Using Native Mass Spectrometry Combined with

Orbitrap Technology to Decipher Micro- and Molecular

Heterogeneity of Intact Antibodies

Natalie J. Thompson Biomolecular Mass Spectrometry and Proteomics

Utrecht University N.J.Thompson@uu.nl

CASSS MS Symposium 25 September 2013

Antibodies as Biotherapeutics

• IgG-based Abs used to target many diseases resulting in anti-cancer, anti-neurodegenerative disease, anti-infection, and anti-toxin treatments

• Advantages – High target specificity – Slow clearance

• Over 30 mAbs have been introduced for clinical use since

1986 – Humira (anti-TNFa) – Remicade (anti-TNFa) – Rituxan (anti-CD20) – Herceptin (anti-HER2) – Avastin (anti-VEGF)

EvaluatePharma 2013 Report 2

Increasing Complexity of Therapeutic Antibodies

Mixtures of Antibodies

3

Post-Translational Modifications Complex Glycosylation

Antibody-Drug Conjugates

Increasing Complexity of Therapeutic Antibodies

4

Sam

ple

Com

plex

ity

Mass Differences

Glycosylation

PTMs

Ab Mixtures

ADCs

Native Mass Spectrometry for Protein Analysis

5

peptides small molecules

1 kDa 10 kDa 100 kDa 1 MDa 18 MDa

(not to scale)

small proteins antibodies viruses large assemblies

• Study of intact protein complexes in the gas phase – “Gentle” ionization from a

volatile buffer – Theoretically infinite mass

analyzer • Accurate masses of intact

protein complexes • Advantages

– Non-covalent complexes retained in the gas phase

– Condensed signal leads to simpler interpretation

– Sample preparation is straightforward

Heck, A.J.R. Nature Methods 2008, 5(11), 927-933

100

100 0

0

Inte

nsity

In

tens

ity

Native (ammonium acetate buffer)

Denatured (5% FA)

2000 4000 6000 8000 m/z

Native MS of Intact Ab Characterization

6 Thompson, N.J. et al. Methods 2013, DOI: 10.1016/j.ymeth.2013.05.003

Native MS on Modified Orbitrap Instrument

Rose, R. J. et al. Nat. Methods 2012, 9(11), 1084-1086 Rosati, S. et al. Angew. Chem. Int. Ed. 2012, 51(52), 12992-12996

7

i) Ion storage in HCD cell ii) Manual control of gas pressure and composition iii) Altered transmission voltages iv) Application in-source dissociation energy

Resolving Power with Native Mass Spectrometry

5*dill pb1881p08 7*exch in 150mM AmAc pH 7.5

m/z5998 5999 6000 6001 6002 6003 6004 6005 6006 6007 6008 6009 6010 6011 6012 6013 6014 6015 6016 6017 6018 6019 6020 6021 6022 6023 6024 6025 6026 6027 6028 6029 6030 6031 6032 6033 6034 6035

%

0

100

6000 6025 m/z

Inte

nsity

To get 50% mass resolution: Δmmin = 146 Da

RP = 1200

Low resolving power due to inefficient desolvation rather

than to TOF mass analyzer

8

Improving Resolving Power

9

Δm = 42 Da Δm = 21 Da

Experimental Approaching Theoretical

10

Improved desolvation

Longer transient time

Bare ions are detected yielding supreme mass accuracy

High Mass Accuracy Reveals Unanticipated PTM

• N-terminal pyroglutamate formation

• Mass error reduced to average of 7 ppm

• Mass error falls into three equidistant categories

• Miscalculation of theoretical mass

11

High Technical Reproducibility

12

Theoretical Mass (Da)

Exp1 Mass (Da)

Exp2 Mass (Da)

Exp3 Mass (Da)

Average Mass (Da)

Mass Error (Da)

144381.8356 144382.9688 144382.6094 144383.7813 144383.12 0.60 144871.5653 144873.4531 144871.8438 144871.9531 144872.42 0.90 145361.2950 145362.7969 145362.8438 145362.7656 145362.80 0.04 145531.2412 145530.9063 145532.5313 145531.75 145531.73 0.81 145573.5124 145575.5000 145573.5469 145575.000 145574.68 1.01 145673.5278 145672.7969 145673.2969 145672.7031 145672.93 0.32 146020.9709 146022.5313 146022.2813 146021.0781 146021.96 0.78 146063.2421 146063.5938 146063.6719 146064.25 146063.84 0.36 146163.2575 146163.2656 146162.3125 146163.8125 146163.13 0.76 146680.6468 146680.5625 146680.5938 146680.375 146680.51 0.12 146722.9180 146722.6250 146721.7813 146722.0156 146722.14 0.44 146765.1892 146766.7031 146766.3906 146766.3594 146766.48 0.19 146822.9334 146821.4219 146822.3438 146821.9844 146821.92 0.46 146865.2046 146864.8281 146863.4219 146864.375 146864.21 0.72 146965.2200 146962.6563 146963.3594 146962.3281 146962.78 0.53

For baseline-resolved Abs:

Quantitative deviation <1.2%

Mass precision <1.4 Da (7.5 ppm)

CH3 Mutations Affect Stoichiometry

Rose, R. J. et al., mAbs, 2013, 5(2), 219-228 13

0.1 μM

1 μM

10 μM

R409K WT F405Q

IgG4Δhinge Glycosylation

100

0

50

abun

dance

146300 146500 146700 146900

100

0

50

abun

dance

73200 73400 73600 73800

14

CH3 Mutations Affect Stoichiometry AND Glycosylation

15

4040 4090 4140m/z

4040 4090 4140m/z

Y407K Y407A 4040 4090 4140

m/z 4040 4090 4140

m/z 4040 4090 4140

m/z

Y407Q Y407E

Glycosylation pattern of WT IgG4Δhinge

18+

most variants

↑ 1-3 sialic acid ↑ galactose ↑ triantennary

Rose, R. J. et al., mAbs, 2013, 5(2), 219-228

Highly Complex Glycosylation Profiles IgG4Δhinge mutants

5000m/z

0

20

40

60

80

100

Rel

ativ

eAb

unda

nce

Y407A+18

4040 4060 4080 4100 4120 4140m/z

30001000 7000 9000

+18

4040 4060 4080 4100 4120 4140 4160 4180m/z

0

20

40

60

80

100R

elat

ive

Abun

danc

e

Y407E

5000m/z

30001000 7000 9000

Y407Q

0

20

40

60

80

100

Rel

ativ

eAb

unda

nce

+18

4040 4060 4080 4100 4120 4140 4160m/z

5000m/z

30001000 7000 9000

+18

4040 4060 4080 4100 4120 4140 4160m/z

Y407K

0

20

40

60

80

100

Rel

ativ

eAb

unda

nce

5000m/z

30001000 7000 9000

16

Mass Differences Used to Determine Terminal Sugars

17

87.54%

12.46%

Y407A

83.37%

16.63%

Y407K

86.61%

13.39%

Y407Q

90.41%

9.59%

Y407E

GLY TRUNCATION non-truncated

truncated

Quantitation of Modifications

28.61%

25.80%

3.15%

42.44%

Y407E

18.60%

12.03%

0.58% 68.78%

Y407K

28.96%

25.23%

2.27%

43.19%

Y407Q

9.03% 7.71%

0.37%

82.89%

Y407A

SIALYLATION 3 sialic

no sialic

1 sialic

2 sialic

18

desialydase

Selective Deglycosylation Confirms Assignment

19

Simplified Sample Prep with HRMS

20

For simple Ab mixtures, deglycosylation is

unnecessary

Antibody-Drug Conjugates (ADCs) ADCETRIS®

(Brentuximab vedotin)

21

02468

DAR:

deglycosylated

abun

danc

e

Increasing Sample Complexity: Glycosylated ADCs

5400 5600 5800 6000 6200 6400 6600 6800m/z

0

10

20

30

40

50

60

70

80

90

100

abun

dan

ce

02468

DAR:

ADCETRIS®

(Brentuximab vedotin)

22

Conclusions

• Resolution between closely related Abs – Baseline resolution of Δm = 42.271 Da – Resolution of Δm = 20.941 Da

• Unequivocal identification – Mass accuracy of average 7 ppm – Mass precision < 1.4 Da

• Reproducible quantitation – Deviation less than 1.2 %

• Analysis of mixtures containing non-covalent and covalent interactions – Characterization of glycoforms in combination with

antibody:antigen or ADC heterogeneity 23

Acknowledgements • Utrecht University

– Albert J.R. Heck – Hans Kamerling – Sara Rosati

• ThermoFisher Scientific

– Eugen Damoc – Eduard Denisov

• Merus

– Mark Throsby – John de Kruif – Linda Kaldenberg

• Genmab

– Ewald van den Bremer – Rob de Jong – Janine Schuurman – Paul Parren

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