Announcment: Integration of SimGlycan 2 into Waters · PDF fileAnnouncment: Integration of...
Transcript of Announcment: Integration of SimGlycan 2 into Waters · PDF fileAnnouncment: Integration of...
©2010 Waters Corporation | COMPANY CONFIDENTIAL
Announcment: Integration of SimGlycan 2 into Waters Glycan/Glycopeptide Analysis Workflows
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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.
©2010 Waters Corporation | COMPANY CONFIDENTIAL
SimGlycan Searching
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SimGlycan Result Browsing
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SimGlycan Structure Window
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SimGlycan Fragmentation Viewer
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Translating New Technologies into ImprovedWorkflows for Biotherapeutic Analysis
Scott J. Berger
Biopharmaceutical Sciences
Waters Corporation
©2010 Waters Corporation | COMPANY CONFIDENTIAL
Salt Lake City: The Best Place to Preach to the Choir
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Applying multiple analytical workflows to understand a candidate biosimilar drug
=
Candidate biosimilar
drug
Innovator drug
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Antibodies represent the next biotherapeutic class open to European biosimilar competition
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Combinatorial Heterogeneity
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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
= ?
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Desalting LCMS Intact Mass Spectra for Biosimilar and Innovator Samples
Innovator mAb
“Biosimilar” mAb
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Zoomed Mass Spectra of Intact AntibodyT
Differences observed between Biosimilar mAb and Innovator mAb
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Intact mAb data (BiopharmaLynx 1.2)
Innovator mAb
“Biosimilar” mAb
Result: Biosimilar has altered glycoform profile, shifted by ~ -62 Da
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LCMS Analysis of reduced antibodies
Result: Light Chains are identical
Innovator mAb
“Biosimilar” mAb
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Deconvoluted Spectra of the Heavy Chain
Result: Biosimilar Heavy chains -32 Da shift, consistent with intact data
Innovator mAb
“Biosimilar” mAb
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LC-FLR-MS analysis of 2-AB labeled released glycans
Biosimilar glycans were qualitatively consistent with the innovator mAb
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LCMSE Peptide Map Comparison
Innovator mAb
“Biosimilar” mAb
Result: No gross differences between antibody maps
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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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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.
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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. $$$
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
Xevo G2 Application Note
©2010 Waters Corporation. Printed in the U.S.A.May 2010 720003493 EN
©2010 Waters Corporation | COMPANY CONFIDENTIAL
Xevo G2 Technical Note
©2010 Waters Corporation. Printed in the U.S.A.May 2010 720003531 EN
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
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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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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)
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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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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
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Beyond Single Protein Characterization
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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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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
64.7258089234507Hemaglutinin A1
100.0407981353873Hemaglutinin B
Hi3 sumHi3 sumIntensity Measure
Relative Resp%
Inj2Inj1Injection Number
35.9153095120487Hemaglutinin A3
64.7258089234507Hemaglutinin A1
100.0407981353873Hemaglutinin B
Hi3 sumHi3 sumIntensity Measure
Relative Resp%
Inj2Inj1Injection Number
Quantification of Subunits(3:2:1)
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
Instrumentation
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Electrospray Mass Spectra of Human Insulin
1162.339 2324.084
5+
4+
3+
5+7+
3+/ 6+
©2010 Waters Corporation | COMPANY CONFIDENTIAL
IMS separation of monomeric insulin from dimeric insulin
637 Å2
1231 Å2
3+
6+
IMS Separation ofmonomeric insulin from dimeric insulin
3+
6+
Drift
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Analysis of Human Insulin Analogs by Synapt G2 HDMS
Apidra
Lantus Novolog Levemir
Humulin Humalog
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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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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
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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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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
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Fetuin – Peak Assignments
T12-13N-Link
T13 N-Link& T3
T2-3N-terminalProcessed
T8N-Link
T21O-Link
T14N-Link
©2010 Waters Corporation | COMPANY CONFIDENTIAL
Fetuin – Glycoforms Found
T12-13: KLCPDCPLLAPLNDSR— Hex6HexNAc5NeuNAc3
T13: LCPDCPLLAPLNDSR— Hex5HexNAc4NeuNAc2
— Hex6HexNAc5NeuNAc3
— Hex6HexNAc5NeuNAc4
T8: RPTGEVYDIEDTLETTCHVLDPTPLANCSVR— Hex5HexNAc4NeuNAc2
— Hex6HexNAc5NeuNAc2
— Hex6HexNAc5NeuNAc3
— Hex6HexNAc5NeuNAc4
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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