Routine Characterization of mAbs and Other Routine Characterization of mAbs and Other Proteins

Patrick Boyce

Biopharmaceutical Marketing Manager

Europe and IndiaEurope and India

©2011 Waters Corporation 1

Agendag

Why?

What scientific challenges?

Technology

Example application—Sequence confirmationq

—Identification and quantification of glycans & other PTMs

©2011 Waters Corporation 2

Why the need to confirm sequence, identify PTMs and quantify PTMs?y q y

Regulatory guidelines such as ICH Q6B, and EMEA/CHMP/BWP/49348/2005 k it l th t EMEA/CHMP/BWP/49348/2005 make it clear that the primary structure should be confirmed, heterogeneity defined, product related substances heterogeneity defined, product related substances characterized, and consistency demonstrated.

©2011 Waters Corporation 3

Scientific challengesg

Confirming primary structure— LC/MS of peptide map complicated by sub-stoichiometric

difi ti d i d l hi h lt i modifications, and missed cleavages, which may result in a very large quantity of peptides.

Detecting, locating & quantifying modificationsQualitative & quantitative analysis of low mass modifications to — Qualitative & quantitative analysis of low mass modifications to high molecular mass samples

— Dynamic range challengeSpecific MS challengesSpecific MS challenges— Multiple charge states, isotope patterns, co-eluting peptides, in-

source fragmentation, data interpretation— Isobaric peptides in peptide mapp p p p p— Detecting the monisotopic ions becomes more demanding as

mass increases.

©2011 Waters Corporation 4

Overview of Waters Technology for Biopharmaceutical Analysisp y

ACQUITY UPLC H-Class Bio— UV and fluorescence detectors

fXevo G2 QTof MSBiopharmaLynx 1.2Column chemistriesColumn chemistries— AccQTag Ultra BEH C18— BEH130 & BEH300 C18 PST — BEH300 C4 PrST columns— BEH Glycan columns— BEH200 SEC columnsBEH200 SEC columns— Protein-PakTM Hi Res IEX columns— MassPREP Micro Desalting column

©2011 Waters Corporation 5

ACQUITY UPLC H-Class BioQ

Ultimate chromatographic performance— Low dispersion system sub-2 µm columns

Biocompatible system— Eliminate system corrosion— Best sample recovery

Flexibility, robustnessSuitable for RP IEX SEC HILIC— Suitable for RP, IEX, SEC, HILIC

— Robustness tested for high salt— AutoBlend+ for dial-up buffersp

Needle in flow path design— Low carryover performance

©2011 Waters Corporation 6

Mass accuracy, resolution and dynamic rangey g

Accurate mass of monoisotopic peptide for identificationidentification

QuanTof technology: Improved quantitation

Enables identification of larger peptides—Disulfide-linked peptides

Missed Cleavage

QuanTofTM

Low ppm MMA over

TOF >20,000 RESOLUTION

QuanTofTM

Low ppm MMA over

TOF >20,000 RESOLUTION

—Missed Cleavage

—Digests: AspN, LysC, GluC

pp>104 Dynamic

Range

pp>104 Dynamic

Range

©2011 Waters Corporation 7

Xevo G2 Technical Note

2,000

Peptide Mass

31

Monoisotopic peak (% of total)1

93

Relative Monoisotopic Peak

Height2

1.1

Isotopic EnvelopeDynamic Range3

2,000

Peptide Mass

31

Monoisotopic peak (% of total)1

93

Relative Monoisotopic Peak

Height2

1.1

Isotopic EnvelopeDynamic Range3

15,000

10,000

5,000

0.018

0.3

5.7

0.14

2.1

26

714.2

47.6

3.8

15,000

10,000

5,000

0.018

0.3

5.7

0.14

2.1

26

714.2

47.6

3.8

20,000 0.001 0.01 1000020,000 0.001 0.01 10000

~9:1~9:1

The largest peptide in most antibody tryptic peptide maps: HC T15 (DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK). Mass 6712.3071 Da.

©2011 Waters Corporation 8

Dynamic Range Required ~1400

UPLC/MSE to Comprehensively Catalog Complex Samples p p

UPLC/MSE comprehensively catalogs complex samples in a single analysis. It delivers high quality, unambiguous exact mass precursor and fragment ion data quickly and easily

©2010 Waters Corporation

MS

TIMETIME

MSE

©2011 Waters Corporation 10

What BiopharmaLynx™ Doesp y

Automates data processing

Facilitates comparisons between a reference standard and batches of reference standard and batches of experimental samples. Useful for:

—demonstrating consistency of batch with material used for clinical studies

—demonstrating comparability of reference product with potential new biosimilar

©2011 Waters Corporation 11

Application - Introduction and Contextpp

Company X is an international company with a powerful track record in small moleculespowerful track record in small molecules.

They wish to expand their product range and plan to make a biosimilar of an antibody

They sent us two samplesThey sent us two samples— The innovator product— Their copy of that product

They wished to verify that they had successfully mimicked the innovator product. http://en.wikipedia.org/wiki

/File:Antibody2.JPG

©2011 Waters Corporation 12

p

UPLC/MS of Intact Control and Check SamplesUPLC/MS of Intact Control and Check SamplesWith Data Processed by BiopharmaLynx 1.2

©2011 Waters Corporation 13

Intact Protein Analysis Workflow

Sample Preparation

+ +

UPLC® Xevo QTof MS Bioinformatics

©2011 Waters Corporation 14

Control and Check Antibodies Processed automatically by BiopharmaLynx 1.2y y p y

CONTROL

Consistent 64 Da difference in antibody masses for each form

CHECK

©2011 Waters Corporation 15

High Resolution Separation of a Reduced Monoclonal Antibodyy

Mass SpectrumLC

Mass Spectrum HC

LC ChromatogramW t BEH300 C4 1 7 HC Waters BEH300 C4 1.7 µm2.1 x 50 mmA: 0.1% Formic in WaterB 0 1% F i i ACN B: 0.1% Formic in ACN

25-35 %B over 15 min

©2011 Waters Corporation 16

Automatically Deconvoluted Spectrum of the Light Chains in BiopharmaLynx g p y

The average masses of

CONTROLmasses of the Light Chains of the

Light Chains are identical

antibodies are identical and it is a d t sreasonable to suggest that they share

CHECKthey share the same chemical

©2011 Waters Corporation 17

composition

Automatically Deconvoluted Spectrum of the Heavy Chains in BiopharmaLynx y p y

The difference between The difference between the CONTROL and CHECK sample is -32.3 D h h iCONTROL Da per heavy chain.

This corresponds to

CONTROL

papproximately -64.6 Da in the intact antibody of the CHECK sample the CHECK sample when compared with the CONTROL antibody.

CHECK

©2011 Waters Corporation 18

Glycan Characterization

ACQUITY UPLC H-Class Bio with FLR detectionQ

BEH Glycan Column

Released glycans, 2-AB Labelledg y ,

©2011 Waters Corporation 19

HILIC-FLR-MS analysis of 2-AB labeled released glycansg y

Biosimilar glycans were qualitatively consistent with the innovator mAb therefore differences are not due to glycoforms

©2011 Waters Corporation 20

Confirmation of SequenceqBy UPLC-MSE of Trypsin Digest of Control and

Check Samples With Data Processed by Biopha maL n 1 2BiopharmaLynx 1.2

©2011 Waters Corporation 21

Company X Samples and Analysis for Peptide Mapp p

2 samples – CONTROL (reference) and CHECK (copy)

RapiGest-assisted 4-hour tryptic digestion

ACQUITY UPLC separation (BEH130 C18 column)

MSE detection – for simultaneous qualitative & quantitative analysis

Data Interpretation performed by BiopharmaLynx

©2011 Waters Corporation 22

—CONTROL mAb Light Chain (LC) and Heavy Chain (HC) sequences loaded

Chromatograms of Control and Check Samplesp

CONTROL

CHECK

©2011 Waters Corporation 23

Sequence CoverageLight Chaing

Coverage Map for Light Chains for CONTROL and

©2011 Waters Corporation 24

g p gCHECK samples is identical (100%)

Mirror View of HT34-35s in Biopharmalynx Processed MS tracep y

HT34-35 in CONTROL sample

Missing peptide Unknown peak in CHECK

Missing peptide in CHECK sample

©2011 Waters Corporation 25

CHECK sample

Mirror View of HT35s in Biopharmalynx Processed dataProcessed data

HT35 i C t l S lHT35 in Control Sample

UnknownMissing peptide Unknown peak in Check S l

Missing peptide in CHECK sample

©2011 Waters Corporation 26

Sample

MSE Spectra of Control mAb HT35 (EEMTK) and HT34-35 (EPQVYTLPPSREEMTK)

T35 (EEMTK) Control Sample Fragment Ion Data

MSE collects all the exact mass precursor and mass precursor and fragment ion data for every peak

( ) l lT34‐35 (EPQVYTLPPSREEMTK) Control Sample Fragment Ion Data

©2011 Waters Corporation 27

MSE Spectra of Control mAb HT35 (EEMTK) and HT34-35 (EPQVYTLPPSREEMTK)

T35 (EEMTK) Control Sample Fragment Ion Data

( ) l l

BiopharmaLynx automatically interprets the T34‐35 (EPQVYTLPPSREEMTK) Control Sample Fragment Ion Datainterprets the MSE data and provides

sequence confirmation

©2011 Waters Corporation 28

MSE Spectra of Check mAb HT35 (DELTK) and HT34-35 (EPQVYTLPPSRDELTK)

T35 (DELTK) Check Sample

T34 35 (EPQVYTLPPSRDELTK) Check SampleSequence confirmation T34‐35 (EPQVYTLPPSRDELTK) Check Sample confirmation of unknown peaks in peaks in CHECK sample

©2011 Waters Corporation 29

p

Conclusions regarding Datag g

Primary amino acid sequence is different between CONTROL and CHECK

3-amino acid stretch where two amino acids are different and resultant mass difference is 32Dadifferent and resultant mass difference is 32DaEEMTK vs DELTK

Clone appears to have been manufactured using the sequence indicated in Drugbank which is different to the CONTROL mAbdifferent to the CONTROL mAb

A simple LCMSE peptide map saved much time and mone in a oiding scale p fo that clone

©2011 Waters Corporation 30

money in avoiding scale-up for that clone

Simultaneously Confirming Sequence, Identifying y g q y gPTMs and Quantifying PTMs with UPLC-MSE of

Trypsin Digest of Control and Check Samples With D t P d b Bi h L 1 2 Data Processed by BiopharmaLynx 1.2

©2011 Waters Corporation 31

Identifying PTMs with BiopharmaLynxy g p y

Scrambled disulfidesdisulfides

©2011 Waters Corporation 32

Identifying PTMs with BiopharmaLynxy g p y

List of allowed fixed

List of available

or variable modifiers

modifiers –custom

d f

©2011 Waters Corporation 33

modifiers can be added

Chromatograms of Control and Check Samplesp

CONTROL

CHECK

©2011 Waters Corporation 34

Automated PTM identification & quantification of PTMs e.g. HT21 (DTLMSIR) g ( )

CONTROL: 9% M-oxidized HT21Unmodified

CHECK: 18% M-oxidized HT21

Unmodified HT21

©2011 Waters Corporation 35

Summaryy

Routine CharacterizationRoutine Characterization

— UPLC/MS on intact and reduced proteins

— UPLC with FLR detection on released glycans

— UPLC/ MSE analysis of peptide map

S ti f tid ACQUITY UPLC o Separation of peptide map on ACQUITY UPLC

o MSE data acquisition mode to get the exact mass precursor and fragment ion data on every p g ydetectable peak across an entire chromatogram with a single analysis

A t t d d t i ith Bi h L

©2011 Waters Corporation 36

— Automated data processing with BiopharmaLynx

Search www.waters.com for:

— Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced LC and MS techniques

— Development of Mass Spectrometric and Informatics Workflows for the Development of Mass Spectrometric and Informatics Workflows for the Automated Assessment of Biosimilarity for a Candidate BiosimilarAntibody

— Fast and Automatic Mapping of Disulfide Bonds in a Monoclonal A tib d i SYNAPT G2 HDMS d Bi h L 1 3 Antibody using SYNAPT G2 HDMS and BiopharmaLynx 1.3

— Comprehensive and Routine Characterization of Proteins and Peptides using an Integrated Waters LC/MS Workflow

— High Sequence Coverage Peptide Mapping of a Monoclonal Antibody High Sequence Coverage Peptide Mapping of a Monoclonal Antibody with UPLC/MSE

— Application Solutions for Biopharmaceuticals: A Focus on Protein Therapeutics

— RapiGest SF— ACQUITY UPLC H-Class Bio— BiopharmaLynx

X G2 QT f

©2011 Waters Corporation 37

— Xevo G2 QTof

Acknowledgementsg

Scott Berger

Asish Chakroborty

Weibin Chen

St John SkiltonSt John Skilton

©2011 Waters Corporation 38