New Innovations in UHPLC and LC-MS Workflows for the Comprehensive Ch t i ti f M l lCharacterization of Monoclonal Antibodies (mAbs) and Antibody Drug Conjugates (ADCs)Conjugates (ADCs)

Ken CookEU Bio-separations Support Expert

November 2014

1 The world leader in serving science

Antibody Therapeutics – An Analytical Challenge

Deamidation (+1 Da)

Oxidation (+16 Da)

Di lfid b d bliDisulfide bond scrambling

Sequence truncation

Change in glycosylation

Low abundance host cell proteins

Low abundance sequence qvariants

Aggregation, protein complex

Quantification in matrix• To ensure proper biomanufacturing of the therapeutic occurs

Characterization

Quantification in matrix

Small molecule measurements

Drug Antibody Ratios

• To ensure critical quality attributes (CQAs) of a product are identified

modifications, impurities, immunogenicity, efficacy, DMPK Drug Antibody Ratios

Linker measurements

Bound vs unbound drug

, p , g y, y,• To ensure these CQAs can be routinely measured

mass spectrometers, separations, software, reagents • To study the ADME of the biotherapeutic

2

• To study the ADME of the biotherapeutic

New Benchmark for Bio-Separations

Thermo Scientific™ Vanquish™ UHPLC System

• Integrated modularityIntegrated modularity

• Viper-based, tool-free fluidic connections

• Biocompatible, iron-free flow pathp p

• Support for latest innovations in column technology

N l th t tti t h l ith• New column thermostatting technology with up to 3 column compartments in one system

• Enhanced LC-MS support and integration

• Portable system control with tablet PC

• Reduced system height

• Revolutionary module drawer system for repairs

• Removable doors for easy access

3

Vanquish Binary Pump

1500 bar / 22 000 psiUp to 5000 µL/minUp to 5000 µL/minNew parallel dual piston principle

• Independent piston drives andIndependent piston drives and variable stroke volume

Unmatched retention time performanceperformanceLowest baseline noise using intelligent piston controlUltra low GDV (as low as 35 µL)( µ )Thermal compensation (ATEC™) compensates compression heat for lowest pulsation2 × 3 solvent channels

• 9 different solvent combinations

5

Finger tight check valve design

Vanquish Split Sampler

Proprietary switching valve with “no maintenance”with no maintenance philosophyBiocompatible fluidicsAdjustable GDV for easy j ymethod transferNew air-to-air cooling concept

• 4 – 40 ºCHigh throughput capabilitiesAutomated barcode featuresIncreased sample capacity with 4 racks or well plates

i i 216 i l• minimum 216 vialsHuge capacity with Vanquish Charger

up to 20 well plates

6

• up to 20 well plates

Vanquish Column Compartment

Unique actively regulated Pre-Heater• for perfect control of mobile phasefor perfect control of mobile phase

and column temperatureExtended temperature range 5–120 ºCCTwo thermostatting modes for easier method transfer and better peak shape managementshape management• still air mode to boost separation

speed and resolution• forced air mode to enable

seamless method transfer and mimic other column thermostats

Up to three independent column compartmentsNew column ID system

7

y

Vanquish Diode Array Detector

LightPipe technology for an unmatched detection experience• 10 mm standard flow cell• 60 mm high sensitivity flow cellB i l i fBest signal-to-noise performanceLowest baseline noiseUltra-wide dynamic rangeUltra-wide dynamic range• up to 3000 mAU• for simultaneous detection of highly

concentrated compounds and trace impurities

Variable slit width for optimum resolution 1–8 nmLow peak dispersionReduced RI and thermal effects

8

Reduced RI and thermal effects

Vanquish System Delivers – Exceptional Sensitivity

• Impurities as low as 0.008 % of the active ingredient (API)

• Exceptional linearity for API d i iti tit ti

3,500

3000Nevirapine

5

1

2

3 4 and impurities quantitation

• Very fast using 800 µL/min with 890 bar maximum

R² = 0.9998

20000

mAU

4

5 678

R2 = 0.999with 890 bar maximum pressure

0

100018

-2

secondsmA

U

72

0

0 0.4 0.8Nevirapine [mg/mL]

0 60 72-500

30seconds

9

Overlay of 13 Runs of a Trypsin Digest mAb with Retention Time Precision

162 5

175.0

187.5

201.5mAU

peak # RT (min)

RT-RSD (%)

125.0

137.5

150.0

162.5(%)3 3.315 0.0829 5.231 0.065

14 6.532 0.01715 6.937 0.023

75.0

87.5

100.0

112.519 10.290 0.02123 12.013 0.01231 14.011 0.01339 15.177 0.01242 15 589 0 010

25 0

37.5

50.0

62.5

42 15.589 0.01051 17.511 0.00755 17.969 0.01161 18.546 0.01083 20.798 0.010

-12.5

0.0

12.5

25.0

85 21.095 0.01287 22.386 0.00996 24.774 0.012103 26.155 0.009106 26 155 0 009

2.5 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 31.3-45.2

-37.5

-25.0

min

106 26.155 0.009109 27.529 0.010

10

Thermo Scientific™ Trypsin Digestion

• Heat stable Trypsin• 70 ºC heat denaturation of proteins so no need for70 C heat denaturation of proteins so no need for

detergents or urea• Immobilized Trypsin in throw away PCR tubeImmobilized Trypsin in throw away PCR tube• No autolysis• Less steps and no clean up – much quicker and easier to• Less steps and no clean up – much quicker and easier to

use• Less modificationsLess modifications• More reproducible

11

Up to 96 Samples, ≤ 60 Minute No PretreatmentNecessary

Undigested ≤ 90 ºC Filt LC/MSUndigested Protein RT: 0.00 - 30.02

80

85

90

95

100

8.21575.80

9.08547.83

≤ 90 C,≤60 Minutes

Filter LC/MS

35

40

45

50

55

60

65

70

75

Rel

ativ

e A

bund

ance

4.95395.65 7.15

464.70 9.17501.28

3.49441.13

14.92683.01

12.17657.02

10.53490.561.96

538.2613.62672.57

0 2 4 6 8 10 12 14 16 18 20 22Time (min)

0

5

10

15

20

25

3017.23802.27

19.36813.3115.38

812.42 21.321398.410.63

515.64 20.05823.40

17.78821.23

22.6974.7

• Complete digest of native protein in ≤ 60 minutes• Pretreatment (reduction and alkylation) – optional

E i t i d th l l• Equipment required – thermal cycler• Multiple patents pending

12

Time for Digestion

Overall time for digestion is fasterActual hands on preparation time isActual hands on preparation time is drastically reducedLess repetitive manual steps reducesLess repetitive manual steps reduces inaccuracy

Sample preparation

Protein assay

Digestion

SPE + dry/reconstituteSpin down

13

Three Individual mAb Digestions Using a UHPLC Vanquish System and an Acclaim C18 2 x 250 mm, 2 q y ,µm Column

14

Slide 14

JH5 Please format in title-case text and remove bold formatting.Jim Hegarty, 15/10/2014

Overlay of Different mAb Digests on the Vanquish System

15

Overlay of 10 Runs of a Trypsin Digest mAb Sample

16

Overlay of 13 Runs of a Trypsin Digest mAb with Retention Time Precision

162 5

175.0

187.5

201.5mAU

peak # RT (min)

RT-RSD (%)

125.0

137.5

150.0

162.5(%)3 3.315 0.0829 5.231 0.065

14 6.532 0.01715 6.937 0.023

75.0

87.5

100.0

112.519 10.290 0.02123 12.013 0.01231 14.011 0.01339 15.177 0.01242 15 589 0 010

25 0

37.5

50.0

62.5

42 15.589 0.01051 17.511 0.00755 17.969 0.01161 18.546 0.01083 20.798 0.010

-12.5

0.0

12.5

25.0

85 21.095 0.01287 22.386 0.00996 24.774 0.012103 26.155 0.009106 26 155 0 009

2.5 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 31.3-45.2

-37.5

-25.0

min

106 26.155 0.009109 27.529 0.010

17

Slide 17

JH8 Please format with title-case text.Jim Hegarty, 15/10/2014

pH gradient Ion Exchange with the Vanquish System

• 5 commercial mAbs• Bevacizumab, Cetuximab, Infliximab, Rituximan, and Trastuzumab, , , ,

• Targetg• Separate main charge variant• Focus on very fast separation and method optimizationy p p

• Generic linear gradient method using Thermo Scientific™g gCX-1 pH buffers (pH 5.6 to 10.2)

• Method speed up based on generic linear gradient

18

Next-Generation Dionex MAb IEC Chemistry – Application to Beat

Lysine variantsYYYY 4x250 mm MabPac SCX-10

60 min. Total Analysis Time

Lysine variants

YYKK

YYKAcidic variants

YYKLysine

Basic variants

YYKK

yvariants

ProPac WCX-10, 4x250 mm

20 0mAUAcidic variants

10.0

15.0

20.04x150 mm MabPac SCX-1015 min. Total Analysis Time

Acidic variants

Basic variants

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

0.0

5.0

min

2MAbPac SCX-10, 4x250 mm

19Improve resolution or sample throughput through chemistry

Slide 19

JH10 Please format with title-case text and use font size that conforms to the PPT template.

Jim Hegarty, 15/10/2014

Important Points to Consider

• Speed of analysisSpeed of analysis• Resolution• Time to develop the method• Time to develop the method• Global applicability

R b t• Robustness

20

Mechanism of Salt and pH Elution of Proteins

21

Salt Versus pH Gradient IEC of mAb Sample

10.0

15.0

30.0

5.0

0.0 5.0 10.0 15.0 20.0 25.0 30.0

0.0

min%B: 10.0

Salt gradient

10 0

15.0

50.0

5.0

10.0

25 0

30 i di t MAbP SCX 10 10 4 250

0.0 5.0 10.0 15.0 20.0 25.0 30.0

0.0

min%B: 25.0

25.0pH gradient

22

30 min gradient, MAbPac SCX-10, 10 µm, 4×250 mm

MAb Charge Variant Separation, 0–100% B

100% B0% B

40.0 10.50pH trace(a)

20 0

30.0

8 00

9.00

ce [m

AU

]

(a)

10.0

20.0

7.00

8.00

Abs

orba

nc

0 5 10 15 20 25 30 35 40-5.0 5.00

6.00

0 5 10 15 20 25 30 35 40

Retention Time [min]

The pH trace at elution was obtained with the Thermo Scientific™ Dionex™ UltiM t ™ 3000 H d C d ti it M it i M d l (PCM 3000)

23

UltiMate™ 3000 pH and Conductivity Monitoring Module (PCM-3000)

MAb Charge Variant Separation, 25–50% B

25% B 50% B

16.0 8.00

10 0

7.75

mAU

]

(c) pH trace

5.0

10.0

7.25

7.50

bsor

banc

e [m

5.0

7.00

Ab

0 5 10 15 20 25 30 35 40-2.0 6.60

Retention Time [min]

24

Generic Linear pH Gradient with the Vanquish System

MAB 1

MAB 2

MAB 3MAB 3

MAB 4

MAB 5

25 pH 5.6 to 10.2 in 10 minutes

Slide 25

JH14 Please format with title-case text.Jim Hegarty, 15/10/2014

Example 1/5: Cetuximab – Vanquish Fast Gradients

5 min gradient

10 min gradient

3 steps method development1. 10 minutes 0100% B in 10 minutes2 1035% B i 5 i t

2.5 min gradient

2. 1035% B in 5 minutes3. 1035% B in 2.5 minutes

Number of charge variants and resolution maintained for

26

2.5 min gradient

Example 2/5: Infliximab – Vanquish Fast Gradients

10 min gradient

5 min gradient

3 steps method development1. 10 minutes 0100% B in 10

minutes

0.8 min gradient

minutes2. 2040% B in 5 minutes3. 1827% B in 0.8 minutes

Number of charge variants and resolution maintained for b i t di t

27

sub-minute gradient

Conclusions

• Very fast separation of mAb charge variants can be achievedVery fast separation of mAb charge variants can be achieved with the Vanquish UHPLC System through initial global methodology onto rapidly developed optimized methods• Run-time comparable or faster than cIEF

• First intact protein UHPLC Ion Exchange application successfully implemented using the Vanquish UHPLC System

28

Structure of IgG and Typical Forms of Heterogeneity

29

Hydrophobic Interaction Chromatography (HIC)

• Principle

• Adsorption to a weakly hydrophobic surface at high salt concentrations

• Elution by a decreasing salt gradient

• Advantages

• Preserves biological activity of proteins during purification and native conformation

• Reversed phase solvents often denature, especially the most hydrophobic proteins

• Ideal mode after salt precipitations and ion exchange• Ideal mode after salt precipitations and ion exchange

• Excellent first step in purification due to its high capacity and low carryover

• Primary application• Primary application

• Purification of proteins and peptides

30

Thermo Scientific™ HIC Column Selection Guide

Resin Type Particle Size Pore Size

MAbPac HIC 10 Silica 5 µm 1000 Å

Resin data

MAbPac HIC-10 Silica 5 µm 1000 Å

MAbPac HIC-20 Silica 5 µm 1000 Å

MAbPac HIC-Butyl Polymer 5 µm Nonporous

ProPac HIC-10 Silica 5 µm 300 Å

Application guideGeneral Proteins

Intact mAbs

mAbAggregates

mAbFragments

Oxidized Variants

ADCCys & Lys

MAbP HIC 10 E ll t E ll t E ll t G d G d G d

Application guide

MAbPac HIC-10 Excellent Excellent Excellent Good Good Good

MAbPac HIC-20 Good Good Good Excellent Excellent Good

MAbPac HIC-Butyl Good Good Poor Good OK Excellent

ProPac HIC-10 OK OK Poor Good Good Poor

31

ProPac HIC 10 OK OK Poor Good Good Poor

Separation of Proteins

80

Column: MAbPac HIC-10, 5 µmFormat: 4.6 ×100 mm4

60

Mobile phase A: 2 M ammonium sulfate, 100 mMsodium phosphate, pH 7.0

Mobile phase B: 100 mM sodium phosphate, pH 7.0

Gradient:Ti ( i ) %A %B

3

40

Time (min) %A %B-5.0 100 00.0 100 01.0 100 0

15.0 0 10020 0 0 100

12

nce

(mA

U)

20

20.0 0 100

Temperature: 30 ºCFlow rate: 1.0 mL/minInj. volume: 10 µL Detection: UV (280 nm)

Abs

orba

n

Detection: UV (280 nm)Sample: Protein mixturePeaks: 1) Myoglobin

2) Ribonuclease A 3) Lysozyme4) α-Chymotrypsinogen A

0 4 8 12 16 20

04) α-Chymotrypsinogen A

32

Retention Time (min)

Loading of Monoclonal Antibody (mAb)

100.0Column: ProPac HIC-10, 5 µmFormat: 4.6 ×100 mmMobile phase A: 0.5 M ammonium sulfate, 0.1 MU

)

70.0

2 0 mg

3.0 mg

Mobile phase A: 0.5 M ammonium sulfate, 0.1 M sodium phosphate, pH 7.0

Mobile phase B: 0.1 M sodium phosphate, pH 7.0 / Acetonitrile (90:10 v/v)

Gradient: 70−100% B in 7 minTemperature: 30 ºCnc

e (m

AU

0 50 mg

1.0 mg

2.0 mg pFlow rate: 0.5 mL/minInj. volume: 10−20 µL Detection: UV (280 nm)Sample: mAb (25 mg/mL)

Abs

orba

n

0.0 0.25 mg

0.50 mgA

0 5 10Minutes

33

Analysis of Intact mAbs

60Column: MAbPac HIC-10, 5 µmFormat: 4.6 ×100 mm

40

50Mobile phase A: 2.0 M ammonium sulfate, 100

mM sodium phosphate, pH 7.0Mobile phase B: 100 mM sodium phosphate, pH

7.0Gradient:

Ti ( i ) %A %B

30

40 Time (min) %A %B-5.0 60 400.0 60 401.0 60 40

15.0 0 10020 0 0 100nc

e(m

AU

)

20

20.0 0 100

Temperature: 30 ºCFlow rate: 1.0 mL/minInj. volume: 2 µL (4 mg/mL)Detection: UV (280 nm)

Abs

orba

n

10

Detection: UV (280 nm)Sample: mAb1

mAb2mAb3mAb4mAb5

0 4 8 12 16 20

0

mAb5

34

Retention Time (min)

Separation of mAb Aggregates

220Column: MAbPac HIC-10, 5 µmFormat: 4.6 ×100 mm

Monomer

150

Mobile phase A: 2 M ammonium sulfate, 100 mMsodium phosphate, pH 7.0

Mobile phase B: 100 mM sodium phosphate, pH 7.0

Gradient:Ti ( i ) %A %B150 Time (min) %A %B-5.0 60 400.0 60 401.0 60 40

29.0 0 10034 0 0 100nc

e (m

AU

)

100 34.0 0 100

Temperature: 30 ºCFlow rate: 0.5 mL/minInj. volume: 10 µL Detection: UV (280 nm)

VariantsAbs

orba

n

50Detection: UV (280 nm)Sample: mAb

Aggregates

5 10 15 20 25

0

35

Retention Time (min)

Separation of mAb Fragments (Papain Digestion)

Column: MAbPac HIC-20, 5 µmFormat: 4.6 ×100 mmMobile phase A: 2 M ammonium sulfate, 100 mM

sodium phosphate, pH 7.0

30a Intact mAb

p p , pMobile phase B: 100 mM sodium phosphate, pH

7.0Gradient:

Time (min) %A %B-5.0 60 400.0 60 401.0 60 40

15.0 0 10020.0 0 100

0

e (m

AU

)

Temperature: 30 ºCFlow rate: 1.0 mL/minInj. volume: Intact mAb: 12 µL (1 mg/mL)

Papain digest: 12 µL (1 mg/mL)Detection: UV (280 nm)S l ) I Ab

30

Abs

orba

nce

b Fab

Fc Sample: a) Intact mAbb) Papain digested mAb

Fc

Papain

0 4 8 12 16 20

0

Retention Time (min)

36

Retention Time (min)

Analysis of Oxidized mAb Variants

60Column: MAbPac HIC-20, 5 µmFormat: 4.6 ×250 mm

Non-oxidized mAb

50Mobile phase A: 2 M ammonium sulfate, 100 mM

sodium phosphate, pH 7.0Mobile phase B: 100 mM sodium phosphate, pH 7.0Gradient:

Time (min) %A %B6 0 50 50

mAb

30

40 -6.0 50 500.0 50 502.0 50 50

30.0 0 10035.0 0 100

e (m

AU

)

Oxidized

variants

20

Temperature: 30 ºCFlow rate: 0.5 mL/minInj. volume: Untreated mAb: 20 µL (1.25 mg/mL)

Oxidized mAb: 20 µL (1.25 mg/mL)Detection: UV (280 nm)

Abs

orba

nce

10

Detection: UV (280 nm)Sample: Untreated mAb

H2O2 oxidized mAb

10 14 18 22 26 30

0

37

Retention Time (min)

Wide Range of Bioanalytical Characterization Methods

38

New Members of the Thermo Scientific™ Orbitrap™ Family

39 The world leader in serving science

Summary: Intact mAb Characterization Workflow

Intact protein/protein complex analysis denatured or native

Top-down or middle-down

mAb and ADCs • Molecular massmAb and ADCsAntibody complexes, aggregates

Molecular mass• DAR of ADC• Binding ratio of complex• Sequence

00

1122 33

4455

66 77

DAR: 2.5 DAR: 2.5 0

12 3

45

6 7

DAR: 2.5

mAb mixtures Any protein therapeutics,

• Structural information• Relative quantification

MS / Native MS00

11 2233 44

55

DAR: 1.5DAR: 1.50

1 23 4

5

DAR: 1.5homogeneous or heterogeneous

40

Monolithic RP Column for Fraction Desalting

Mi i i d i P S ift lithi l41

Minimized carry-over using ProSwift monolithic columns

1 x 50 mm ProSwift Column, Intact Rituximab

90

100 3201.80

3205.31100

50

60

70

80

e A

bund

ance

3198.28

60

70

80

90bu

ndan

cezoom150 ng

10

20

30

40

Rel

ativ

e 3208.84

3212.3810

20

30

40

50

Rel

ativ

e A

b

70

80

90

1000

3201.80

3205.3180

90

1000

10 4750.60

zoom30 ng

30

40

50

60

70

3198.28

3208.84

30

40

50

60

70g

3200m/z

0

10

20

30

3212.38

2000 3000 4000 5000m/z

0

10

20

30

4607.37

42

m/zm/z

mAb (Herceptin) – Direct Infusion Analysis

Intact HerceptinIntact Herceptin Reduced HerceptinReduced Herceptin

Mixture of both 100 3901.5

390 8

1303.0100

Light & Heavy Chain

No LC20

40

60

80

Rel

ativ

e A

bund

ance

3905.83897.2

3910.03893.3

3914.3 Heavy

danc

e

1234.5 1379.7

1205.61100.9 1465.9

1077 5

3880 3900 3920

m/z

0

80

90

100 4118.23901.5

4235.83801.53706.5

4360.43530.0 60

80

100

ndan

ce

5028.2

5022.85030.7

Light Chain

Heavy Chain

Rel

ativ

e A

bun 1077.5

1055.1 1563.5

1033.5 1675.21803.91012.9

993.11954 2

50

50

60

70

80

Abu

ndan

ce

3447.93369.6

4492.43294.8

3223.24632 9

5020 5040

m/z

0

20

40

60R

elat

ive

Abu

n5020.1 5033.4

5017.55038.8

1200 1400 1600

m/z

1954.2974.0923.9

800 20001000

0

30

40

50

Rel

ativ

e A 4632.9

3154.6

4782.34941.7

5490.6

m/z

Intact Protein ModeLight & Heavy Chain

2000 3000 4000 5000 6000/

0

10

20

43

m/z

Accurate Monoisotopic Mass Measurement Using Ultra High Resolution

LC-MS of Antibody Light Chain and Heavy Chain

+23

70

80

90

100

ce

90

100

926.0595z=25

890.4806964 5199

100G0F G1F+45 +45 G1F

+45

80

90

100

1005.5 1006.0 1006.5 1007.0 1007.5m/z

0

10

20

30

40

50

60

Rel

ativ

e A

bund

anc

30

40

50

60

70

80

Rel

ativ

e A

bund

ance

z=26 964.5199z=24

857.5000z=27 1006.4988

z=231102.3079

z=211052.2487z=22

798.5352z=29

1157.3224z=20771.8840

z=30 1218.1823 45

50

55

60

65

70

75

80

85

90

95

elat

ive

Abun

danc

e

+451135.0 1135.2 1135.4 1135.6 1135.8 1136.0 1136.2 1136.4 1136.6 1136.8 1137.0

m/z

0

10

20

30

40

50

60

70

Rel

ativ

e Ab

unda

nce

800 900 1000 1100 1200 1300 1400m/z

0

10

20

30 z=30 1218.1823z=19

1285.9696z=18 1361.4380

z=17

m/z

1126 1128 1130 1132 1134 1136 1138 1140 1142 1144 1146m/z

0

5

10

15

20

25

30

35

40Re

G2F+45

0.7 ppmG0F G1F

G2F

2.3 ppm 1.8 ppm

20.2 ppm

Q Exactive Plus, 280K, protein modeQ Exactive Plus, 140K

44

, , p,

MS of Intact Antibody Drug Conjugate (ADC)

2867.3693

2886.0424

2848.90492904.9884

2924.6023

2860 2880 2900 2920

D1D3100

D2D3

D0

∆M

∆M

D4

D6

D5∆M∆M ∆M

∆M

DAR=3.23

Abun

danc

e

∆M D7∆M

∆M0

mass145000 146000 147000 148000 149000 150000 151000 152000

Q Exactive Resolution Setting = 17 5 K

45

Q Exactive, Resolution Setting = 17.5 K

Comparison of two Different Levels of Antibody-Drug Ratio

Mirror plot of two ADC samples

12

34DAR: 2 5DAR: 2 5

04

56

DAR: 2.5DAR: 2.5

TY 6 7

5TIV

E IN

TEN

SIT

34

5

RE

LAT

01 2DAR: 1.5DAR: 1.5

MASS

46

Native MS Using Exactive Plus EMR

• An ESI compatible volatile solution with near neutral pH is

• Extended mass range up to m/z 20,000p

used to prepare protein samples. • Under these conditions, the

ionized proteins carry fewer

• Unmatched Desolvation using in-source CID and in HCD cell

• Improved high massp ycharges than those produced by ESI in acidic, denatured condition.

Improved high mass transmission for large protein assemblies

• Therefore, in native MS, protein ions are detected at a significantly higher m/z range (>5000 10 000 m/z for antibody

mAb, Acidic pH, Denatured 40

60

80

100

ative Abu

ndan

ce

3276.463350.953071.72

3428.83

3510.48

2948.91

3596.112835.51

3685 95(>5000 -10,000 m/z for antibody complexes) than conventional MS in acidic condition. mAb, Neutral pH, Native

20

40

60

80

1000

20Rela 3685.95

3780.492730.54

3984.74

6410.47

6701.82

5897.61

7020.98

5670.63

47

3000 4000 5000 6000 7000m/z

0

Native MS of Antibody-Antigen Complex

24

mAb/Ag complexes 1:1 mAb:JAM-A

37%

Raw spectrum Deconvoluted spectrum

26+6704.8

24+6260.8

25+6973.1

J10.4 : JAM‐A (1:0)J10.4 : JAM‐A (1:1)J10.4 : JAM‐A (1:2)

Free mAb33%

1:2 mAb:JAM-A

30%174304.4 “ 2.0 Da

150237.1 “ 1.1 Da

198369.6 “ 2.3 DaJAM-AMWtheo 24065.3 Da

J10.4 (G0F/G0F)MWtheo 150234.0 Da

25+6010.4 28+

7085.7

5067.127+

7348.0

J10.4 : JAM A (1:2)

Free antigen + 24065.2 Da+ 24067.3 Da

MWtheo 24065.3 Da

5348.626+

7630.6

4813.9

3000 4000 5000 6000 7000 8000 9000 m/z

Exactive Plus EMR, 35K resolution

48 Yue Xuan et al 2014 ASMS WP265

Native MS of An ADC Brentuximab Vedotin

Raw spectrum Deconvoluted spectrum

DAR4151163.2 “ 0.9 Da

DAR2DAR6

DAR8DAR0 +2635.4 Da

148527.8 “ 0.8 Da

153797.5 “ 0.7 Da

DAR8DAR0

146000144000 Mass (Da)150000148000 154000152000 158000156000

+2634.2 Da +2634.3 Da

+2635.1 Da145893.6 “ 2.2 Da

156432.6 “ 1.8 Da

8

A = 3.8

8

0

0

n

n

DAR

DAR

A

nADAR = 4.2

Exactive Plus EMR, 35K resolution Reference DAR = 4.0 from HIC profile

49 Yue Xuan et al 2014 ASMS WP265

Top-down Sequencing of mAb Light Chain and Heavy Chain

6065707580859095

100

ance

9.40

9.43

Heavy ChainHeavy Chain

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2005

1015202530354045505560

Rel

ativ

e A

bund

a

9.51

9.54

8.68 9.588.64

11.6011.39 11.679.81 12.1112.4313.04 13.48 14.26 18.3814.875.25 17.8216.80 18.8415.53 19.405.66 6.57 6.88 8.154.792.041.68 4.280.56 3.773.21

Light ChainLight Chain

95

100 926.0421795

100 943.70939

926.56803 964.53605912.94204 983 07214

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

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

Rel

ativ

e A

bund

ance

890.435231006.48304

857.54731 1052.15994

826.95971 1102.21393

1157.31923798.43798

1218.24686

1285.76463

1361.37097

1446.36757 1928.219261542 77334

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

Rel

ativ

e A

bund

ance

983.07214

896.92702

1022.39706878.71644

1039.90923

863.82377

1064.91630849.42853

1087.596651132.24767

835.53171

824.69843 1157.98015

1188.64441809.06373 1246.60252

796.420031277.75291

784.14411 1310.472931340.65991 1381 20972

1358.4633R=63229

1381.5299R=61180654 8088

800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

5

10 1542.773341652.91434 1779.96801

1402.74939 1714.18271 1978.920591500.87635 1620.24752

750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400m/z

0

5

101340.65991 1381.20972

1419.60570

Top-Down ETDTop-Down ETD Top-Down HCDTop-Down HCD Top-Down ETDTop-Down ETD Top-Down HCDTop-Down HCD

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100m/z

0123456789

10111213141516171819202122232425

Rel

ativ

e A

bund

ance

2113.6096R=50197

z=2727.4096R=87578

z=11710.8310R=52319

z=?957.7885R=75456

z=3814.4418R=83020

z=1 2053.1886R=48925

z=51203.3486R=65878

z=4

598.3671R=95258

z=11776.6622R=54444

z=4 1995.4757R=51193

z=61436.1799R=63602

z=21577.9014R=60803

z=1976.7954R=75485

z=31922.2693R=52609

z=6

1637.8022R=53772

z=4

1166.3300R=67418

z=41465.1915R=61782

z=21362.7739R=66235

z=51039.5523R=73268

z=11837.2082R=55489

z=8

1287.6322R=59804

z=5

1496.7342R=53712

z=8

2125.5108R=49229

z=4

1095.1932R=71210

z=3708.3556R=91164

z=?943.7979R=76929

z=?

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200m/z

05

101520253035404550556065707580859095

100

Rel

ativ

e A

bund

ance

R 63229z=5

1163.8148R=67305

z=4

1460.9685R=61888

z=41234.6791R=67883

z=11132.2208R=64880

z=6366.1392R=118389

z=1

227.1031R=140795

z=1

1059.6033R=69601

z=2931.2534R=73831

z=51498.9186R=59293

z=5851.4588R=78433

z=3740.4205R=82441

z=3280.1659R=120206

z=1555.3135R=98241

z=1456.2451R=109172

z=1 1657.8372R=54573

z=5

1324.2470R=59037

z=5

1760.8878R=53365

z=7669.0434R=87777

z=3

1560.7824R=46417

z=12

1876.2770R=53228

z=9 2110.8164R=49444

z=?2019.9175R=50055

z=?

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900m/z

05

101520253035404550556065707580859095

100

Rel

ativ

e A

bund

ance

z=6357.1764R=123346

z=1

1237.8547R=62254

z=121176.0188R=64872

z=9

1058.5184R=67410

z=10996.4778R=73386

z=6 1163.4464R=66387

z=71350.3870R=57750

z=11943.9542R=76169

z=6589.2645R=92757

z=1488.2168R=103466

z=1804.3552R=81419

z=1 861.4256R=77459

z=51485.3243R=56303

z=10

1423.3610R=60219

z=3

416.2135R=109524

z=1717.3232R=85684

z=? 1657.6358R=54770

z=5630.3452R=93390

z=1329.1816R=125123

z=1546.2191R=95806

z=11531.7690R=57807

z=51715.6677R=57200

z=11779.8795R=52968

z=?

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

05

101520253035404550556065707580859095

100

Rel

ativ

e A

bund

ance

654.8088R=93049

z=2562.2153R=95417

z=1 780.6289R=80809

z=4 991.7209R=72442

z=4 1126.5516R=69306

z=4943.7977R=75893

z=3

1023.7354R=72418

z=41321.9569R=59790

z=31224.9176R=66411

z=3821.6454R=78519

z=4

901.2427R=76984

z=5

1364.6411R=58634

z=3448.1725R=107575

z=1 698.3248R=86222

z=2

1499.2345R=53814

z=2374.2027R=119210

z=1

1441.0831R=56542

z=5

470.2141R=106556

z=2 1609.7778R=52835

z=2203.0852R=166002

z=1 1726.8511R=57394

z=?1826.3965R=58552

z=?

50

Top-down Sequencing of mAb Light Chain and Heavy Chain

5 ppm mass tolerance ETD fragmentation HCD fragmentation

51 Terry Zhang et al 2014 ASMS WP263

Middle-Down Sequencing of mAb

Intact Fab(50kDa)

F bRICATOR R d ti LC MS/MS

(25kDa)Fab light

Fab heavy

PTM id tifi d d l t d

1gG(150kDa)

Fc(50kDa)

FabRICATOR Reduction LC-MS/MS(25kDa)

Fab heavy

(25kDa)Fc

PTMs identified and located• N –terminal PyroGlu• C-terminal K loss

52

• N-glycosylation

Jie Qian et al 2014 ASMS WP264

Summary: Bottom-up Workflow

EEnzyme Digestion

• Peptide map

• Sequence coverage 1573.7y13

W406 double oxidation, Relative abundance = 0.09%

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900m/z

133.0

173.1

b2

204.1y2

242.2

270.1

326.2b6-H2O++

b6++

361.2b3

y3

381.2

424.2y4

b8++

446.2

y8++

474.2b4

b9++

511.3y5

b10++

571.3b5

y10++b11++

622.3

y6

654.3

y11++

707.4

b13++

735.4

b7

771.3y13++

b14++

827.9y14++

854.4b8

913.4y8

b16++

947.5

y16++

967.5b9

1008.5

1042.5y9

y19++

1095.6b10

1139.0y20++

y10

b21++y21++

1208.6b11

1329.6y11

1444.6y12

1541.7y13

1654.8y14

y15

• Sequence coverage

• Site and type of PTMs

• Site and type of glycosylation120 4.5M[4+]

160 6.0M[3+]

ETD S t

G102 –R129, N103 glycosylation,  Relative abundance = 13.85%

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

145.1

y1

173.1

b2

204.1y2

242.2

270.1

361.2b3

y3

381.2

397.2

424.2y4

474.2b4

b9++

494.3

511.3y5

571.3

622.3

y6

661.2 707.4

735.4

b7 y7

787.3y13++

y14++

854.4b8

895.4

913.4y8

967.5b9

1009.5

1042.5y9

1077.5b10-H2O

1095.6b10

y10

1155.0y20++

1190.6b11-H2O

1208.6b11

1361.6y11 1476.6

y12

1686.7y14

y15

0 00%

80.00%

90.00%

100.00%

• Site and type of glycosylation

• Disulfide mapping

I iti

200 300 400 500 6 00 700 8 00 900 1 000 1100 1 200 1300 14 00 1500 160 0 1700 1800 1900m/z

159.1

z·2

303.2z·3

466.2z·4

z·10++ z·11++ z·12++ z·1 4++

838.9z·15 ++

z'15++88 9.4z·16++

z'1 6++

z·25[3 +]

9 24.5

963.6

1034.0z·18 ++

z'18++z19++z·19++

c·3++c3++z2 0++z·20++z'20++

1113.2

z2 1++z·21 ++

y·21++1266 .5c·7++c7++

c·8++c8++c·9++

13 39.0c9++

c·10++1374.5c10++

c·11++

1439.1c11++

c·24[3 +]c24[3 +]

1484.0

a ·25 [3+]

c·25 [3+]

c2 5[3 +]

c·12++c12++

c·26[3 +]c26[3+]a27 [3+]a·27[3+]

c·27[3+]

c27[3+]

c·13++

c13++

z27[3+]z·27[3+]

z'27[3+]

y·27[3+]

y27[3+]

a28[3+]

a·28[3+]

z2 8[3+]

z·28 [3+]

c14++

1677.8z·15

z'16c16++ c1 7++ c·18++c18++

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900m/z

138.1

(G)

168.1

186.1

204.1(Gn)

366.1(GGn)

528 .2(GGnM)

579.3y5

y12++ (Bn-1)-GGnMy13++707.4

y6

y14++ y7y15++ y16++906.5y8

y26[3+]978 .0y17++

1009 .81042 .5y18++

y19++Y1-F[3+]

1106.5y20++ 1150.0

y21++

Y2-F[3+]

1214 .6y22++

y12

1300 .6y24++ 1344.1

y25++

1437.2y26++

1522.7

1564.2

1624.2Y1-F++

1697.3Y1++

Y2-F++1806.8M1++

M2++

ETD Spectrum 

HCD Spectrum 

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

15min 30min 60min 90min 120min Unquenched

M49+Oxidation

M49+Double Oxidation

N60+Deamidation

M304+Oxidation

M393+Oxidation

• Impurities

• Sequence variants

53

• Qualitative and quantitative

Resolution with Q Exactive Plus vs Q Exactive HF

Transient Resolution ResolutionTransient Length

ResolutionQ Exactive / Q Exactive Plus

ResolutionQ Exactive HF

32 ms na 15 k64 ms 17 5 k 30 k64 ms 17.5 k 30 k

128 ms 35 k 60 k256 ms 70 k 120 k256 ms 70 k 120 k512 ms 140 k 240 k

1024 280 k* **1024 ms 280 k* na*** available for Q Exactive Plus with configured license ꞌEnhanced Resolutionꞌ** 1 sec long transient is not available for Q Exactive HF

54

1 sec long transient is not available for Q Exactive HF

Peptide Maps of Herceptin Digest; 6−40 min Gradients

100

6/16 min6/16 min

time

0100

0100

8/16 min8/16 min

Abun

danc

e

0%B

/ to

tal r

un t100

0100

11/28 min11/28 min

15/28 min15/28 min

Rel

ativ

e A

adie

nt ti

me

to 3

0

0100

15/28 min15/28 min

20/38 min20/38 min

Gra0

100

0100

30/48 min30/48 min

0 05 10 15 20 25 30 35 40 45 50 55 60

100

0

40/60 min40/60 min

55

0 05 10 15 20 25 30 35 40 45 50 55 60

Time (min)

Can Use Shorter Gradients

Complete Characterization of Glycopeptides Using HCD and ETD

C441-R449, N448 glycosylationC441-R449, N448 glycosylation

Relative abundance = 0.52%

• Unique HCDpdETD method features on-the-fly identification of glyco-peptides

798.3M[4+]

1064.1M[3+]

1596.2M++

ETD Spectrum ETD SpectrumETD Spectrum

ETD fragmentation HCD fragmentation

using diagnostic fragment ions from sugar fragmentation.

• A high quality HCD spectrum is178.1

c1279.1

c2

366.1c3

494.2c4

631.3c5 845.0 913.1

z6[3+]

966.7

a·9[3+]z9[3+]z·9[3+]

1214.01267.5

z4++z·4++

z5++z·5++z'5++y5++

z6++

1413.1z·6++

z'6++

y·6++y6++

1450.1

z7++z·7++

z'7++y7++ a·8++z8++

z·8++

b·8++

c·8++

c8++

a·9++

1574.7

z9++

z·9++

• A high quality HCD spectrum is generated for each peptide.

• An additional ETD spectrum is

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z

138.1

204.1(Gn)(Gn)

274.1

366.1(GGn)

HCD SpectrumHCDHCDgenerated for each glycopeptide.

• For each glycopeptide, ETD provides information of peptide sequence and

168.1

186.1

292.1(S) 666.3

Y1-F++

767.9Y2-F++ 848.9

M1++

929.9M2++

1002.9M2F++

M3M3F++

1331.6-GGnM++

1477.7Y1

HCD Spectrum HCD SpectrumHCD Spectrum

information of peptide sequence and site of glycosylation - HCD provides information of glycan structure and additional peptide sequence

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

528.2(GGnM)

(SGGn)(Bn-1)-SGGnMY1++b7

Y2++

M1F++

M3++

A1G0M2++

1084.5

A1G0M2F++

A1G1M2++ 1186.0-SGGnM++

A1G1++ 1267.0-SGGn++

A2G1++ -G-F++1534.7

Y2-FY21696.8

M1M1F1858.8

M2

56

additional peptide sequence.

Zhiqi Hao et al 2014 ASMS TP264

Trend Toward HR/AM MS for Intact IgG Mass Measurement e.g. Glycoform Analysis

80859095

100 2745.7720

2851.3544

75

80

85

90

95

100 2745.7720

2797.56972695.8919

R: 17.5K

4045505560657075

ativ

e A

bund

ance

2907.25952556.4844

2965.37642471.3405

3025 86322391 628825

30

35

40

45

50

55

60

65

70

Rel

ativ

e A

bund

ance

510152025303540

Rel 3025.86322391.6288

2680 2700 2720 2740 2760 2780 2800 2820m/z

0

5

10

15

20

25

1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000m/z

0

-7 ppmG0F+G1F

-0 7 ppm 8 5G0F+G0F G0F+G2F (or 2G1F)

0.7 ppm -8.5 ppm

-0.9 ppm

Protein Deconvolution 1.0

G1F+G2FG2F+G2FG0+G0F

G0+G02xMan5

5.0 ppm0.9 ppm

Q ExactiveO bit MS

57

G1F+G2F+SA

In-depth characterization, comparability studies

Orbitrap MS