imppprovement in full workflow capabilities to provide ...€¦ · imppprovement in full workflow...
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Improvement in Full Workflow Capabilities to Provide p pIncreased Reproducibility, Speed and Throughput for the Characterization of BiosimilarsMike OliverSample Preparation and Accucore LC Product ManagerThermo Fisher Scientific
The world leader in serving science
Thermo Fisher Scientific
Thermo Scientific™ Biopharma Peptide Analysis Workflows
Discovery
Development HRAM Identification& Relative Quan
SMART Digest™ KitsSOLAµ™ Plates
Vanquish UHPLC ™ SystemsAccucore Vanquish™ Columns
Q Exactive™ Plus SystemsQ Exactive™ HF Systems
QAQC
Targeted AbsoluteSOLAµ Plates Accucore Vanquish Columns Q Exactive™ HF SystemsLCMS
Targeted Absolute Quan
QAQC
SMART Digest KitsSOLAµ Plates
Vanquish SystemsAccucore Vanquish Columns
ChromeleonChromatogrpahy Data SystemUHPLC
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Thermo Scientific™ Peptide Workflow Challenges
• Sample preparation:• Multifaceted & complex• Multifaceted & complex • Time consuming• Issues with reproducibilityIssues with reproducibility
• Separation & Detection:• Highly complex samplesg y p p• High resolution separations• High levels of reproducibility• Low abundant peptides/PTM’s• High throughput workflows• Compliance to regulation
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Peptide MappingPeptide Workflow Challenges
RT: 0.00 - 60.00
90
10025.9920.752.12
23.1733.83
19.32
• Peptide Map• PTMs
Peptides
Digestion
0
10
20
30
40
50
60
70
80
Rel
ativ
e A
bund
ance
26.7418.64
16.57 27.00 38.57
39.1139.20
14.24
2.37 31.7413.53 36.553.7912.77
4.208.48
8.4029.92 39.638.72 51.17
48.516.25 9.22 40.72 48.03 52.50 5
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDNKALPAPIEKTISKAKGQ
• Impurities
Intact Ab B tt 0 5 10 15 20 25 30 35 40 45 50 5Time (min)
0 PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Intact Ab Bottom up
S /PTMSequence/PTMs unknown or need to be confirmed Peptide identification
by MS and MS/MSFast analysis
Method Transfer to LC-UV
Sequence and PTMs known. No further information
Peptide identification by unknown and reference sample
High degree of confidence on No further information
required Stability studies, QA/QC, batch release)
reference sample chromatogram comparison (retention time comparison)
retention time determination is
required!
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How to Address These Challenges?
The world leader in serving science
Simple Digestion
Simple & easy to implement
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Method development
T= 15 minT 15 min
T= 30 min From 60 minutes
T= 45 min
T= 60 min
u esonwards the peak area is constant
T= 60 min
T= 75 min
Digestion time optimisation for IgG in one hour!
T= 90 min
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Digestion time optimisation for IgG in one hour!
Reproducible Digestion
Overlay of two BSA digest done by two different users
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Reproducible clean up and pre-concentration
• SOLAµ Fritless Design provides:• Less chance of blocking• Less chance of blocking• Better flow through characteristics• High reproducibilityHigh reproducibility• 20 fold pre-concentration
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Cetuximab
The world leader in serving science
Biopharma Peptide workflow
Discovery
Development PepFinderSoftware
SMART Digest Kits Vanquish H SystemsAcclaim PepMap
Q Exactive HFS t SII for Xcalibur
QAQC
Acclaim PepMapRSLC Columns
Systems SII for XcaliburSoftware
• Flow rate 0.05 - 1.5 ml/min• A: water + 0.1% FA• B: 20% water + 80% ACN +
0.1% FA
Cetuximab (drug product)
• 5 mg mL-1 Cetuximab/Erbitux ®
• 146 kDa
• MS1: R=30kMass range: 200-2000 m/zAGC target: 3e6Maximum IT: 100 ms
• MS2: R=15k• Gradient: 4-55% B in
5/8/10/15/30 min• Column temperature: 60º C• Acclaim PepMap RSLC,
• From Eli Lilly
SMART Digest
• MS2: R=15kMass range: 140 -2000 m/zAGC target: 1e5Maximum IT: 100 msNCE: 27%p p ,
1 x 150 mm, C18, 2 µm, 100Å2.1 x 250 mm, C18, 2.2 µm, 120Å
• Injection volume: 1 – 3 µl
• 1:4 dilution with Digest Buffer• Digestion: 60 min, 70°C, 1400 rpm• Post Digest Reduction: 5 mM TECP
, 30 min, 60°C, 1000 rpm
• Calibrated with classical positive CalMix
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Erbitux is a registered trademark of Eli Lilly.
201.5mAU
Cetuximab - High Reproducibility
150.0
162.5
175.0
187.5
100.0
112.5
125.0
137.5
62.5
75.0
87.5
100.0
12.5
25.0
37.5
50.0
-37.5
-25.0
-12.5
0.0
Acclaim™ RSLC 120 column (RP18, 2.2 µm, 2.1 x 250 mm) digested with SMART™ digest kit.
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
37.5min
• Cetuximab – SMART digestionCetuximab SMART digestion• 13 overlaid chromatograms• High reproducibility
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• High reproducibility
Cetuximab - High Reproducibility
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Cetuximab – Increased speed and throughput
8.0e9
counts
3 8e10
4.3e10
counts
4.0e9
6.0e9
1500 µL/min 5 min
3.3e10
3.8e10
1.00 1.50 2.00 2.50 3.00 3.50 4.00
2.0e9
min
1100 µL/min 5 min
2.3e10
2.8e10
1000 µL/min, 10 min
1.3e10
1.8e10
400 µL/min
600 µL/min, 20 min
2.5e9
7.5e9
300 µL/min 30 min
400 µL/min 30 min
2.50 3.75 5.00 6.25 7.50 8.75 10.00 11.25 12.50 13.75 15.00 16.25 17.50 18.75 20.00 21.25 22.50 min
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SMART digestVanquish AND Smart Digest - High Reproducibility
Smart Digest reduced vs. unreducedDisulfide Bridge assignment S-S linked peptides
200
250
6.0e9
8.0e9
4nm
C
VDAD vs. Q Exactive HF
50
100
150
0.0e0
2.0e9
4.0e9
UV@
214
TIC
15
4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 min
Characterization of Disulfide linkage
• 2 Samples:• Tryptic digest under reducing conditionsTryptic digest under reducing conditions• Tryptic digest under non-reducing conditions
• Comparison of the two samples with PepFinder and• Comparison of the two samples with PepFinder and characterization of the disulfide bridges in the molecule.
• Goal: To Characterize the native disulfide bonds.Result: The light chain is coupled to the HC in three• Result: The light chain is coupled to the HC in three different ways (LC/HC 1-3), so there is just one effective internal S-S bridge and one unlinked Cys.
S‐S link Protein Peptide Charge Confidence Time m/z Avg Mass Monoisotopic Mass
LC2 2: Cetuximab light chain 2:S127‐R142/2:H189‐K207 = 3820.903m[1ss] LC2 6 0.994476 16.4239 638.158 3822.9 3820.90283
HC2 1: Cetuximab heavy chain 1:S136‐K149/2:V191‐K207 = 3079.532m[1ss] HC2 5 0.994716 13.5157 617.114 3080.53 3079.53271
HC3 1: Cetuximab heavy chain 1:C326‐K327/1:T261‐K279 = 2328.098m[1ss] HC3 4 0.999336 13.2676 583.28 2328.93 2328.10547
HC4 1: Cetuximab heavy chain 1:N366‐K375/1:W422‐K444 = 3844.823m[1ss] HC4 6 0.995241 15.0965 642.145 3846.83 3844.82861
LC/HC1 1: Cetuximab heavy chain 1:S224‐K227/2:S127‐R142 = 2189.024m[1ss] LC/HC1 4 0.989217 19.0262 548.514 2190.03 2189.02417LC/HC1
LC/HC2 1: Cetuximab heavy chain 1:S224‐K227/2:V191‐K207 = 2267.056m[1ss] LC/HC2 4 0.982561 9.17067 568.02 2268.05 2267.05127
LC/HC3 1: Cetuximab heavy chain 1:S224‐K227/2:V19‐R24 = 1146.480m[1ss] LC/HC3 3 0.998258 5.81022 383.167 1146.48 1146.47827
Hi 1 C t i b h h i 1 T228 K253/1 T228 K253 5454 783 [2 ] hi 7 0 979177 23 715 780 691 5457 79 5454 78564
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Hinge 1: Cetuximab heavy chain 1:T228‐K253/1:T228‐K253 = 5454.783m[2ss] hinge 7 0.979177 23.715 780.691 5457.79 5454.78564
Rituximab
The world leader in serving science
Biopharma Peptide workflow
Discovery
Development PepFinderSoftware
SMART Digest Kits Vanquish Flex SystemsA l i RSLC C l
Q Exactive HFS t SII for Xcalibur
QAQC
Acclaim RSLC Columns Systems SII for XcaliburSoftware
• Flow rate: 0.3 mL min-1
• A: water + 0.1% FA• B: 20% water + 80% ACN +
0.1% FA
MabThera® (drug product)
• 10 mg mL-1 rituximab 147 kDa
• From F. Hoffmann-La Roche Ltd
• MS1: R=30kMass range: 200-2000 m/zAGC target: 3e6Maximum IT: 100 ms
• MS2: R=15k• 2 – 45% B in 30 min• Column temperature: 50º C• Injection volume: 1 µL• Thermo Scientific™ Acclaim™ 120
(Basel, Switzerland)
SMART Digest
• 1:4 dilution with Digest Buffer
• MS2: R=15kMass range: 140 -2000 m/zAGC target: 1e5Maximum IT: 100 msNCE: 27%
• Thermo Scientific Acclaim 120 C18 2.2 µm 120Å (2.1 x 250 mm) column
• Digestion: 75 min, 70°C, 1400 rpm• Post Digest Reduction: 5 mM TECP
, 30 min, 60°C, 1000 rpm
• Calibrated with classical positive CalMix
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MabThera is a registered trademark of F. Hoffmann-La Roche, Ltd.
Method Transfer: LC-UV-MS to Robust and Reliable LC-UV
MS-TIC
UV@214 nm
Overlaid chromatograms of the Total Ion Current (TIC) and the UV trace at 214 nm of a SMART digested Rituximab sample
5.0 10.0 15.0 20.0 25.0 30.0 min
UV@214 nm
digested Rituximab sample.
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Peak assignment with PepFinder™ of the tryptic peptides from Rituximab.
Advantage of HRAM for Peptide Mapping
Wrong Answer for Both Peptides Right Answer for Both Peptides
RP = 15,000 RP = 56,700
516.77581 (observed)
nsity
(%)
nsity
(%)
Inte
Inte
(correct) (correct)
Mass [amu]
Mass [amu]
Peptide mixture: [Val5]-Angiotensin II Lys-des-Arg9-BradykininSequence: DRVYVHPF KRPPGFSPF Formula: C49H69N13O12 C50H73N13O11Exact mass: [M+2H]2+ = 516.76671 [M+2H]2+ = 516.78490Dm (mmu): 18.2 mmu
20 Joshua J. Coon, et al. ASMS 2012 oral, MOB pm
Peptide Mapping with LC-MS
The Sequence Coverage Map shows the overlap of the different
tid id tifi d i diff tpeptides identified in different intensities and in different lengths due to missed cleavages with a sequence coverage for heavy and light chain of 99.2%.g
Protein Modification Recovery AbundanceRituximab_LC Q1+NH3 loss Good 87.81%Rituximab_LC W90+Oxidation Good 2.06%Rituximab_HC ~Q1+NH3 loss Good 100.00%Rituximab_HC W281+Oxidation Good 4.98%Rituximab_HC N301+A1G0F Fair 2.87%Rituximab_HC N301+A1G1F Fair 1.22%Rituximab_HC N301+A2G0 Fair 1.30%Rituximab_HC N301+A2G0F Fair 37.69%
The Modification Summary shows the confidently identification and relative quantification of a subset of
Rituximab_HC N301+A2G1F Fair 44.86%Rituximab_HC N301+A2G2F Fair 10.77%Rituximab_HC N301+M5 Fair 1.07%Rituximab_HC N365+Deamidation Good 2.72%Rituximab_HC W385+Oxidation Good 5.37%
i i b d
qmonitored modifications on light and heavy chain of Rituximab respectively. The selected modifications are deamidations, oxidations, pyro-Gln formations on the N-terminus of heavy and light chain, glycosylation of the N301 on the heavy chain and sequence variants like C terminal Lys (K+ variant)
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Rituximab_HC G450+Lys Good 3.2683% and sequence variants like C-terminal Lys (K+ variant).
Rituximab Peptides Reduced and Non-Reduced
150
188 _ _mAU WVL:214 nm
42.7
%C: 0.0 %
Reduced peptides
100
125
50
75
0
25
2
1
-50
-25
-100
-75
-168
-125
min
Flow: 400 µl/min
%B: 10.0 % Di-Sulphides
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3.9 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.3168
Summary
The world leader in serving science
Biopharma Peptide Workflows
Discovery
Development
SMART Digest KitsSOLA Pl t
Vanquish SystemsA l i RSLC C l
Q ExactiveS t
InformaticsS ft
QAQC
SOLAµ Plates Acclaim RSLC Columns Systems SoftwareLCMS
• Simplified & Faster sample preparationHi hl d ibl & f t ti• Highly reproducible & fast separations
• Increased throughput• Confidence in results
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Bio-Pharma Peptide Workflows
Discovery
Development
SMART Digest KitsSOLA Pl t
Vanquish SystemsA l i RSLC C l
Q Exactive PlusS t
QAQCInformaticsS ft
Acknowledgments:
SOLAµ Plates Acclaim RSLC Columns Systems Software
Acknowledgments:• Ken Cook, Bio-Separations Support Expert• Carsten Paul HPLC Solution Specialist Germering• Carsten Paul, HPLC Solution Specialist, Germering• Martin Samonig, Application Scientist, Germering
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Questions?
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