©2015 Waters Corporation 1 Mass Spectrometry work flows for Biopharmaceuticals Denis Calnan Biopharma Business Development Manager (Northern Europe)
©2015 Waters Corporation 1
Mass Spectrometry work flows for Biopharmaceuticals
Denis Calnan Biopharma Business Development
Manager (Northern Europe)
©2015 Waters Corporation 2
Aims
Waters dedicated Biopharma LCMS workflows
Characterisation of Biotherapeutics
UNIFI Practical data examples - mAb
©2015 Waters Corporation 4
Antibody compared to Asprin
©2015 Waters Corporation 5
Antibody characterisation
©2015 Waters Corporation 6
Biopharmaceutical Applications for Protein Characterization
Protein Characterization
Intact/Subunit MS
Top Down
Peptide Mapping
Released Glycan Analysis
Protein Separation Charge Variant Aggregation…
Higher Order Structure
HDX
Host Cell Protein Analysis
Adv
ance
d M
S A
naly
sis
Wor
kflo
ws
©2015 Waters Corporation 7
Application Solution
Application Solution
Application Specific
ASR
Methods & Library Content
User Training
Engineer Training
Biopharmaceutical Platform Solution
H-Class/ Bio + TUV/ FLR
Xevo G2-XS Qtof UNIFI v1.8 ASR
©2015 Waters Corporation 8
MS Products Positioning for BioPharm
Routine LCMS Platform
Analysis of all biotherapeutic primary structure attributes
Automated streamlined workflows
Compliant Ready
Biopharmaceutical Platform Solution with UNIFI
Option
Research LCMS Platform
• Analysis of all biotherapeutic primary structure attributes
• HCP analysis with 2D ACQUITY M-Class inlet and HDMSE.
• HDX workflow with HDX inlet and HDMSE and ETD.
Process Only
Research Platform with SYNAPT G2-Si (HDMS)
©2015 Waters Corporation 9
Routine Mass Detection for Biopharma
Peptide Mapping & Synthetic Peptides
RFMS Labeled Glycans
Detergents & Formulants
Oligonucleotides
ADC Free Drug Analysis
Subunit Analysis
©2015 Waters Corporation 10
The Waters Biopharmaceutical System Solution
XEVO G2XS QTof
UNIFI Scientific Information System
An analytical platform for UPLC/UV and UPLC/MS biotherapeutic analysis deployable across unregulated and GxP
laboratories
TUV & FLR Detectors
ACQUITY UPLC H-Class BIO
BioSeparations Chemistries
©2015 Waters Corporation 11
Built on decades of analytical data and informatics experience.
©2015 Waters Corporation 12
1. Focus on Routine Characterization Efficiencies of automation and workflow
2. Workgroup Benefits Data/Instrument Access and Management
3. High resolution analytics across an organization NonCompliant, GxP, Compliant-Ready, Qualification, QC?
4. Reporting, Reporting, Reporting Goal is to communicate results, not collect/process data
5. Universal Interface for Chromatography and MS Reduce Training Costs and Increase Accessibility
Unifi 5 Key Messages
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Intact Mass Analysis
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LC/MS Intact Mass Analysis of Antibody on Xevo G2-XS
+50 Charge state
Mass Spectrum
1 µg on column
MaxEnt1 Deconvoluted Mass Spectrum
A fast, simple, and highly sensitive method for intact mass assessment
G0F/G0F
G0F/G1F
G1F/G1F
G1F/G2F
G2F/G2F
Improved feature/glycoform
definition
Glycoform Mass Error (ppm)
G0F/G0F 0.7
G0F/G1F 0.0
G1F/G1F 4.0
G1F/G2F 8.0
G2F/G2F 20.0
©2015 Waters Corporation 15
UNIFI: Intact Mass Analysis (Compare Mode)
©2015 Waters Corporation 16
Simple Intact Antibody LCMS Analysis Report
©2015 Waters Corporation 17
Peptide Mapping
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Confirming primary structure – data acquisition
Digested sample Generic UPLC® gradient Generic MSE method
Control: 011009_32.raw
0.0302 19.9835Retention Tim e (m ins)
5 10 15
% (m
ax =
673
61.0
Cou
nts)
0
25
50
75
100
1:T73*11.84 1:T79
13.921:T71*9.87
1:T829.38
1:T578.55
1:T2413.12
1:T66*14.95
1:T55*7.95
1:T43*5.57
1:T396.68
1:T25*7.27
1:T11*3.30
1:T54*6.50
1:T67*5.67
1:T46*4.45
1:T5210.67
1:T65*1.89
1:T133.64
1:T79*11.631:T72
3.041:T80*0.87
1:T9*17.36
12.19
19.7718.0216.76
T
Min Intensity Threshold : 0 counts
Comprehensive unbiased MS/MS acquisition of exact masses of all precursor ions and fragment ions from a single injection
©2015 Waters Corporation 19
UPLC/MSE Comprehensively Analyzes Complex Samples
UPLC/MSE is a simple method of unbiased data acquisition that comprehensively analyzes all components in a single analysis.
©2015 Waters Corporation 20
Low energy MS spectrum
MSE
Collision energy ramp 10-40 V
Underlying spectra
©2015 Waters Corporation 21
Xevo G2-XS QTof
The increased sensitivity and XS collision cell provide better fragment ions for peptide sequence
conformation.
0.9 pmol on column
LC/MS Peptide Mapping of NIST mAb NIST mAb
Denature & Alkylate
Trypsin Digest
LC/MSE
Peptide Map
Time (min)
Coverage Map
Fragment Spectrum Low abundant Oxidized Peptide T1-2 (0.9%)
Fragment Spectrum high abundant Peptide T1-2
©2015 Waters Corporation 22
LCMSE Peptide Map Comparison
Innovator mAb
“Biosimilar” mAb
Result: No gross differences between antibody maps
©2015 Waters Corporation 23
Peptide Mapping: Zeroing in on Biosimilar Protein Differences
New Peak in BIOSIMILAR sample at m/z 1872.96
Peptide T34-35 (missed cleavage) in INNOVATOR not in BIOSIMILAR
INNOVATOR (TIC)
BIOSIMILAR (TIC)
INNOVATOR (spectrum)
BIOSIMILAR (spectrum)
©2015 Waters Corporation 24
Glycoprotein Characterization Multiple Strategies – Complementary Information
©2015 Waters Corporation 25
RapiFluor-MS: Enhanced Workflows for Glycan Monitoring
©2015 Waters Corporation 26
What is new from Waters? Novel RapiFluor-MSTM (RFMS) Reagent
Patent Pending
©2015 Waters Corporation 27
16 hrs
Conventional
3 hrs
0.1 hrs
(Pre-Labeling Clean-Up) (Drying) 1-3 hrs
(Drying) 2 hrs
<15 min
5 min
30 min
GlycoWorks RapiFluor-MS N-Glycan Kit
10 min
Glycoprotein
Released N-Glycans + Protein
Labeled N-Glycans + Reagents
Labeled Glycans
LC-FLR LC-FLR-MS
Deglycosylation
Labeling
Clean-up
Analysis
Reaction Byproducts
Simplified Sample Preparation
>24 HOURS Patent Pending
©2015 Waters Corporation 28
1: TOF MS ES+BPI
6.93e6
Conventional 2-AB Glycan from 10 µg of Protein 1.5 Day Sample Prep
RapiFluor-MS Glycan from 0.1 µg Protein <1 hr Sample Prep
10 20 min
800 1000 1200 1400 1600
800 1600 m/z
1130.921, 2+
Xevo G2-XS QTOF
FA2G2Ga1
Direct Sensitivity Comparison using UPLC/FLR/Xevo G2-XS QTOF
©2015 Waters Corporation 29
Released N-Glycan UPLC Analysis Workflows
SAMPLE PREP SEPARATION DETECTION & INFORMATICS
GlycoWorks Kits RapiFluor-MS N-Glycan Kit ACQUITY FLR/QDa
and Empower 3 Software
FLR/Xevo G2-XS QTof MS and UNIFI Scientific Information System
ACQUITY UPLC Glycan BEH Amide
Column
Deglycosylation, Labeling and Clean-up in 30 min
Unmatched sensitivity
for FLR and MS detection
FLR Quantification GU Retention
MS Confirmation
FLR Quantification GU Retention
Accurate Mass Confirmation MS/MS Fragmentation
FLR
TIC
MS/MS
©2015 Waters Corporation 30
The ACQUITY® QDa™ Mass Detector - a pioneering product with mass appeal
Revolutionary innovative design focused on ease of use for analysts
Orthogonal mass detection – added
information with every sample Compact, robust and affordable -
built for constant use with a wide variety of chromatographic conditions
Seamlessly integrates with Empower based HPLC & UPLC™ systems
©2015 Waters Corporation 31
Familiar Graphical User Interface for Ease-Of-Use and Fast Adoption
Empower GUI “identical” to that of a PDA for setup, data viewing and reporting – Very little training needed – Quick addendum to current lab SOP
©2015 Waters Corporation 32
extracted spectrum
Acquity QDa
ACQUITY® QDa™ Detects Peptides Over a Wide Mass Range
3 µg injection
Peak Peptide average mass [M+] M+2] [M+3] [M+4] [M+5]
1 Angiotensin frag. 1-7 899.0 900.0 450.5 300.6 225.7 180.8 2 Bradykinin 1060.2 1061.2 531.1 354.4 266.0 213.0 3 Angiotensin II 1046.2 1047.1 524.1 349.7 262.5 210.2 4 Angiotensin I 1296.5 1297.4 649.2 433.1 325.1 260.3 5 Renin substrate 1759.0 1760.0 880.5 587.3 440.7 352.8 6 Enolase T35 1873.2 1874.2 937.6 625.4 469.3 375.6 7 Melittin 2847.5 2848.4 1424.7 950.1 712.8 570.5
Charge State
1 2
3
4
5
6 7
[M+4]
[M+3]
712.7
949.9
[M+5] 570.4
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Specificity – Resolving Co-Elutions
XIC
SIR
Peak 2
Peak 2
Peak 1
Peak 1&2
Peak 1 [M+4H]+4
536.3 m/z
Peak 2 [M+2H]+2
426.5 m/z
TUV
Peak 2 426.5 m/z
Key Takeaway MS reveals co-elutions and enables you to independently monitor both species
©2015 Waters Corporation 34
CDR Peptide Monitoring
TIC
XIC
1
2 3
4
5
6
7
Key Takeaway MS enables targeted monitoring of peptides for product ID testing
Light Chain 1: ASQDVNTAVAWYQQKPGK 2: LLIYSASFLYSGVPSR 3: SGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTK Heavy Chain 4: DTYIHWVR 5: IYPTNGYTR/(6)YADSVKG 7: WGGDGFYAMDYWGQGTLVTVSSASTK
Harris, R.J., Kabakoff, B., Macchi, F.D., Shen, F.J. Kwong, M., Andya, J.D., Shire, S.J., Bjork, N., Totpal, K., Chen, A.B., Identification of multiple sources of charge heterogeneity in a recombinant antibody. J. Chromatogr. B 752 (2001) 233-245.
©2015 Waters Corporation 35
Biopharmaceutical Platform Solution with UNIFI Analysis of key biotherapeutic primary structural attributes
Biopharmaceutical Platform Solution with UNIFI
Intact Protein Peptide Mapping Glycan Application Solution
Bioseparations (IEX, RP, HIC, SEC)
• Infusion MS • LC/MS • LC/MS/MS (Top-Down) • IMS-MS • LC/UV/MS • UV Only
• LC/MSE • LC/UV/MSE • LC/MS/MS(DDA) • LC/HDMSE
• UV Only
• LC/MS • LC/FLR/MS • LC/FLR/MS/MS(DDA) • FLR Only
©2015 Waters Corporation 36
Addressing bottlenecks in the analytical process?
Sample Generation
Sample Preparation Acquisition Data
Analysis Report
Generation
Routine
ASR Standards
©2015 Waters Corporation 37
Vion IMS QTof
IMS
• MS • MSE
• Targeted MSMS • DDA MSMS • HDMS • HDMSE
• HS-Targeted MSMS • HS-DDA MSMS
Dynamic range
File Size
2nd Generation ADC
©2015 Waters Corporation 38
Ion mobility
Improving the Performance – HDMSE Workflow
©2015 Waters Corporation 40
2
IMS Increases Peak Capacity: The Datacube
Peak capacity = NLC x NIM x Nm/z x F F = fraction of bins occupied
Nm/z > NLC > NIM
However, LC, m/z and IM not completely orthogonal
©2015 Waters Corporation 41
Rapid HDMSE Map Screening
5 min map 89% Coverage
10 min map 97% Coverage
20 min map 98% Coverage
Trastuzumab Tryptic Digest data on Vion IMS QTof
©2015 Waters Corporation 42
Antibody targeted to cancer tumor
Potent cytotoxin chemotherapeutic drug
• Tubulin polymerization inhibitors • Maytansines (DM1, DM4) • Auristatins (MMAE, MMAF)
• DNA damaging agents • Calicheamicins • Duocarmycins • Anthracyclines (doxorubicin)
• Humanized monoclonal Ab (IgG1)
Anatomy of an Antibody-Drug Conjugate (ADC)
42
Linker stable in circulation
• Linker biochemistry • Acid labile (hydrazone) • Enzyme dipeptides (cleavable) • Thioether (uncleavable) • Hindered disulfide (uncleavable)
Site of conjugation • IgG Lysine's or interchain
Cysteines
Melissa M. Schutten, DVM, PhD, DACVP, Genentech, Safety Assessment Pathology NorCal Society of Toxicology Meeting, September 27, 2012
©2015 Waters Corporation 43
ADC Raw Materials mAb, Drug,
Linker
LC/MS Methods
Intact Mass Neutral pH
Peptide Mapping
Bio-transformation
LC/UV Methods
HIC/SEC/IEX
2D LC Method HIC/RP
Established LC & MS Methods for low
molecular weight drugs and monoclonal
antibodies
Higher Order IMS, HDX
Antibody Drug Conjugate (ADC) Characterization Needs (LC/MS)
©2015 Waters Corporation 45
Cysteine Conjugation Location Possibilities “Positional Isomers”
0
4
6
8
2
©2015 Waters Corporation 46
Cysteine Conjugate Samples
Samples Analyzed
Critical Quality Attributes • Drug to antibody ratio (DAR) • Drug load distribution • Drug conjugation localization on the mAb • Drug Occupancy of conjugation Sites (specific amino acid)
Sample Modification extent DAR
Cys-mAb none N/A Cys-Low Low 2.88 Cys-Med Medium 4.55 Cys-High High 6.07 { Cysteine
conjugated
©2015 Waters Corporation 47
HIC LC/UV Characterization
Hydrophobic Interaction Chromatography (HIC)
HIC 4.6mm x 100 mm, 2.5 µm Non-porous
©2015 Waters Corporation 48
Low conjugation
Medium conjugation
High conjugation
2
0
4
6 8
2
0
4
6 8
2 0
4
6
8
HIC/UV of 3 Conjugation Levels
Low Expected DAR
Retention time (min) Area % Area (%) DAR
0 4.33 2090606 16.67 0 2 5.22 5113001 40.76 0.82 4 6.32 3577936 28.52 1.14 6 8.06 1577722 12.58 0.75 8 10.55 184257 1.47 0.12
Total DAR 2.83
Med Expected DAR
Retention time (min) Area % Area (%) DAR
0 4.60 240058 2.74 0 2 5.43 1647907 18.83 0.38 4 6.45 3658413 41.8 1.67 6 9.71 2348552 26.83 1.61 8 10.52 858297 9.81 0.78
Total DAR 4.44
High Expected DAR
Retention time (min) Area % Area (%) DAR
0 4.94 37284 0.38 0 2 5.47 341717 3.49 0.07 4 6.45 3036393 31 1.24 6 8.20 2796647 28.55 1.71 8 10.41 3583253 36.58 2.93
Total DAR 5.95
©2015 Waters Corporation 49
UNIFI Report of LC/UV (HIC) of ADC Automated ADC characterization using UNIFI • Adaptable
workflow and reporting increases productivity in ADC development
©2015 Waters Corporation 51
Conclusion • UPLC and Mass Spectrometry are powerful techniques provide: specificity, sensitivity, quantitation, flexibility and future proofing Waters Instruments and methods available for Biopharma
applications Extensive applications support and dedicated service organisation Chemistry Consumables ISO , CE manufacturing, Single vendor complete solution [email protected] www.waters.com
©2015 Waters Corporation 52
Thank you