Announcment: Integration of SimGlycan 2 into Waters Glycan ... · ©2010 Waters Corporation | COMPANY CONFIDENTIAL Xevo G2 Technical Note ©2010 Waters Corporation. Printed in the

©2010 Waters Corporation | COMPANY CONFIDENTIAL

Announcment: Integration of SimGlycan 2 into Waters Glycan/Glycopeptide Analysis Workflows


Informatics for Released Glycan and Glycopeptide Mass Analysis

Acquire LC/MS/DDAMS or MALDI MS/DDAMS

SimGlycan® assigns the likely structure of glycans and glycopeptides using mass spectrometry data.

MS/MS data (mass and intensity) is searched against a glycan database (8500+ Entries)

A scored list of the most probable structures is returned.


SimGlycan Searching


SimGlycan Result Browsing


SimGlycan Structure Window


SimGlycan Fragmentation Viewer


Translating New Technologies into ImprovedWorkflows for Biotherapeutic Analysis

Scott J. Berger

Biopharmaceutical Sciences

Waters Corporation


Salt Lake City: The Best Place to Preach to the Choir


Applying multiple analytical workflows to understand a candidate biosimilar drug

=

Candidate biosimilar

drug

Innovator drug


Antibodies represent the next biotherapeutic class open to European biosimilar competition


Combinatorial Heterogeneity


Collaboration

Company X is recognized for a proven track record in generic small molecule pharmaceuticals.

They wished to expand to produce biosimilar antibodies.

They collaborated with Waters to exploit UPLC and TofMS technologies to support this new effort.

They were expecting only minor differences from the innovator company mAb.

Graphic: http://en.wikipedia.org/wiki/File:Antibody2.JPG

= ?


Desalting LCMS Intact Mass Spectra for Biosimilar and Innovator Samples

Innovator mAb

“Biosimilar” mAb


Zoomed Mass Spectra of Intact AntibodyT

Differences observed between Biosimilar mAb and Innovator mAb


Intact mAb data (BiopharmaLynx 1.2)

Innovator mAb


Result: Biosimilar has altered glycoform profile, shifted by ~ -62 Da


LCMS Analysis of reduced antibodies

Result: Light Chains are identical

Innovator mAb



Deconvoluted Spectra of the Heavy Chain

Result: Biosimilar Heavy chains -32 Da shift, consistent with intact data

Innovator mAb



LC-FLR-MS analysis of 2-AB labeled released glycans

Biosimilar glycans were qualitatively consistent with the innovator mAb


LCMSE Peptide Map Comparison

Innovator mAb


Result: No gross differences between antibody maps


Difference Due to Unknown Peak in Check Sample

Changes in TIC mirror plot correlate with

detected component results

Peptide T34-35 (missed cleavage) detected in innovator, missing in

biosimilar, mass 1903.98

32.02 Da Mass Difference

New peak in biosimilar of mass 1871.96


Unknown peak in biosimilar, -32 Da difference from T35

T35 (EEMTK)

LCMSE Peptide map difference also found for T35 (but not T34)

Result: Difference mapped to T35 peptide


Sorting out the T35 differences

Drugbank Herceptin™ HC T35 sequence is DELTK — Drugbank database entry DB00072

— Ref: www.drugbank.ca/drugs/DB00072

Genentech Herceptin™ HC T35 sequence is EEMTK— Mass is 32 Da heavier.

— Consistent with the marketed product— REF: Journal of Chromatography B, 752 (2001) 233–245; Identification of multiple

sources of charge heterogeneity in a recombinant antibody. Harris et al.


MSE Spectra confirm innovator mAb T35 and T34-35 as Herceptin sequences

T35 (EEMTK), RT = 3.86 min, Control Sample

T34-35 (EPQVYTLPPSREEMTK), RT=32.21 min, Control Sample


MSE Spectra confirm “biosimilar” mAb T35 and T34-35 as incorrect Drugbank sequences

T35 (DELTK), RT=6.81 min

T34-35 (EPQVYTLPPSRDELTK), RT = 34.90 min


Sorting out the T35 differences

Drugbank Herceptin™ HC T35 sequence is DELTK — Drugbank database entry DB00072

— www.drugbank.ca/drugs/DB00072

Genentech Herceptin™ HC T35 sequence is EEMTK— Mass is 32 Da heavier.

— Consistent with the marketed product— REF: Journal of Chromatography B, 752 (2001) 233–245; Identification of multiple

sources of charge heterogeneity in a recombinant antibody. Harris et al.

Result: Wrong protein sequence was cloned and expressed by Company X.

Impact: The biosimilar candidate is not the same protein as the innovator and would be considered a new drug. $$$


For Further Details

mAbs 2:4, 1-16; July/August 2010;© 2010 Landes Bioscience

Thursday Poster 026 (Proteins: Recombinant)

MS analysis reveals differences in a humanized mAb protein drug and a biosimilar version

Martin Gilar, Hongwei Xie, Asish B. Chakraborty, Deepalakshmi P. Dakshinamoorthy, Weibin Chen, Ying-Qing Yu, StJohn Q. Skilton, Scott J. Berger, Jeffrey R. Mazzeo


How does the Xevo G2 QTof improve biotherapeutic characterization workflows?

QuanTof: low ppm mass accuracy for peptides over >104

dynamic range — Load more sample to see lower level variants

QuanTof: Improved quantitation over dynamic range

QuanTof + Higher Resolution: routine detection of larger peptides— Disulfide-linked peptides

— Missed Cleavage

— Digests: AspN, LysC, GluC QuanTofTM

Low ppm MMA over >104 Dynamic

Range

TOF >20,000 RESOLUTION

QuanTofTM

Low ppm MMA over >104 Dynamic

Range

TOF >20,000 RESOLUTION


Xevo G2 Technical Note

©2010 Waters Corporation. Printed in the U.S.A.May 2010 720003494 EN

• Spiked in a SIL peptide at 1:10,000 vs. protein digest

• Detect both at low ppm mass accuracy

• BiopharmaLynx detected the spiked in peptide


Xevo G2 Application Note



Xevo G2 Technical Note


20,000

15,000

10,000

5,000

2,000

Peptide Mass

0.001

0.018

0.3

5.7

31

Monoisotopic peak (% of total)1

0.01

0.14

2.1

26

93

Relative Monoisotopic Peak

Height2

10000

714.2

47.6

3.8

1.1

Isotopic EnvelopeDynamic Range3

20,000

15,000

10,000

5,000

2,000

Peptide Mass

0.001

0.018

0.3

5.7

31

Monoisotopic peak (% of total)1

0.01

0.14

2.1

26

93

Relative Monoisotopic Peak

Height2

10000

714.2

47.6

3.8

1.1

Isotopic EnvelopeDynamic Range3

The largest peptide in most antibody tryptic peptide maps: HC T15

(DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH

KPSNTK). Mass 6712.3071 Da.

~9:1~9:1

Dynamic Range Required ~1400


Direct Comparison of Xevo G2 vs Xevo QTof (same load)

Waters Xevo G2 Qtof MS Waters Xevo Qtof MS

Hybrid ADC Detection TDC Detection

Resolution mode ~24,000 for (GluFib)2+ (measured)

Resolution mode ~12,000 for (GluFib)2+ (measured)

Dynamic range >104 Dynamic range >103

Roughly Equivalent Peptide Sensitivity

UPLC/MSE Peptide Map25 pmol Trastzusumab

Thursday Poster 040 (Proteins: Recombinant)

Comparison of detector technologies for LC/QTof MSE biotherapeutic protein peptide mapping studies.

Jeff Mazzeo; Weibin Chen ; Scott Geromanos; Scott Berger


Electrospray ionization (not MS detection) primarily controls peptide MS response

0

10

20

30

40

50

0 10 20 30 40 50hADC Intensity Rank (Fully Tryptic Unmodified Peptides)

TD

C I

nte

nsit

y R

an

k (F

ully T

rypti

c U

nm

odif

ied

Pe

pti

des

)

Most Intense -> Least Intense

Most In

tense

-> Le

ast In

tense


Detector improvements = Better mass accuracy

0

5

10

15

20

25

-10 -8 -6 -4 -2 0 2 4 6 8 10 12 140

2

4

6

8

10

12

14

-10 -8 -6 -4 -2 0 2 4 6 8 10 12 14

Std Dev 1.8 ppm

Std Dev 3.5 ppm

hADC TDC

No.

of

PEPT

IDES

Mass Accuracy (ppm)


Comparable Coverage and Variant Detection (Heavy Chain)

Description: HCControl coverage: 99.6%Protein: Traz_HC

1:1 EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR1:51 IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG1:101 GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK1:151 DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT1:201 YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP1:251 KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN1:301 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ1:351 VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV1:401 LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGDescription: HC

Control coverage: 99.1%Protein: Traz_HC

1:1 EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR1:51 IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG1:101 GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK1:151 DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT1:201 YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP1:251 KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN1:301 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ1:351 VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV1:401 LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG

hADC

TDC

PEPTIDE = ID confirmed with 3+ MSE Fragments

= Alkylated Cys, MetOx, Deamidated N C M N


hADC

TDC

Equivalent MSE fragmentation detections

0

5

10

15

20

25

30

35

THTCPPCPAPELLGGPSVFLFPPKPK

TPEVTCVVVDVSHEDPEVK

TVAAPSVFIFPPSDEQLK

GPSVFPLAPSSK

EVQLVESGGGLVQPGGSLR

VVSVLTVLHQDWLNGK

TTPPVLDSDGSFFLYSK

VYACEVTHQGLSSPVTK

SGTASVVCLLNNFYPR

FNWYVDGVEVHNAK

ASQDVNTAVAWYQQKPGK

DIQMTQSPSSLSASVGDR

STSGGTAALGCLVK

DSTYSLSSTLTLSK

GFYPSDIAVEWESNGQPENNYK

SGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTK

FTISADTSK

VDNALQSGNSQESVTEQDSK

NQVSLTCLVK

WQQGNVFSCSVMHEALHNHYTQK

LSCAASGFNIK

SLSLSPG

TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK

GLEWVAR

AEDTAVYYCSR

DTYIHWVR

DTLMISR

ALPAPIEK

YADSVK

NTAYLQMNSLRADYEK

FSGSRVTITCR

WGGDGFYAMDYWGQGTLVTVSSASTKVQWK

TISK

EPQVYTLPPSRLTVDK

QAPGKVEPK

SFNR EYKEEMTK

GQPRVEIK

LLIYSASFLYSGVPSR VDKVSNK

SCDKTKPR APK

IYPTNGYTRGEC

EEQYNSTYR

hADC BYTDC BY

No.

of b

- and

y-io

n d

ete

ctio

ns

Peptide


Beyond Single Protein Characterization


Analysis of Multivalent Flu Vaccine

Hongwei Xie2, St John Skilton2; Joseph Rininger1; Weibin Chen2, Joomi Ahn2, Yinqing Yu2.

1: Protein Sciences 2: Waters Corporation

SIMULTANEOUS CHARACTERIZATION AND QUANTIFICATION OF HEMAGLUTININ PROTEINS IN AN

INFLUENZA VACCINE CANDIDATE WITH AN LC-MSE ASSAY

Session: Thursday P02, Proteins: Recombinant Poster Number: 041

HA1

HA3


Analysis of Flu Vaccine using Proteomics Workflows in PLGS

1 MTQFTDIDKL AVSTIRILAV DTVSKANSGH PGAPLGMAPA AHVLWSQMRM 51 NPTNPDWINR DRFVLSNGHA VALLYSMLHL TGYDLSIEDL KQFRQLGSRT 101 PGHPEFELPG VEVTTGPLGQ GISNAVGMAM AQANLAATYN KPGFTLSDNY 151 TYVFLGDGCL QEGISSEASS LAGHLKLGNL IAIYDDNKIT IDGATSISFD 201 EDVAKRYEAY GWEVLYVENG NEDLAGIAKA IAQAKLSKDK PTLIKMTTTI 251 GYGSLHAGSH SVHGAPLKAD DVKQLKSKFG FNPDKSFVVP QEVYDHYQKT 301 ILKPGVEANN KWNKLFSEYQ KKFPELGAEL ARRLSGQLPA NWESKLPTYT 351 AKDSAVATRK LSETVLEDVY NQLPELIGGS ADLTPSNLTR WKEALDFQPP 401 SSGSGNYSGR YIRYGIREHA MGAIMNGISA FGANYKPYGG TFLNFVSYAA 451 GAVRLSALSG HPVIWVATHD SIGVGEDGPT HQPIETLAHF RSLPNIQVWR 501 PADGNEVSAA YKNSLESKHT PSIIALSRQN LPQLEGSSIE SASKGGYVLQ 551 DVANPDIILV ATGSEVSLSV EAAKTLAAKN IKARVVSLPD FFTFDKQPLE 601 YRLSVLPDNV PIMSVEVLAT TCWGKYAHQS FGIDRFGASG KAPEVFKFFG 651 FTPEGVAERA QKTIAFYKGD KLISPLKKAF

LC/MSE

Data

Match to: H1A Score: 589

MSE: Discovery and Characterization of Glycopeptides

35.9153095120487Hemaglutinin A3


100.0407981353873Hemaglutinin B

Hi3 sumHi3 sumIntensity Measure

Relative Resp%

Inj2Inj1Injection Number



100.0407981353873Hemaglutinin B

Hi3 sumHi3 sumIntensity Measure

Relative Resp%

Inj2Inj1Injection Number

Quantification of Subunits(3:2:1)


Identifying Unknown Process Impurity Proteins (HCP’s)

1 MTQFTDIDKL AVSTIRILAV DTVSKANSGH PGAPLGMAPA AHVLWSQMRM 51 NPTNPDWINR DRFVLSNGHA VALLYSMLHL TGYDLSIEDL KQFRQLGSRT

101 PGHPEFELPG VEVTTGPLGQ GISNAVGMAM AQANLAATYN KPGFTLSDNY 151 TYVFLGDGCL QEGISSEASS LAGHLKLGNL IAIYDDNKIT IDGATSISFD 201 EDVAKRYEAY GWEVLYVENG NEDLAGIAKA IAQAKLSKDK PTLIKMTTTI 251 GYGSLHAGSH SVHGAPLKAD DVKQLKSKFG FNPDKSFVVP QEVYDHYQKT 301 ILKPGVEANN KWNKLFSEYQ KKFPELGAEL ARRLSGQLPA NWESKLPTYT 351 AKDSAVATRK LSETVLEDVY NQLPELIGGS ADLTPSNLTR WKEALDFQPP 401 SSGSGNYSGR YIRYGIREHA MGAIMNGISA FGANYKPYGG TFLNFVSYAA 451 GAVRLSALSG HPVIWVATHD SIGVGEDGPT HQPIETLAHF RSLPNIQVWR 501 PADGNEVSAA YKNSLESKHT PSIIALSRQN LPQLEGSSIE SASKGGYVLQ 551 DVANPDIILV ATGSEVSLSV EAAKTLAAKN IKARVVSLPD FFTFDKQPLE 601 YRLSVLPDNV PIMSVEVLAT TCWGKYAHQS FGIDRFGASG KAPEVFKFFG 651 FTPEGVAERA QKTIAFYKGD KLISPLKKAF

LC/MSE

Data

ProteinLynx Global Server or MASCOT or Other Bioinformatics Tools

.PKL Export File

Match to: Protein_X Score: 189

Unassigned Components


Creating a database of HCP’s and MRM transitions for HCP monitoring

Thursday Poster 042 (Proteins: Recombinant)Quantitative Analysis of Host Cell Proteins in Biologics Derived from Different Cell Lines and Purification Schemes by a LC-MS Assay Catalin Doneanu*, Manbir Labhan1, Alex Xenopoulos2, Holly Prentice2, Keith Fadgen*, Weibin Chen*, Martha Stapels*, St John Skilton*, William Haskins1 and Jeff Mazzeo** Waters 1 University of Texas, San Antonio, TX2 Millipore Corporation, Bedford, MA

LC/MSE analysis for ID and absolutequantification of HCP contaminantsin biopharmaceuticals

Samples: IgG after Protein A

2D RP/RP LC enabled HCPsdetections down to 10 ppm

Transition to high-throughput MRMassay for monitoring/quantification

Goal: Creating a database of HCPs and MRM transitions for monitoring

them


Characterizing Therapeutic Protein 3D Structures by Electrospray Ion-Mobility Mass Spectrometry

- Biological Significance and Comparison with X-ray Crystallography and NMR Measurements

Weibin Chen, Asish Chakraborty, Iain Campuzano, Scott Berger, StJohn Skilton and Jeff Mazzeo

ASMS 2010 Session MOD_4:10 PM Room 155

Qualitative Analysis of Protein Therapeutics by MS

May 24, 2010


Instrumentation


Electrospray Mass Spectra of Human Insulin

1162.339 2324.084

5+

4+

3+

5+7+

3+/ 6+


IMS separation of monomeric insulin from dimeric insulin

637 Å2

1231 Å2

3+

6+

IMS Separation ofmonomeric insulin from dimeric insulin

3+

6+

Drift


Analysis of Human Insulin Analogs by Synapt G2 HDMS

Apidra

Lantus Novolog Levemir

Humulin Humalog


ESI spectra of INF α2b before and after high temperature stress

INF α2b at pH 7.0

INF α2b at pH 7.0Incubated at 55oC for 20 min

Indistinguishable by MS


Probing conformation changes of Interferon under high temperature stress

Drift Time (ms)

Inte

nsi

ty

INFα2b @ pH 7.0INFα2b after

Incubated for 20 min@ 55oC

Dimer

70◦C For 30 min

From A. Diress et al. J. Chromatogr. A 1217 (2010) 3297–3306

Mass Selected Arrival Time Distribution (5+)

IFN α2bStd

SEC


Development of a Hyphenated Ion Mobility –Mass Spectrometry Technique for the

Characterization of Glycosylated Peptides

Craig Dorschel, Jim Langridge, Scott Geromanos

ASMS 2010 Session WOD_2:50 PM Room 155

MS of Glycoproteins

May 26, 2010


Mobility, m/z Selection:Gas Phase Fractionation by Ion Mobility

050510_UAA114_Fetuin_04.raw : 1

Glycopeptides have high m/z,

Large collisional cross section,Long drift time,

High charge states

Ions in this region have these characteristics


Mobility, m/z Maps for Other Glycoproteins

050510_UAA114_HERcn_01.raw : 1

050510_UAA114_a1AGP_02.raw : 1050510_UAA114_Fetuin_04.raw : 1

050510_UAA114_TRFE_01.raw : 1

Fetuin α1-Acid Glycoprotein

Transferrin Trastuzumab


Fetuin – Peak Assignments

T12-13N-Link

T13 N-Link& T3

T2-3N-terminalProcessed

T8N-Link

T21O-Link

T14N-Link


Fetuin – Glycoforms Found

T12-13: KLCPDCPLLAPLNDSR— Hex6HexNAc5NeuNAc3

T13: LCPDCPLLAPLNDSR— Hex5HexNAc4NeuNAc2

— Hex6HexNAc5NeuNAc3


T8: RPTGEVYDIEDTLETTCHVLDPTPLANCSVR— Hex5HexNAc4NeuNAc2





T8 Hex5HexNAc4NeuNAc2

5+ Charge StateRs = 31,000

RT

Drift Time

m/z

Glycoform 1:RT = 39.36 min.MH+ = 5876.533Drift Time = 6.59Intensity = 16776

Glycoform 2:RT = 39.34 min.MH+ = 5876.537Drift Time = 5.72Intensity = 3751


Conclusions

Innovations in analytical workflows have enabled the routine application of LCMS for biotherapeutic characterization

— UPLC and Chemistries: Reproducibility, Resolution, Speed

— QTof MS: Universal detection, Engineered simplicity, Dynamic range, Mass accuracy, Mass stability, MSE

— Informatics: Biopharmaceutical and Proteomic Analysis

The combination of routine and advanced workflows can reveal exquisite detail about complex biotherapeutic products.

— Better development decisions can be made faster

— New drugs can reach market sooner

— Expensive mistakes can be corrected earlier

Documents

Announcment: Integration of SimGlycan 2 into Waters Glycan ... · ©2010 Waters Corporation | COMPANY CONFIDENTIAL Xevo G2 Technical Note ©2010 Waters Corporation. Printed in the