mass spectrometry work flows for biopharmaceuticals · ©2015 waters corporation 1 mass...

©2015 Waters Corporation 1

Mass Spectrometry work flows for Biopharmaceuticals

Denis Calnan Biopharma Business Development

Manager (Northern Europe)


Aims

Waters dedicated Biopharma LCMS workflows

Characterisation of Biotherapeutics

UNIFI Practical data examples - mAb


Antibody compared to Asprin


Antibody characterisation


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


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


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)


Routine Mass Detection for Biopharma

Peptide Mapping & Synthetic Peptides

RFMS Labeled Glycans

Detergents & Formulants

Oligonucleotides

ADC Free Drug Analysis

Subunit Analysis


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


Built on decades of analytical data and informatics experience.


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


Intact Mass Analysis


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


UNIFI: Intact Mass Analysis (Compare Mode)


Simple Intact Antibody LCMS Analysis Report


Peptide Mapping


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


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.


Low energy MS spectrum

MSE

Collision energy ramp 10-40 V

Underlying spectra


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


LCMSE Peptide Map Comparison

Innovator mAb

“Biosimilar” mAb

Result: No gross differences between antibody maps


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)


Glycoprotein Characterization Multiple Strategies – Complementary Information


RapiFluor-MS: Enhanced Workflows for Glycan Monitoring


What is new from Waters? Novel RapiFluor-MSTM (RFMS) Reagent

Patent Pending


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


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


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


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


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


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


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


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.


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


Addressing bottlenecks in the analytical process?

Sample Generation

Sample Preparation Acquisition Data

Analysis Report

Generation

Routine

ASR Standards


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


Ion mobility

Improving the Performance – HDMSE Workflow


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


Rapid HDMSE Map Screening

5 min map 89% Coverage



Trastuzumab Tryptic Digest data on Vion IMS QTof


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


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)


Cysteine Conjugation Location Possibilities “Positional Isomers”

0

4

6

8

2


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


HIC LC/UV Characterization

Hydrophobic Interaction Chromatography (HIC)

HIC 4.6mm x 100 mm, 2.5 µm Non-porous


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


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


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


UNIFI Report of LC/UV (HIC) of ADC Automated ADC characterization using UNIFI • Adaptable

workflow and reporting increases productivity in ADC development


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

mailto:[email protected]�


Thank you

mass spectrometry work flows for biopharmaceuticals · ©2015 waters corporation 1 mass...

Documents