intact monoclonal antibody characterization using a bench...

Intact Monoclonal Antibody

Characterization Using A Bench-Top

Orbitrap Mass Spectrometer

Zhiqi Hao Thermo Fisher Scientific

San Jose, CA

2

Image from Kozlowski et al. n engl j med 365;5 nejm.386 org august 4, 2011

Aspirin (180 Da)

MAb (150,000 Da)

Monoclonal Antibody (mAb) – an Analytical Challenge

3

mAb Characterization – HR/AM Mass Spectrometry a Natural Fit

• Some of the analytical challenges:

Deamidation (+1 Da)

Oxidation (+16 Da)

Disulfide scrambling

Sequence truncation

Change in glycosylation

Aggregation

etc…

• Accurate characterization requires high resolution and accurate mass MS

• Characterizations are performed at intact protein, sub-structure and peptide levels

• Mass ~150 kDa • 2 light chains (~20 kDa each) • 2 heavy chains (~50 kDa each) • 16 disulfide bonds • Glycans

4

Outline

• The new generation OrbitrapTM mass analyzer • Intact antibody characterization using the Q ExactiveTM

quadrupole Orbitrap mass spectrometer • Introducing Q Exactive quadrupole Orbitrap mass spectrometer

• Intact mAb analysis • Mass measurement

• Relative quantification of glycoforms

• Structure characterization

• Fab

• Summary

5

• The new generation OrbitrapTM mass analyzer

Exactive Plus

Q Exactive

Orbitrap Elite

6

Intact Protein Detection in Orbitrap Analyzer

0 1000 2000 3000 t, ms

Myoglobin (16.9 kD)

z: +16

0 1000 2000 3000 t, ms

0 1000 2000 3000 t, ms

0

Transferrin (78kD)

z: +40

Enolase (46kD)

z: +24

Solutions: • Higher Vacuum • Hardware Innovation • Software Innovation

Fast Signal Decay: • Collision of ions • Fragmentation of ions • De-phasing of ion packets

7

#29216 IT: 500.000 ST: 1.05 uS: 1 NL: 7.15F: FTMS + p ESI Full ms [0.00-384.00]Info: Ufill=0.01 MCal=24h RF=1810V TmpDiff

0 1 2 3 4 5 6 7 8 9 10m/z

0

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Rela

tive

Inte

nsity

6.56290.3585

6.4464

6.40610.50106.34466.3241

6.2629

6.24326.10036.0396

5.83635.77555.63274.96222.14822.0607 4.51544.08912.18082.0204

3.68333.52083.2571 9.90649.55888.95501.1884 8.84477.86337.2506

#27955 IT: 10.000 ST: 0.28 uS: 1 NL: 7.15E2F: FTMS + p ESI Full ms [0.00-96.00]Info: Ufill=0.01 MCal=9h RF=1430V Stable=2min

0 1 2 3 4 5 6 7 8 9 10 11m/z

0

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Relat

ive In

tensit

y

0.1758

0.3435

0.4006

0.5435 4.7990 7.63040.6147 4.32664.0796 7.36875.5774 10.38089.58811.0611 10.63228.87952.9427 6.8835 7.7499 10.9545

ms ms

8.9 ms < 0.6 ms

Earlier Orbitrap Instruments

Q Exactive Orbitrap Elite Exactive Plus

Reduced Detection Delay Improves Intact Protein Measurement

0 1000 2000 3000 t, ms

0

Transferrin (78kD)

z: +40

<10ms Efficient Capture of Initial Transient

Stronger Signal

Better Mass Accuracy

8

0

20000

40000

60000

80000

100000

120000

140000

160000

0 5 10 15

Re

solu

tio

n

Scan Speed [Hz]

Exactive

Q-Exactive Resolution

at m/z 200

Resolution

at m/z 400

Max. scan

speed (Hz)

17,500 12,500 12

35,000 25,000 7

70,000 50,000 3

140,000 100,000 1.5

Resolution increased by 2X

Speed increased by 2X

Improved Resolution and Scan Speed

9

• Intact antibody characterization using the Q ExactiveTM

quadrupole Orbitrap mass spectrometer

• Introducing Q Exactive quadrupole Orbitrap mass spectrometer

• Intact mAb analysis • Mass measurement

• Relative quantification of glycoforms

• Structure characterization

• Fab

10

Q ExactiveTM - Innovations and Features

The first benchtop Quadrupole-Orbitrap mass spectrometer

ORBITRAP

Resolution: 140,000

Mass Accuracy: < 3 ppm

QUADRUPOLE MASS FILTER

Mass Range: 50 – 6000 amu Prec Isolation: 0.4 – 10 amu

HCD cell and C-Trap

ION SOURCE

S-Lens

11

AntiHer2_2ug_5000 4/23/2012 4:35:25 PM

RT: 0.00 - 20.02

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Time (min)

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Rel

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5.89

0.37 6.71 7.23 7.96 15.518.35 15.949.08 11.069.55 11.45 16.6212.10 13.07 13.4110.00 14.990.85 17.215.713.743.15 5.071.70 18.762.53 4.35 19.77

NL:2.79E9TIC MS AntiHer2_2ug_5000

AntiHer2_2ug_5000 #439-512 RT: 5.73-6.07 AV: 74 NL: 3.04E7T: FTMS + p ESI sid=45.00 Full ms [1800.00-5000.00]

1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200m/z

0

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Rel

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bund

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2907.25

2797.59 3025.87

3088.892745.82

2695.90

2968.59

3154.57

2647.78 3223.152601.35

3294.722556.54

2513.21 3369.59

2430.84 3447.922353.71

3529.932246.72 3616.002149.04 3801.46 3901.522003.93 4006.911899.12 4122.56 4235.94 4365.32


2940 2960 2980 3000 3020 3040 3060 3080 3100 3120m/z

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Rel

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2965.3810

3025.8740

3088.8882

2971.84453032.5158

3095.5851

3019.5958

2959.2434

3082.4699

2977.68572996.3881

3038.23963055.3179

3101.77532933.2498 2953.0596

3013.3330 3075.9493

AntiHer2_2ug_5000 4/23/2012 4:35:25 PM

RT: 0.00 - 20.02

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Time (min)

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Re

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bu

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5.89

0.37 6.71 7.23 7.96 15.518.35 15.949.08 11.069.55 11.45 16.6212.10 13.07 13.4110.00 14.990.85 17.215.713.743.15 5.071.70 18.762.53 4.35 19.77



1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200m/z

0

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Re

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2907.25

2797.59 3025.87

3088.892745.82

2695.90

2968.59

3154.57

2647.78 3223.152601.35

3294.722556.54

2513.21 3369.59

2430.84 3447.922353.71

3529.932246.72 3616.002149.04 3801.46 3901.522003.93 4006.911899.12 4122.56 4235.94 4365.32

Total Ion Chromatogram

Averaged full MS spectrum across 1 min elution peak

Intact Monoclonal Antibody Mass Measurement

G0F+G1F

G1F+G2F

G0F+G0F

G0+G0F

G0F+G2F

• 2 ug mAb on column • 5 min gradient • Instrument resolution setting = 17.5K

12

Deconvoluted Spectrum

-7 ppm G0F+G1F

G0F+G0F G0F+G2F (or 2G1F)

G1F+G2F

G2F+G2F G0+G0F

G0+G0 2xMan5

G1F+G2F+SA

5.0 ppm

-0.7 ppm -8.5 ppm

-0.9 ppm

• 9 glycoforms identified, all within ~12 ppm of theoretical average mass (+/-2 Da mass accuracy)

• Deglycosylated form detected with high mass accuracy at low relative abundance

• Deconvoluted spectrum is free of artifacts and the various forms of the protein are well resolved

13

Reproducible Mass Measurement for IgG Glycoforms

• -6.9 +/- 6.4 ppm mass error across all measurements

• Mass measurement is highly reproducible, even across instruments

RAW file Q Exactive G0+G0F G0F+G0F G0F+G1F G0F+G2F G1F+G2F

1 1 -10.5 0.7 -10.5 -13.8 -18.0

2 1 -11.6 -1.1 -8.8 -11.2 -12.0

3 1 5.1 -5.0 -2.6 5.1 5.6

4 2 -14.3 3.0 -6.9 -5.4 -5.9

5 2 -8.6 -2.2 -12.2 -12.5 -12.9

6 2 -14.3 -6.6 -12.3 -14.8 -10.1

-7 ppm

G0F+G1F

G0F+G0FG0F+G2F (or 2G1F)

G1F+G2F

G2F+G2FG0+G0F

G0+G02xMan5

G1F+G2F+SA

5.0 ppm

-0.7 ppm -8.5 ppm

-0.9 ppmIgG glycoform mass measurement accuracy (error in ppm)

14

• Relative abundances are highly reproducible across runs

Reproducible Relative Abundances for IgG Glycoforms

IgG glycoform relative abundances (%)

RAW file Q Exactive G0+G0F G0F+G0F G0F+G1F G0F+G2F G1F+G2F

1 1 12.9 74.1 100.0 67.0 23.4

2 1 12.0 72.8 100.0 66.2 22.0

3 1 12.2 75.0 100.0 67.0 23.6

4 2 12.7 75.7 100.0 63.6 21.6

5 2 13.2 75.4 100.0 64.8 21.0

6 2 12.9 76.6 100.0 64.7 21.6

CV 3.4% 1.6% N.A. 3.9% 4.4%

-7 ppm

G0F+G1F


G1F+G2F

G2F+G2FG0+G0F

G0+G02xMan5

G1F+G2F+SA

5.0 ppm

-0.7 ppm -8.5 ppm

-0.9 ppm

15

Characterization of Fab

Papain_digest_no_cys_nonlinear-2ul #525-556 RT: 18.97-19.51 AV: 32 NL: 1.86E6T: FTMS + p ESI Full ms [800.00-3500.00]

1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300m/z

0

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25

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Relat

ive A

bund

ance

1475.30

1430.59 1522.86

1348.93

1573.57

1311.46

1627.82

1276.061685.91

1748.311242.55

1815.511888.07

1966.741210.68

2052.18

1180.472145.45

2247.55

2008.66 2097.89

1900.37

2160.00 2318.22

intact Fab Fab light Fab heavy

Reduction

Intact Fab

Fab light

LC MS

Fab heavy

47177.6 Da 23038.8 Da 24148.3 Da

PPN_red_60C_02 #1157-1169 RT: 20.34-20.47 AV: 13 NL: 4.38E4T: FTMS + p ESI Full ms [1000.00-2700.00]

1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750m/z

0

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25

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Relat

ive A

bund

ance

1510.311421.47

1610.80

1342.58

1725.83

1272.02

1208.29

1689.821440.80

1619.461487.38

1579.531547.13 1661.981466.57

1714.22

1760.17

1733.09

1385.811356.36

1323.971281.05

1412.83

1263.861222.44

PPN_red_60C_02 #1138-1144 RT: 20.13-20.18 AV: 7 NL: 1.30E5T: FTMS + p ESI Full ms [1000.00-2700.00]

1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800m/z

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25

30

35

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Relat

ive A

bund

ance

1440.89

1356.12

1537.021280.95

1646.581213.53

1153.05 1773.26

1328.25 1689.951475.811388.321272.79 1413.63

1562.471162.23 1613.361507.74

1288.45

1657.88

1256.64 1723.071748.38

RT: 7.95 - 20.71

8 9 10 11 12 13 14 15 16 17 18 19 20Time (min)

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Relati

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ndance

13.95

14.18

16.78 17.64 18.18 18.71 19.36 20.4310.72 11.14 12.118.25 9.32 9.648.89

NL:2.04E8TIC MS PPN_noCys_Red_140K

Fc

Preparation and identification of Fab light and heavy chains

• The 10 Da mass difference between the intact and the sum of the reduced Fab suggests 5 disulfide bonds, just as expected

16

High Resolution Measurement of Fab on LC Time Scale

20111109_Reduced_140k_Dionex_03 #893-919 RT: 36.12-36.86 AV: 27 NL: 5.71E5T: FTMS + p ESI Full ms [600.00-3000.00]

1440.2 1440.4 1440.6 1440.8 1441.0 1441.2 1441.4 1441.6 1441.8m/z

0

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Rela

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danc

e

1440.85 1440.97

1441.101440.78

1441.16

1440.72

1441.22

1440.661441.29

1440.60

1441.411441.481440.53

1440.091440.23 1440.48 1441.54

1441.661441.78

20111109_Reduced_140k_Dionex_03 #894-919 RT: 36.15-36.86 AV: 26 NL: 7.12E5T: FTMS + p ESI Full ms [600.00-3000.00]

800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

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Relativ

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dance

1213.61z=19 1281.03

z=181098.03

z=211048.16z=22 1356.21

z=171152.93

z=20 1440.85z=16

1536.97z=15

1002.68z=23

960.98z=24

1646.75z=14

922.62z=? 1773.28

z=13

887.06z=?

854.32z=?

823.77z=?

798.98z=?

1707.55z=?

1403.22z=?

1467.62z=?

1836.75z=?

1587.56z=?

1784.51z=?

Monoisotopic mass measured at 140 K

Fab Measured Expected Error (ppm)

H 24133.8700 24133.7937 2.7

L 23025.3342 23025.3125 0.9

Baseline resolved isotopic peaks of light chain

+15 charge

17

Identification and Relative Quantification of Impurities


1100 1200 1300 1400 1500 1600 1700 1800m/z

0

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Rel

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bund

ance

1430.63

1475.31

1388.57

1522.871348.92

1573.56

1311.491627.82

1685.911276.09

1242.53 1748.291815.54

1210.67

1180.47

1151.64

1609.46

1124.301654.55

1716.84 1780.78 1831.26


1516 1518 1520 1522 1524 1526 1528 1530 1532m/z

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Rel

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1522.87

1523.49

1521.461517.50

1520.54 1526.181519.51 1524.86 1528.041516.941518.56

1530.381529.26 1532.30

Sub-structure Resolution Impurity Delta Mass (Da) Relative intensity (%)

Fab 17.5K 16.3 18.1

Fab heavy 140K 16.0158 21.9

PPN_red_37C_140K_120504084725_XT_00001_M_ #1 RT: 1.00 AV: 1 NL: 3.57E4T: FTMS + p ESI Full ms [1000.00-2700.00]

24120 24130 24140 24150 24160 24170 24180m/z

0

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Rel

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24147.93

24146.94 24149.94

24145.91

24150.94

24151.9424144.89

24152.9524143.90

24153.93

24154.9724156.0024141.95

24163.9024166.90

24140.89 24169.9224129.93 24138.85 24173.0624123.9724116.87

Oxidized Species of Fab

Intact Fab Fab heavy Resolution =140 K Resolution =17.5 K

Average mass Monoisotopic mass

18

20111109_Reduced_tHCD20light_Dionex03 #94-111 RT: 36.20-36.80 AV: 18 NL: 6.63E4T: FTMS + p ESI Full msx ms2 [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [ ...

200 400 600 800 1000 1200 1400 1600m/z

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324.19z=1

437.28z=1

739.40z=1159.09

z=1 524.31z=1

216.10z=1

812.34z=1 1175.91

z=9409.28

z=11322.90

z=8974.97

z=6303.13

z=11076.53

z=4839.48

z=3703.74z=3 1435.71

z=6619.30

z=21512.03

z=7

1377.71z=3

1751.88z=5

Targeted HCD on six precursors of different charge states

Top-down Sequencing Using Unique Spectrum Multiplexing Mode

Six Individual isolation and fragmentation events

One Orbitrap detection event

High resolution top-down MS/MS

Full MS of light chain 20111109_Reduced_140k_Dionex_03 #894-919 RT: 36.15-36.86 AV: 26 NL: 7.12E5T: FTMS + p ESI Full ms [600.00-3000.00]

800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

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Relative

Abundan

ce

1213.61z=19 1281.03

z=181098.03

z=211048.16z=22 1356.21

z=171152.93

z=20 1440.85z=16

1536.97z=15

1002.68z=23

960.98z=24

1646.75z=14

922.62z=? 1773.28

z=13

887.06z=?

854.32z=?

823.77z=?

798.98z=?

1707.55z=?

1403.22z=?

1467.62z=?

1836.75z=?

1587.56z=?

1784.51z=?

Parallel Filling and Detection Spectrum Multiplexing

19

30% backbone fragments

Sequence Coverage Achieved for Fully Reduced Fab

Light chain Fab heavy chain

34% backbone fragments P Score = 2.2E-34

• ‘Signal protein’ mode • 5 ppm fragment ion tolerance

P Score = 4.9E-19

20

Top-down Approach Can Provide Structural Information

Mapping disulfide linkage in partially reduced light chain PPN_noCys_Red_140K #129-133 RT: 13.79-14.10 AV: 5 NL: 8.83E4

T: FTMS + p ESI Full ms [1000.00-2700.00]

1151.5 1152.0 1152.5 1153.0 1153.5 1154.0m/z

0

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1152.73z=20

1152.78z=20

1152.63z=20 1152.83

z=20

1152.58z=20

1152.93z=20

1152.98z=20

1152.53z=20

1153.03z=201152.48

z=201153.13

z=201151.98z=201151.48

z=?1152.38

z=201151.77

z=20 1153.53z=?1153.18

z=201153.68

z=? 1153.98z=? 1154.32

z=?

• Deconvoluted monoisotopic mass was 23021.4185 Da, 4 Da less than expected.

• Top-down sequencing mapped two internal disulfide bonds

• Further sample processing produced completely reduced light chain with monoisotopic mass of 23025.3342 Da, only 0.9 ppm off expected.

+20 charged

partially reduced light chain completely reduced light chain

21

•Summary

22

Summary

• Accurate, reproducible and fast mass measurement of intact mAb was achieved using the Q Exactive MS and Protein Deconvolution software.

• The Q Exactive high resolution top-down analysis and ProSight PC sequence assignment generated extensive, accurate sequence information for Fab light chain and heavy chain, offering a fast way to confirm amino acid sequence and detect unexpected modifications.

• Both, precise mass measurements and extensive, high confident sequence and structural information can be obtained for intact mAb using this workflow solution that combines fast chromatography, high resolution MS, high throughput msx HCD and high fidelity data analysis.

23

Intact Monoclonal Antibody Characterization Workflow

Cell culture

Affinity purification

Charge separation

Desalting /

H/L chain separation

AntiHer2_2ug_5000 4/23/2012 4:35:25 PM

RT: 0.00 - 20.02

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Time (min)

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Re

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5.89

0.37 6.71 7.23 7.96 15.518.35 15.949.08 11.069.55 11.45 16.6212.10 13.07 13.4110.00 14.990.85 17.215.713.743.15 5.071.70 18.762.53 4.35 19.77



1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200m/z

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Re

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Ab

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2907.25

2797.59 3025.87

3088.892745.82

2695.90

2968.59

3154.57

2647.78 3223.152601.35

3294.722556.54

2513.21 3369.59

2430.84 3447.922353.71

3529.932246.72 3616.002149.04 3801.46 3901.522003.93 4006.911899.12 4122.56 4235.94 4365.32

20111109_Reduced_tHCD20light_Dionex03 #94-111 RT: 36.20-36.80 AV: 18 NL: 6.63E4T: FTMS + p ESI Full msx ms2 [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [ ...

200 400 600 800 1000 1200 1400 1600m/z

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324.19z=1

437.28z=1

739.40z=1159.09

z=1 524.31z=1

216.10z=1

812.34z=1 1175.91

z=9409.28

z=11322.90

z=8974.97

z=6303.13

z=11076.53

z=4839.48

z=3703.74z=3 1435.71

z=6619.30

z=21512.03

z=7

1377.71z=3

1751.88z=5

MS of intact antibody

Top-down HCD of light chain

-7 ppm

G0F+G1F


G1F+G2F

G2F+G2FG0+G0F

G0+G02xMan5

G1F+G2F+SA

5.0 ppm

-0.7 ppm -8.5 ppm

-0.9 ppm

from crude cell culture to HR/AM characterization

• Intact Mass

• Relative quan of glycoforms

• Sequence confirmation

• Structural information

Poster : ThP15,

Shanhua Lin et. al

http://portal.thermo-brims.com

24

Acknowledgement

• Thermo Fisher Scientific, San Jose and Bremen Yi Zhang David Horn Seema Sharma Xiaoyue Jiang Andreas FR Huhmer Patrick K Bennett Markus Kellmann Thomas Moehring

• Barnett Institute, Northeastern University, Boston Shiaw-Lin Wu

• CGMP BPPF, Development Center for Biotechnology, Taipei Irene, Ae-Ning Lin

Yi-Hsuan Pan Ya-Fen Yang

intact monoclonal antibody characterization using a bench...

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