Biopharmaceuticals: From the Top Down, Bottom Up, Outside ...€¦ · Biopharmaceuticals: From the...

Biopharmaceuticals: From the Top Down,

Bottom Up, Outside In, and Falling to Pieces

Scott Berger

Waters Users Meeting Presentation

ASMS 2015

Discussion Today

Vion IMS QTof: Biopharmaceutical Applications

UNIFI 1.8 Biopharmaceutical Enhancements

Routine Mass Detection for Large Molecules

HDX MS System Extensions

Vion IMS QTof

• MS • MSE

• Targeted MSMS • DDA MSMS • HDMS • HDMSE

• HS-Targeted MSMS • HS-DDA MSMS

Dynamic range

File Size

2nd Generation ADC

Intact Mass Analysis XIC Chromatogram, 0.1 µg on column

Waters mAb Std

Trastuzumab

Xevo XS and Vion Resolution Mode Performance

Resolution=38K

Resolution=48K

Resolution Mode

mAb A Light Chain

mAb B Light Chain

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

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

UNIFI Top Down Analysis Workflow

UNIFI Platform Solution Xevo G2-XS

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

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

Multiply Charged (Non-Resolved)

(e.g. Peptides) (e.g. Larger Proteins)

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)

Rapid HDMSE Map Screening

5 min map 89% Coverage

Trastuzumab Tryptic Digest data on Vion IMS QTof

High quality fragmentation from the 5 min HDMSE map

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

UNIFI Peptide Mapping Workflow Sequence Variant Search (SVS)

ASMS POSTER ThP367

UNIFI Peptide Mapping Workflow De Novo Sequencing

Data Acquisition Peptide Mapping MSE

FastDDA MS/MS

Data Processing

Review

Investigate: Elucidation Tools

DeNovo Sequencing Tools

PEAKS (3rd party)

Report

Top match results

UNIFI Peptide Mapping Workflow De Novo Sequencing

RapiFluor-MSTM (RFMS) Technology

Patent Pending

RapiFluor-MS

POSTERS MP590, MP600

Enhanced chemical properties for glycan analysis: • Rapid Tagging • Efficient Fluorescence • Enhanced Ionization Efficiency

8-16 hrs

Conventional

0.1 hrs

(Pre-Labeling Clean-Up) (Drying)

1-3 hrs

(Drying)

<15 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

Unparalleled FLR and MS Sensitivity

Fluorescence MS (BPI)

Instant AB Labeled

RapiFluor-MS Labeled

2-AB Labeled

tive P

ance (

0.1 0.6

Procainamide Labeled

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

GlycoWorks RapiFluor-MS N-Glycan Kit: Product Information

Kit formats: Large Volume (4 x 24 Samples) & Small Volume (4 x 8 Samples)

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

Glycan FLR +MS Confirmation Workflow

Dextran ladder

Retention time Calibration Curve

Analyte

Scientific Library Search

CFG and Oxford structures available.

Glycan DDA Workflow UNIFI Processed Glycan DDA Data

.mzml and .LCS are compatible with SimGlycan

Data Export

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

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

25 26 27 28 29 30 31 32 33 34 35 36 37 38

Time (min)

Glycopeptides0.01

8 9 10 11 12 13 14 15 16 17 18

Time (min)

Subunits

‘Middle-Up’ ‘Middle-Down’

Glycopeptides

ACQUITY UPLC Glycoprotein BEH Amide 300Å 1.7 µm

Fc/2 Profile

GlycoNinja App In ASMS Suite and Downloadable Post-ASMS

Variant of “Fruit Ninja”

Consumables discounts

Downloadable PC Game Apple App Store Android App Store

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

ACQUITY® QDa™

for Biopharma

The Power of Routine Mass

Detection

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

Existing stack +1 upgrade

A Mass Detector that Starts-Up, Sets-up, and is Maintained Like and Optical Detector

Disposable Source Aperture

Routine Mass Data for Every Peak

FA2B -

1070.7

1070.6

1050.3

1151.8

1114.6

1196.0

240.00

480.00

Minutes

10.00 15.00 20.00 25.00 30.00

…confidence in results….ease of method development…quick diagnosis of problems

LysC digest - Herceptin ® SYNAPT vs. QDa (30-60 minutes)

QDa Empower

SYNAPT MassLynx

Quantification T21-Oxidized T21

MS provides the specificity and sensitivity for relative quantification of peptides

QDa = 96.8%

QDa = 3.2%

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”

Enabling Mass Detection for N-Glycan Routine Monitoring and Quantitation

Minutes

10 20 30 40

ACQUITY QDa signal for

conventional labeling technology

Minutes

10 20 30 40

ACQUITY QDa signal for RapiFluor-MS

labeling technology

Dextran Mixture

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

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

Initial

Prototype Commercial

Product Launch

Waters

Software

Upgrade

Waters HDX

Software

Launch

UPLC® for HDX

Early Access

HDX System

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

Residue

Analysis

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

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.

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

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

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

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

Biopharmaceuticals: From the Top Down, Bottom Up, Outside ...€¦ · Biopharmaceuticals: From the...

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