identification of antibody fragments in sds- capillary … · max. 4.6397e‐05 4.5987e‐05...

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.

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.

•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?

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

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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

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0.0000

0.0002

0.0004

0.0006

0.0008

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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

Image Ref.: http://www.cartage.org.lb/en/themes/Sciences/LifeScience/GeneralBiology/Immunology/Recognition/AntigenRecognition/Antibodystructure/ab3.gif

Antibody Structure

(CDKTHTC)IgG1

150 kD 100 kD 50 kD

+ +

50 kD50 kD

Hinge Region Breakdown

150 kD 125 kD 100 kD 25 kD25 kD

+ +

Light Chain Loss

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

150 kD 125 kD 75 kD100 kD 50kD 25 kD

Nominal Molecular Weights of Antibody Fragments

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

Determine if cSDS distinguish these fragments of similar mass via adequate resolution and a reproducible migration/mobility measurements?

Goal

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

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.

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

Minutes

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

AU

0.00

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0.00

0.01

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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

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

Non-Reduced Migration Time DataExample with no Migration Time Overlap

Migration Time (min.)

Non-Reduced Migration Time Data

Migration Time (min.)

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

Reduced Migration Time Data

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

Non-Reduced Mobility Data

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

Reduced Mobility Data

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


Non-Reduced Relative MT Data

2.238

2.235

Reduced Relative MT Data

Beginning

End

Ab‐B Ab‐A Ab‐CRMT RMT RMT


Ab‐B Ab‐A Ab‐CRMT RMT RMT


Reduced Relative MT Data

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

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

Resolution of Fragments Within the Same Nominal MW

100 Kd vs

vsvs50 Kd

Examples at 100 Kd and 50 Kd

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

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


17996.0 50398.2 82800.4 115202.6 147604.8 180007.0Mass (m/z)

0

1027.8

0

10

20

30

40

50

60

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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

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

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0.045

AU

0.000

0.005

0.010

0.015

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0.025

0.030

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0.040

0.045

Full scale view

Native IgG1 sample

Fraction

101.1 kD

Fc+Fab


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


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


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



100 Kd Region

Aged Sample

Recent Sample


Fab Fc HeavyChain

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


Directed Degradation to Generated Fragments of Similar Mass50 Kd

50 Kd Region

Aged Sample

Recent Sample

Zoomed-in view

Directed Degradation to Generated Fragments of Similar Mass50Kd (Fc)

Fc

Papain Digestion Protein A

Eluate

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


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



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


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


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

Directed Degradation to Generated Fragments of Similar Mass50Kd (Fab)

Fab

Papain Digestion Protein A

Flow-Thru

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


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

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


Zoomed-in view

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


Zoomed-in view

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

Directed Degradation to Generated Fragments of Similar Mass50Kd (Heavy Chain)

Heavy Chain

Tris pH 8, 8M Urea, 20mM DTT


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


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


cSDS of Non-Reduced Heavy Chain Fragment


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


L

H

HH

cSDS of Non-Reduced Heavy Chain Fragment

Zoomed-in view

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

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

identification of antibody fragments in sds- capillary … · max. 4.6397e‐05 4.5987e‐05...

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