
0.00075
0.00100
0.00125
0.00150
0.00175
0.00200
Two samples, one further along in shelf life, showing fragments at approx. 100Kd
Directed Degradation to Generated Fragments of Similar Mass100Kd
100 Kd Region
Aged Sample
Recent Sample
![Page 40: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/40.jpg)
Directed Degradation to Generated Fragments of Similar Mass50Kd
Fab Fc HeavyChain
![Page 41: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/41.jpg)
Minutes
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
AU
-0.00025
0.00000
0.00025
0.00050
0.00075
0.00100
0.00125
0.00150
0.00175
0.00200
AU
-0.00025
0.00000
0.00025
0.00050
0.00075
0.00100
0.00125
0.00150
0.00175
0.00200
Two samples, one further along in shelf life, showing fragments at approx. 50Kd
Directed Degradation to Generated Fragments of Similar Mass50 Kd
50 Kd Region
Aged Sample
Recent Sample
Zoomed-in view
![Page 42: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/42.jpg)
Directed Degradation to Generated Fragments of Similar Mass50Kd (Fc)
Fc
Papain Digestion Protein A
Eluate
![Page 43: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/43.jpg)
17996.0 50398.2 82800.4 115202.6 147604.8 180007.0Mass (m/z)
0
245.5
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
Voyager Spec #1=>RSM2000[BP = 53060.2, 246]
53057.92
53279.08
53462.15
52728.90
53664.3626642.60
22740.0053831.99
26466.74
26845.19
54164.88
53.0 kD
26.4 kD
22.7 kD
L [1+]
H’Fc[1+]
Fc[1+]
MALDI Spectrum of Non-Reduced Fc Fragment
Directed Degradation to Generated Fragments of Similar Mass50Kd (Fc)
![Page 44: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/44.jpg)
Directed Degradation to Generated Fragments of Similar Mass50Kd (Fc)
Full scale view
Minutes
12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
Original Sample
Fraction
cSDS of Non-Reduced Fab Fragment Full-Scale view
![Page 45: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/45.jpg)
Directed Degradation to Generated Fragments of Similar Mass50Kd (Fc)
Zoomed-in view
Minutes
12 14 16 18 20 22 24 26 28 30 32 34 36
AU
-0.0010
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
AU
-0.0010
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
Original sample
Fraction
cSDS of Non-Reduced Fc Fragment Zoomed-in view
![Page 46: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/46.jpg)
Directed Degradation to Generated Fragments of Similar Mass50Kd
Minutes12 14 16 18 20 22 24 26 28 30 32 34 36
AU
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
AU
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
Zoomed-in view
Original Sample
Spiked with Fraction
cSDS of Non-Reduced Fc Fragment
![Page 47: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/47.jpg)
Directed Degradation to Generated Fragments of Similar Mass50Kd (Fab)
Fab
Papain Digestion Protein A
Flow-Thru
![Page 48: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/48.jpg)
Directed Degradation to Generated Fragments of Similar Mass50Kd (Fab)
17996.0 50398.2 82800.4 115202.6 147604.8 180007.0Mass (m/z)
0
724.8
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
Voyager Spec #1=>RSM2000[BP = 25417.5, 725]
25435.7147998.56
22774.59
48186.77
25630.06
22950.61
24221.20
73266.1295886.65
35,400
L [1+]
H’’Fab[1+]
Fab [1+]
Fab [2+]
48.1 kD
25.4 kD
22.7 kD
MALDI Spectrum of Non-Reduced Fab Fragment
![Page 49: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/49.jpg)
Full scale view
Minutes
12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
Original sample
Fraction
Directed Degradation to Generated Fragments of Similar Mass50Kd
Zoomed-in view
![Page 50: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/50.jpg)
Zoomed-in view
Minutes
12 14 16 18 20 22 24 26 28 30 32 34 36 38
AU
-0.0015
-0.0010
-0.0005
0.0000
0.0005
0.0010
0.0015
AU
-0.0015
-0.0010
-0.0005
0.0000
0.0005
0.0010
0.0015
Original Sample
Fraction
50Fab
Directed Degradation to Generated Fragments of Similar Mass50Kd
Zoomed-in view
![Page 51: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/51.jpg)
Minutes12 14 16 18 20 22 24 26 28 30 32 34 36
AU
-0.00025
0.00000
0.00025
0.00050
0.00075
0.00100
0.00125
0.00150
0.00175
0.00200
AU
-0.00025
0.00000
0.00025
0.00050
0.00075
0.00100
0.00125
0.00150
0.00175
0.00200
Zoomed-in view
Original Sample
Spiked with FractionFab
cSDS of Non-Reduced Fab Fragment
Directed Degradation to Generated Fragments of Similar Mass50Kd Zoomed-in view
![Page 52: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/52.jpg)
Directed Degradation to Generated Fragments of Similar Mass50Kd (Heavy Chain)
Heavy Chain
Tris pH 8, 8M Urea, 20mM DTT
![Page 53: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/53.jpg)
Directed Degradation to Generated Fragments of Similar Mass50Kd
17996.0 50398.2 82800.4 115202.6 147604.8 180007.0Mass (m/z)
0
25.8
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
Voyager Spec #1=>RSM2000=>SM11[BP = 22735.0, 26]
22736.6951990.80
20231.11
21896.70
19087.1226241.74
22973.3720059.95
25559.0618816.85
52341.9718581.4921458.53
26516.4224287.49
23203.5423351.22
27217.7551517.52
27950.1950346.02
30620.48 50652.0829903.35 103398.1352817.0739486.29 84803.11
57187.8643695.71101765.2877158.56 140533.53
51.8 kD
H [1+]
MALDI Spectrum of Non-Reduced Heavy Chain Fragment
![Page 54: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/54.jpg)
Directed Degradation to Generated Fragments of Similar Mass50Kd (Heavy Chain)
Minutes
12 14 16 18 20 22 24 26 28 30 32 34 36
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050Full scale view
Original sample
Spiked with Fraction
cSDS of Non-Reduced Heavy Chain Fragment
![Page 55: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/55.jpg)
Directed Degradation to Generated Fragments of Similar Mass50Kd (Heavy Chain)
Minutes12 14 16 18 20 22 24 26 28 30 32 34 36
AU
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
AU
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
Original Sample
Spiked with Fraction
L
H
HH
cSDS of Non-Reduced Heavy Chain Fragment
Zoomed-in view
![Page 56: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/56.jpg)
cSDS provides impressive resolution for a size-based separation
Resolution was very reproducible
Structures related to Antibody Fragmentation could be Tentatively Identified through spiking experiments
Relative Migration Times appeared to be the best parameter to monitor peaks in cSDS Electropherograms
Relative Migration Time was right at the cusp of being able to track peak unequivocally, but not quite there. Perhaps had the capillary length notbeen purposefully deviated the performance of all parameters would have been better.
Summary
![Page 57: Identification of antibody fragments in SDS- Capillary … · max. 4.6397e‐05 4.5987e‐05 4.4515e‐05 3.7663e‐05 3.7177e‐05 3.5672e‐05 ab‐b lc ab‐c lc ab‐a lc ab‐b](https://reader030.vdocuments.net/reader030/viewer/2022013015/5b9419a509d3f2df3f8c42d6/html5/thumbnails/57.jpg)
Method DevelopmentGroup
Babita SaxenaSusan KantenweinRagani Srikakulam
Peter Kuo
Thank You
Protein CharacterizationGroup
Qinwei ZhouGordon Freckleton (MALDI)
Ming-Ching Hsieh (Purification)Tun Liu
Richard Seipert
CASSS, Organizing Committee, Staff, and AttendeesBioanalytical Science at ImClone