Biopharmaceuticals: From the Top Down, Bottom Up, Outside ...€¦ · Biopharmaceuticals: From the...
Transcript of Biopharmaceuticals: From the Top Down, Bottom Up, Outside ...€¦ · Biopharmaceuticals: From the...
©2015 Waters Corporation 1
Biopharmaceuticals: From the Top Down,
Bottom Up, Outside In, and Falling to Pieces
Scott Berger
Waters Users Meeting Presentation
ASMS 2015
©2015 Waters Corporation 2
Discussion Today
Vion IMS QTof: Biopharmaceutical Applications
UNIFI 1.8 Biopharmaceutical Enhancements
Routine Mass Detection for Large Molecules
HDX MS System Extensions
D2O
©2015 Waters Corporation 3
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 4
Intact Mass Analysis XIC Chromatogram, 0.1 µg on column
Waters mAb Std
Trastuzumab
©2015 Waters Corporation 6
Xevo XS and Vion Resolution Mode Performance
Resolution=38K
Resolution=48K
Resolution Mode
Resolution Mode
mAb A Light Chain
(11+)
mAb B Light Chain
(12+)
©2015 Waters Corporation 7
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 9
UNIFI 1.8 New Functionality and Capabilities
Top Down Protein Analysis Workflow
Peptide Mapping:
Sequence Variant Search (SVS)
De Novo Sequencing (Elucidation tool)
Store peptide XICs and summed spectra
SYNAPT: ETD data processing (Top Down and Peptide DDA)
Release glycan characterization:
GU + Accurate MS workflow supporting new RFMS label
Enhanced Glycan DDA workflow
Waters RFMS Glycan GU Scientific library
©2015 Waters Corporation 11
UNIFI Top Down Analysis Workflow
UNIFI Platform Solution Xevo G2-XS
Vion
Automated UNIFI Data Acquisition
Targeted MSMS (CID)
Data Processing (BayesSpray Deconvolution)
Review and Reporting
UNIFI (Process Only) Xevo G2-XS
Synapt G2-Si HDMS
MassLynx Data Acquisition Targeted MSMS (CID or ETD)
ProSight PTM
Option: Export to ProSightPTM
ASMS POSTER MP205
Data Import
©2015 Waters Corporation 12
BayesSpray Deconvolution
A novel nested sampling Bayesian inference algorithm for deconvolution of electrospray mass spectral data
Output: Component mass-intensity list
Mock Spectral data for each component
Cleaner decovoluted spectral baselines for both peptide and protein data
Processes isotope resolved and Non-resolved data from complex top down protein fragmentation spectra
Maxent 3 MaxEnt 1
BayesSpray Deconvolution
Lower Charge State Isotope Resolved
Data
Multiply Charged (Non-Resolved)
Data
(e.g. Peptides) (e.g. Larger Proteins)
©2015 Waters Corporation 13
UNIFI Review Window Myoglobin ETD (SYNAPT G2-Si HDMS)
Raw Spectrum
BayeSpray Mock Spectrum
BayeSpray Deconvoluted Spectrum
Annotate identified deconvoluted fragment ions
Option to export to ProsightPTM Upload File Format (.PUF)
©2015 Waters Corporation 16
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 17
High quality fragmentation from the 5 min HDMSE map
©2015 Waters Corporation 19
Impacts
• Product related impurity
• Clinical safety and efficacy
• Present in every biotherapeutic
• Critical for cell line development,
process optimization, quality control
Existing Challenges
• Present at very low levels
• Sensitivity and dynamic range
• Prone to high false positive match
• Manual data review needed
Sequence Variant Analysis (SVA)
In Silico Component Generation
• Digest products
• Missed cleavages
• Semi-digest products
• Modifications
Sequence Variant
Generation
• Blosum 62 matrix likelihood
• 1-2 variants per peptide
Sequence Variant Search
• Mass match (+/- Tolerance)
Fragment Ion Confirmation
• In Silico CID or ETD
• Mass match (+/- Tolerance)
Scoring
• Bayesian Analysis
• Best matches returned
©2015 Waters Corporation 20
UNIFI Peptide Mapping Workflow Sequence Variant Search (SVS)
ASMS POSTER ThP367
©2015 Waters Corporation 21
UNIFI Peptide Mapping Workflow De Novo Sequencing
Data Acquisition Peptide Mapping MSE
FastDDA MS/MS
Data Processing
Review
Auto
mate
d W
ork
flow
Investigate: Elucidation Tools
DeNovo Sequencing Tools
PEAKS (3rd party)
Report
©2015 Waters Corporation 22
Top match results
UNIFI Peptide Mapping Workflow De Novo Sequencing
©2015 Waters Corporation 24
RapiFluor-MSTM (RFMS) Technology
Patent Pending
RapiFluor-MS
POSTERS MP590, MP600
WP238
Enhanced chemical properties for glycan analysis: • Rapid Tagging • Efficient Fluorescence • Enhanced Ionization Efficiency
©2015 Waters Corporation 26
8-16 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
Work Flow: Present and Future
16-24 HOUR
Patent Pending
16-24 HOURS
©2015 Waters Corporation 27
Unparalleled FLR and MS Sensitivity
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Fluorescence MS (BPI)
Instant AB Labeled
RapiFluor-MS Labeled
2-AB Labeled
Rela
tive P
erf
orm
ance (
%)
52.5
7.0
0.1 0.6
Procainamide Labeled
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30.0*
7.0*
RapiFluor-MS vs Instant: 2x ↑ in FLR and 1000x ↑ in MS RapiFluor-MS vs Traditional: 14x ↑ in FLR and 160x ↑ in MS RapiFluor-MS vs PCA: 14x ↑ in FLR and 3x ↑ in MS
©2015 Waters Corporation 28
GlycoWorks RapiFluor-MS N-Glycan Kit: Product Information
Kit formats: Large Volume (4 x 24 Samples) & Small Volume (4 x 8 Samples)
©2015 Waters Corporation 29
Released N-Glycan Analysis Workflows
SAMPLE PREP SEPARATION DETECTION & INFORMATICS
GlycoWorks RapiFluor-MS N-Glycan Kit ACQUITY FLR/QDa
Detectors 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 31
Glycan FLR +MS Confirmation Workflow
Dextran ladder
FLR
Retention time Calibration Curve
GU
5
GU
15
GU
10
Analyte
FLR
XIC
Scientific Library Search
CFG and Oxford structures available.
©2015 Waters Corporation 32
Glycan DDA Workflow UNIFI Processed Glycan DDA Data
.mzml and .LCS are compatible with SimGlycan
Data Export
©2015 Waters Corporation 33
FA2
Higher glycan precursor ion intensity better MSMS fragmentation
Simplified fragmentation pattern for easy data interpretation
Enhanced MSMS with RFMS Labeling Information-Rich MS/MS Spectra
©2015 Waters Corporation 34
5.0
2 N-Glycans
1 N-Glycan 21.5%
0 N-Glycans 3.1%
Novel Widepore HILIC Column for Supporting Multiple Analysis Workflows
Intact Glycoprotein
Patent pending
Traditional proteolytic digestion to yield site-specific glycan profiling
Rapid domain specific glycan profiling using IdeS digestion Higher quality MS of glycoforms
Glycan occupancy on intact mAbs Profiling occupancy and heterogeneity of EPO O-glycosylation
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A214
Time (min)
Glycopeptides0.01
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Time (min)
Subunits
‘Middle-Up’ ‘Middle-Down’
Glycopeptides
ACQUITY UPLC Glycoprotein BEH Amide 300Å 1.7 µm
Fc/2 Profile
©2015 Waters Corporation 35
GlycoNinja App In ASMS Suite and Downloadable Post-ASMS
Variant of “Fruit Ninja”
Consumables discounts
Downloadable PC Game Apple App Store Android App Store
©2015 Waters Corporation 36
Glycan Breakfast Seminar
Bringing Glycan Analysis to a New Age of Enlightenment: A discussion of novel LCMS
technologies and workflows.
Ying Qing Yu and Matt Lauber Waters Corporation
Wed Jun 3 7:00 – 8:00 PM Waters Hospitality Suite Majestic E Ballroom, 2nd floor Renaissance St. Louis Grand Hotel 800 Washington Ave
www.waters.com/glycanbreakfast
Registration Desk at Users meeting OR
©2015 Waters Corporation 37
ACQUITY® QDa™
for Biopharma
The Power of Routine Mass
Detection
©2015 Waters Corporation 38
Easily Added to Existing Systems
Existing stack
Easy to deploy Fully integrated with Empower® CDS Minimal training required Qualification documents/ procedures ready-to-go 110/220V operation Minimal maintenance
QDa
Existing stack +1 upgrade
©2015 Waters Corporation 39
A Mass Detector that Starts-Up, Sets-up, and is Maintained Like and Optical Detector
Disposable Source Aperture
©2015 Waters Corporation 40
Routine Mass Data for Every Peak
A2 -
815.1
FA2 -
888.2
FA2B -
989.6
A2G
1a -
896.2
A2G
1b -
896.0
FA2G
1a -
969.3
FA2G
1b -
969.2
FA2BG
1a -
1070.7
FA2BG
1b -
1070.6
A2G
2 -
977.1
FA2G
2 -
1050.3
FA2BG
2 -
1151.8
FA2G
1S1 -
1114.6
FA2G
2S1 -
1196.0
FA2BG
2S1 -
865.3
FA2G
2S2 -
894.7
FA2BG
2S2 -
962.2
0.00
240.00
480.00
Minutes
10.00 15.00 20.00 25.00 30.00
EU
…confidence in results….ease of method development…quick diagnosis of problems
©2015 Waters Corporation 41
LysC digest - Herceptin ® SYNAPT vs. QDa (30-60 minutes)
QDa Empower
SYNAPT MassLynx
©2015 Waters Corporation 42
Quantification T21-Oxidized T21
5.5%
94.5%
SIR
TUV
TIC
MS provides the specificity and sensitivity for relative quantification of peptides
©2015 Waters Corporation 43
QDa = 96.8%
QDa = 3.2%
H21
H21-Ox
SIR H21: M+2 [418.4] H21-Ox: M+2 [426.4]
Relative Quantification Native Vs. Oxidized Peptide
Herceptin ®
Can easily be multiplexed into Multi-Attribute Monitoring Assays
“MAM Assays”
©2015 Waters Corporation 44
Enabling Mass Detection for N-Glycan Routine Monitoring and Quantitation
Inte
nsity
0
Minutes
10 20 30 40
ACQUITY QDa signal for
conventional labeling technology
Inte
nsity
0
2x106
4x106
6x106
Minutes
10 20 30 40
ACQUITY QDa signal for RapiFluor-MS
labeling technology
2x106
4x106
6x106
Dextran Mixture
©2015 Waters Corporation 45
Routine N-Glycan Detection with Matching FLR and MS response
Minutes 5 10 15 20 25 30 35 40
Minutes
5 10 15 20 25 30 35 40
Detection across a broad range of glycoforms:
IgG Simple bi-antennary structures
RNase B High mannose structures
Fetuin Large, complex and highly sialylated structures
RapiFluor-MS Labeled Glycans
©2015 Waters Corporation 49
ACQUITY QDa Mass Detection Key Biopharm Applications…..so far
Peptide Mapping
& Synthetic Peptides
RFMS Labeled Glycans
Detergents & Formulants
Oligonucleotides
ADC Free Drug Analysis
Subunit Analysis
Wed Morning Glycan Breakfast
POSTER ThP601
POSTER MP600, WP238
©2015 Waters Corporation 50
Initial
Prototype Commercial
Product Launch
2.0
2005
2006
2007
2008
2009
2010
2011
2012
2013
Waters
HDX
Software
Upgrade
Waters HDX
Software
Launch
1.0
UPLC® for HDX
Early Access
HDX System
3.0
2nd Gen
HDX System with
ACQUITY UPLC®
M-Class
10 years of Investments, Innovations, Partnerships
Collaboration with Prof. John Engen, Northeastern University
Customer collaborations in HDX Early Access Program 2014
Enzymate™
Pepsin
Column
Global
Local
Residue
Level
Analysis
©2015 Waters Corporation 51
Training & Support
Waters takes responsibility for your success in executing HDX MS assays, not just the performance of the system’s components
Sample Preparation
Online Pepsin Digestion
Liquid Chromatography
• Nano to Microscale
Mass Spectrometry
• QTof MS
Informatics System Check Analytical Standards
Waters HDX MS Solution
Innovations that addresses each step of HDX MS workflow
Automation Chronos
DynamX™ Software
v. 3.0 ACQUITY
UPLC® M-Class System
SYNAPT® G2-Si HDMS™
for IMS, MSE, ETD
Enzymate™ Pepsin Column
MassPREP™ Phosphorylase
B Digest
Xevo G2-XS
Launch ASMS, Ship: 2H 2015 BPR
Beta
©2015 Waters Corporation 52
New HDX Automation Utilizing PAL RTC Robotics
Smaller footprint, faster operation
Improved integration with MassLynx
Flexibility of selectable syringes – choose the
best syringe volumes for each experiment.
Bottom sensing for increased accuracy for
small sample volumes
Higher efficiency tray cooling. No opening of
drawers for stable low-temp quench
User level access control- method templates
can be protected
Come see this in the Waters suite at ASMS 2015.
©2015 Waters Corporation 53
Back Pressure Regulator (BPR) enables Pressurized Online Pepsin Digestion for HDX-MS
Trap mode
Injection mode
Back Pressure Regulator (BPR)
Plumbing Scheme for the Back Pressure Regulator
Back pressure regulator is currently available under beta agreement
The Nuances in Pressurized On-Line Pepsin Digestions of mAbs and the Implications for Hydrogen/Deuterium Exchange MS
ASMS POSTER MP188
©2015 Waters Corporation 54
DynamX™ HDX Data Analysis Software v. 3.0
• Enhanced data visualizations for easier interpretation of structural differences
• Amino acid-level structural information from ETD fragmentation
• Easier to visualize changes against protein sequence
• Coverage maps with enhanced visualization options
• Protein-ligand (drug) binding analysis
• Streamlined IMS data handling & presentation
• Heat map automatically transfers HDX data to the 3D structure in PyMOL
HDX data processing with DynamX 3.0
Global … at protein level
Local … at peptide digest level
Residue … at peptide fragmentation level
©2015 Waters Corporation 55
A collection of HDX-MS review articles and papers highlighting key biotherapeutic HOS applications of HDX-MS.
Supporting documentation that describes the value of choosing Waters HDX MS technologies.
A presentation you can customize to relate the benefits of adding HDX MS capabilities.
How can you educate yourself and your organization on HDX MS?
www.waters.com/HDXtoolbox
©2015 Waters Corporation 56
Thanks again to our speakers, and
thank you very for sharing your
Saturday with us.
Today’s Reception: 4:00 – 6:00 PM
Waters Hospitality Suite:
Mon - Tue - Wed 8:00 – 11:00 PM