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

2

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

3

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!

4

How to Address These Challenges?

The world leader in serving science

Simple Digestion

Simple & easy to implement

6

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

7

Digestion time optimisation for IgG in one hour!

Reproducible Digestion

Overlay of two BSA digest done by two different users

8

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

9

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

11

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

13

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

18

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.

19

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)

21

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

22

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

24

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

25

Questions?

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