Material Measurement Laboratory

Mass Spectral Database of Glycans and Glycopeptides in Therapeutic DrugsMaria Lorna A. De Leoz, Xinjian (Eric) Yan, Xiaoyu (Sara) Yang, Lisa Kilpatrick, Yuxue Liang, Michael Tarlov, Stephen Stein

Therapeutic Monoclonal Antibodies are Widely Used in Medicine

• Used as treatment for cancer and several other diseases

• $20B market/year• >30 approved mAbs• 6/10 best-selling drugs, 2012

2mAbs – monoclonal antibodies

Therapeutic Monoclonal Antibodies Are Glycosylated

• Glycans are attached to the protein molecules

• Changes in glycosylation may affect drug safety and efficacy

• Glycosylation highly heterogeneous

• Glycan analysis remains a formidable challenge

Adapted from Oxford Glycobiology Institute

Glycans

Glycans

Fab

Fc

Monoclonal Antibody Therapeutic Protein

3

Protein denaturation

Glycoprotein

N-glycan releaseN-glycan releaseDecantate

Precipitate

Ethanol precipitation

Solid phase extractionLiquid chromatographyMass spectrometry

N-GLYCANS ARE RELEASED FROM PROTEINS

De Leoz, M.L.A.; Young, L.J.T.; An, H.J.; Kronewitter, S.; et al., Mol Cell Proteomics 2011; 10, M110.002717.De Leoz, M.L.A.; Young, L.J.T.; An, H.J.; Kronewitter, S.; et al., Mol Cell Proteomics 2011; 10, M110.002717.

N-GLYCANS ARE ANALYZED BY MASS SPECTROMETRY

• Can analyze using ion trap or orbitrap MS• Sample injected through LC or direct infusion • Several MS/MS activation methods

• Higher-energy C-trap Dissociation (HCD)• Collisionally-Induced Dissociation (CID by ion trap or orbitrap)• Electron Transfer Dissociation (ETD)

• High mass resolution • Max at 125,000 at m/z 400 (i.e. it can distinguish peaks at m/z

400.0000 and 400.0032)• High mass accuracy

• < 5 ppm error routinely

ESI-LTQ Velos/Orbitrap MS

5

*

*

*

* *

* **

10% ACN (+)

20% ACN (+)

40% ACN (-)

Rela

t ive Inte

ns i

t y

Hexose (Hex) N-acetylhexosamine (HexNAc) Fucose (Fuc) Sialic Acid (NeuAc)

De Leoz, M.L.A.; Young, L.J.T.; An, H.J.; Kronewitter, S.; et al., Mol Cell Proteomics 2011; 10, M110.002717.De Leoz, M.L.A.; Young, L.J.T.; An, H.J.; Kronewitter, S.; et al., Mol Cell Proteomics 2011; 10, M110.002717.

N-Glycan Profiling of Serum Glycoproteins

STARTERGLYCAN MS/MSLIBRARY

NGA42

G0NGA2F1

G0F

NGA3B1,2

G0BNA2G1F1

G1F

A1F1

G2FSNA2F1

G2F

A2F1

G2FS2NGA2B2

G0B

NGA21

G0NGA4B2

G0B

1Purchased; 2Kindly provided by the Consortium for Functional Glycomics through Dr. Jim Paulson

Glycan Consensus Spectrum

Y4, Y4

B4

[M+Na]+

Y3, Y3

Y4/Y4B3, B4/Y4, B4/Y4

0,2A5

0,2A40,2A4/Y40,2A4/Y4

B4/Y3

B4/Y3

B3/Y4, B3/Y4

B3/Y3, B3/Y3

-H2O

2,4A5

[M+2Na]2+-(2H2O+CH2O)

B4

Y4 Y4

Y3Y3 B3

Mannose N-acetylglucosamine

[M+2Na]2+

HCD, 55eV

Glycopeptide Consensus Spectrum

b2y2 y4 b5 b6

[M+2H]2+

-(2H2O+CH2O)

(TKPREEQYNSTYR)=p

y1

0,2A1

oxonium

oxonium

oxonium

p-NH3

-H2O

-H2O -H2O -H2O p-NH3

-NH3

2,4A1

m/z 963.417, z=3HCD, 62eVp p p

p p

p

pp

p

Glycan from Rituximab Matches Library

Rituximab

Glycan Library

Head to Tail Comparison