Peptide Mapping 101: Essential Tools for Effective Development and Characterization. Part 1:Introduction to Peptide Mapping

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Peptide Mapping 101: Essential Tools for Effective Development and Characterization

Part 1:Introduction to Peptide Mapping

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Stephan M. Koza, Ph. D.

Principal Applications Chemist Waters Technologies Corporation

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Agenda Agenda

What is Peptide Mapping and Why Do It?

Protein Digestion

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What is Peptide Mapping? What is Peptide Mapping?

The chemical or enzymatic treatment of a protein to produce peptide

fragments

Separation and identification of these fragments in a reproducible

manner

For biotherapeutic proteins and peptides peptide mapping is:

manner

In-depth analysis that can identify minor and even isobaric differences

in protein primary structure such as errors in the transcription of complementary DNA, point mutations., and PTMs (CQAs)

Due to the complexity and inherent variability of the method peptide

mapping is generally a comparative procedure where the peptide map of the test sample is compared to that of a reference substance

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Uses of Peptide Mapping Uses of Peptide Mapping

Proteomics Studies

Protein Biopharmaceutical Analysis – Structural characterization

o Pattern conforms to primary structure

o Used with MS for primary structure determination o Non-Reduced Mapping for Disulfide Bond Assignment

– Protein modification

o Identify post-translational modifications

• Glycosylation, substitution, truncation

o Determine product related impurities: deamidation, oxidation, etc. o Characterization of variants observed in other methods (IEX, SEC)

– Protein identity

o Confirm presence of “signature peptides” o Product integrity – lot-to-lot analysis

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Biopharmaceutical Classes That Use Biopharmaceutical Classes That Use Peptide Mapping Methods

Peptide Mapping Methods

Peptides/Proteins derived through recombinant

DNA-based processes

– Insulin Diabetes

– Erythropoietin Cancer

– Monoclonal antibodies derived by recombinant DNA

processes, and their derivatives

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Why Do We Develop Peptide Maps Why Do We Develop Peptide Maps for Biotherapeutic Proteins?

for Biotherapeutic Proteins?

Guidance for Industry

Q6B Specifications: Test Procedures and Acceptance Criteria for

Biotechnological/Biological Products

d. Peptide map

Selective fragmentation of the product into discrete peptides is

performed using suitable enzymes or chemicals…….Peptide mapping of the drug substance or drug product using an appropriately

validated procedure is a method that is frequently used to confirm desired product structure for lot release purposes.

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Agenda Agenda

What is Peptide Mapping and Why Do It?

Protein Digestion

Peptide Separations

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Enzymes and Chemistries for Enzymes and Chemistries for Protein Digestion

Protein Digestion

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Enzymes and Chemistries for Enzymes and Chemistries for Protein Digestion

Protein Digestion

EUROPEAN PHARMACOPOEIA 5.0, 2.2.55. PEPTIDE MAPPING

Trypsin, Lys-C,and Asp-N are most commonly used and can provide high fidelity digestions for

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In

In silicosilico DigestionDigestion Tools for Tools for Selecting Selecting an Enzyme (or Chemical):

an Enzyme (or Chemical):MassLynxMassLynx

Protein/Peptide Editor Protein/Peptide Editor

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In

In silicosilico DigestionDigestion Tools for Tools for

Selecting an Enzyme (or Chemical) Selecting an Enzyme (or Chemical)

• Trypsin results in 2 amino acids and 1 di-peptide, Lys-C might

be a better choice as it generates 3 manageable peptides

• Further digestion would be needed to assign disulfide bonds in

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Flow Chart of Peptide Mapping Flow Chart of Peptide Mapping

Protein (e.g. antibody)

Denaturation,

Disulfide Reduction/Alkylation, Buffer Exchange

Enzymatic Digestion (e.g. Trypsin)

Peptide Map Analysis

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What’s RapiGest™ SF

Anionic detergent that improves solubility and digestion of

many proteins for improved enzymatic digests.

Unlike conventional denaturants, RapiGest SF does not inhibit

enzyme activities so it can reduce digestion times and reduces the amount of enzyme used.

It does not cause protein modifications (e.g., urea causing

carbamylation) unlike some other protein denaturants.

It’s an acid labile surfactant whose degradation products do

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Reproducible Peptide Mapping

Pitfalls of Peptide Mapping – that can affect robustness,

reproducibility and accuracy:

– Sample preparation

o Incomplete digestion

o Non-reproducible digestion conditions

Non-specific cleavages (over-digestion)

o Non-specific cleavages (over-digestion)

o Enzyme lot-to-lot variability (activity units or mass?)

– Non-reproducible chromatography

It is critical that SOPs be written clearly and transferred

precisely in order for peptide maps to be reproducible between different labs or even analysts

Preparing a blank digest is always recommended for

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Agenda Agenda

What is Peptide Mapping and Why Do It?

Protein Digestion

Peptide Separations

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Peptide Separations Peptide Separations

Column Selection

– Ethylene Bridged Hybrid (BEH) Particle Technology

– UPLC vs HPLC

– Charged Surface Hybrid Technology

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Bridged Ethanes In Silica Matrix

U.S. Patent No. 6,686,035 B2 and others patent pending

Organo Silica Hybrid Particles

Ethylene Bridged Hybrid - BEH Technology™

Tetraethoxysilane Bis(triethoxysilyl)ethane + 4 Polyethoxysilane Si EtO EtO OEt EtO Si EtO EtO CH₂ EtO CH₂ Si OEt OEt OEt Si EtO O CH2 CH2 Si O Si EtO OEt Si _O O OEt O Si O Si OEt O O OEt Et Et n

Organo Silica Hybrid Particles – pH stability

– Reduced ionic interactions

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Small Particle Size Small Particle Size

Porous Particle Peptides Mobile Phase 1500 Da Peptide 2 3.5 µm

Diffusion distances decrease

– Reduced Eddy diffusion, A-Term

– Improved mass transfer kinetics, C-Term Column efficiency

Narrower peaks

Adsorption Equilibria Diffusion-related band broadening

0 1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 Velocity (mm/sec) H ( m m ) 1.7 µm 2.1 mm ID 40 µL/min µL/min400

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Why UPLC

Why UPLC®® _{for peptide mapping}_{for peptide mapping}

More resolution even using a shorter gradient More resolution even using a shorter gradient

A U 2.0e-2 3.0e-2 4.0e-2 5.0e-2 6.0e-2 7.0e-2 90 min HPLC 2.1 x 300 mm, 3.5 µ ©2013 Waters Corporation 20 Time 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00 54.00 56.00 A U 2.0e-2 3.0e-2 4.0e-2 5.0e-2 6.0e-2 7.0e-2 8.0e-2 9.0e-2 1.0e-1 Time 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00 85.00 90.00 1.0e-2 2.0e-2 55 min UPLC 2.1 x 150 mm, 1.7 µ

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Charged Surface Hybrid (CSH) Technology Charged Surface Hybrid (CSH) Technology

Charged Surface Hybrid (CSH) Technology and Its Use in Liquid Chromatography.

P.C. Iraneta, K.D. Wyndham, D.R. McCabe, and T.H. Walter Waters White Paper 720003929EN 2011

Expands upon the robust BEH particle technology

patent pending

Peptide

Expands upon the robust BEH particle technology CSH130 C18

= BEH130 base particle

+ low level of basic moieties

+ trifunctional C18/end cap

Acidic pH

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100%

Peak Capacity Peak Capacity

Peak Capacity =

– The number of peaks that can be separated within a retention window

Neue, U. D., J Chromatogr A 2005, 1079 (1-2), 153-61.

– The best metric for determining the quality of gradient separations

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A

A Novel Novel Column Column Chemistry: Chemistry: CSH130 C18 (0.1% TFA) CSH130 C18 (0.1% TFA) U V a b so rb an ce ( 2 1 4 n m ) BEH130 C18 Porous (130Å) 1.7 µm 2.1 x 150 mm Competitor’s “Industry Standard” C18 Porous (300Å) 5 µm 2.1 x 250 mm 1 2 Time (min) 10 50 10 20 30 40 50 10 50 Time (min) 10 50 Time (min) 10 50 Time (min) U V a b so rb an ce ( 2 1 4 n m ) CSH130 C18 Porous (130Å) 1.7 µm 2.1 x 150 mm Competitor’s Superficially Porous “Peptide” C18 SPP (100Å) 1.7 µm 2.1 x 150 mm

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Peak Capacity

Peak Capacity -- FA FA vsvs TFATFA

“Industry Standard” Silica C18 5 µm 2.1 x 250 mm

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Peak Capacity

220 270 320 370 c, 4 σ BEH130 C18 1.7 µm 2.1 x 150 mm 20 70 120 170 220 0.00 0.05 0.10 Pc,4 Percent TFA Competitor’s

“Industry Standard” Silica C18 5 µm 2.1 x 250 mm 0.05 0.05 0.10 0.00 0.00 0.10 % TFA % FA FA TFA

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220 270 320 370 c, 4 σ Peak Capacity

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Peak Capacity

220 270 320 370 c, 4 σ Competitor’s SPP “Peptide” C18 1.7 µm 2.1 x 150 mm BEH130 C18 1.7 µm 2.1 x 150 mm CSH130 C18 1.7 µm 2.1 x 150 mm 20% 90% 20 70 120 170 220 0.00 0.05 0.10 Pc,4 Percent TFA Competitor’s

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350 400 450

High Mass Load

CSH C18 BEH C18

A

6 µg of mixture Loadability

Loadability

Attribute – how much analyte can be loaded before peak shape deteriorates

CSH130 C18 1.7 µm

Typical Mass Load

6 µg of mixture

(Equivalent to ~ 45 µg of a mAb)

350 400 450

Low Mass Load CSH C18 BEH C18

B

CSH C18 BEH C18 0.6 µg of mixture BEH130 C18 1.7 µm CSH130 C18 1.7 µm

Low Mass Load

0.6 µg of mixture (Equivalent to ~ 4.5 µg of a mAb) ©2013 Waters Corporation 28 Pc ,4 σ 150 200 250 300 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 Pc ,4 σ Percent TFA 0.05 0.05 0.00 0.10 0.10 0.00 % TFA % FA BEH130 C18 1.7 µm 150 200 250 300 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 Pc ,4 σ Percent TFA 0.10 0.10 0.00 0.05 0.05 0.00 0.10 0.10 0.00 BEH130 C18 1.7 µm *Previously shown 0.05 0.05 0.10 0.00 0.00 0.10 % TFA % FA FA TFA 0.05 0.05 0.10 0.00 0.00 0.10 % TFA % FA FA TFA

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Which Column do I choose CSH130 C18 Which Column do I choose CSH130 C18 or BEH130 C18? or BEH130 C18? 0E+0 1E+6 2E+6 0 10 20 30 40 50 60 In te n s it y BEH C18 1.7 µm Pc,4σ= 399 BEH130 C18 1.7 µm P_c,4σ= 399 0.1% FA 0 10 20 30 40 50 60 Time(min) 0E+0 1E+6 2E+6 0 10 20 30 40 50 60 In te n s it y Time(min) CSH C18 1.7 µm Pc,4σ= 532 CSH130 C18 1.7 µm P_c,4σ= 532

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0E+0 1E+6 5.5 6.5 7.5 8.5 9.5 10.5 11.5 12.5 Time(min) LC LC--MS MS

Retention and Selectivity Retention and Selectivity

BEH130 C18 T10 T3 T5/ T12 T19 T40 T3 SVYDSR T5 GVFR T12 ANIDVK T19 HLADSK T10 GVLHAVK T40 IATAIEK More positive charge ©2013 Waters Corporation 30 0E+0 1E+6 5.5 6.5 7.5 8.5 9.5 10.5 11.5 12.5 13.5 Time(min) CSH130 C18 T10 T3 T19T5/_T12 T40

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UPLC

UPLC andand HPLCHPLC

0.2 0.4 0.6 0.8 1.0 A2 1 4 0.2 0.4 0.6 0.8 1.0 A2 1 4 0.1 % FA 1.7 µm CSH130 C18 2.1 x150 mm 0.1 % TFA ~8000 psi

High peak capacity separations not limited to UPLC

0.0 10 20 30 40 50 Time (min) 0.0 0.2 0.4 0.6 0.8 1.0 14.5 24.5 34.5 44.5 54.5 64.5 74.5 A2 1 4 Time (min) 0.0 10 20 30 40 50 Time (min) 0.0 0.2 0.4 0.6 0.8 1.0 13.5 23.5 33.5 43.5 53.5 63.5 73.5 A2 1 4 Time (min) 2.5 µm XP ~3000 psi Longer Run Time Lower Pressure Method Transfer

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UPLC

UPLC andand HPLCHPLC

0.2 0.4 0.6 0.8 1.0 A2 1 4 0.2 0.4 0.6 0.8 1.0 A2 1 4 0.1 % FA 1.7 µm CSH130 C18 2.1 x150 mm 0.1 % TFA ~8000 psi

High peak capacity separations not limited to UPLC

CSH130 C18 Peptide Separation Technology Columns Available Now: Upcoming:

©2013 Waters Corporation 32 0.0 10 20 30 40 50 Time (min) 0.0 0.2 0.4 0.6 0.8 1.0 14.5 24.5 34.5 44.5 54.5 64.5 74.5 A2 1 4 Time (min) 0.0 10 20 30 40 50 Time (min) 0.0 0.2 0.4 0.6 0.8 1.0 13.5 23.5 33.5 43.5 53.5 63.5 73.5 A2 1 4 Time (min) 2.5 µm XP ~3000 psi Longer Run Time Lower Pressure Method Transfer

Available Now: Upcoming:

Analytical Columns Nano (75, 150, 300 µm ID) 1.7 µm

2.5 µm XP

3.5 µm

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Three Outstanding Three Outstanding

Peptide Separation Technology Columns Peptide Separation Technology Columns

0 0.2 0.4 0.6 0.8 1 1.2 10 15 20 25 30 35 40 45 50 A2 1 4 Time (min) 1.2 CSH130 C18 1.7 µm BEH130 C18 1.7 µm 2 3 4 5 6 1 Peptide/Protein kDa 1 Bradykinin 1.1 2 Renin Substrate 1.8 3 Ubiquitin 8.6 4 Cytochrome C _(Equine) 12.4 5 _(Bovine)Insulin 5.7 6 Melittin 2.8 130 Å Time (min) 0 0.2 0.4 0.6 0.8 1 10 15 20 25 30 35 40 45 50 A2 1 4 Time (min) 0 0.2 0.4 0.6 0.8 1 1.2 10 15 20 25 30 35 40 45 50 A2 1 4 Time (min) BEH300 C18 1.7 µm BEH130 C18 1.7 µm 2 3 4 5 6 1 2 3 5 6 1 4 300 Å

ACQUITY UPLC H-Class Bio UV @ 214 nm / Xevo G2 QTOF 1 µg each component

2.1 x 150 mm columns 2% ACN for 1 min,

then to 50% ACN over 60 min 0.3 mL/min

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New Addition to the Suite of New Addition to the Suite of

Waters Peptide Separation Technology Waters Peptide Separation Technology

Peptide Separation Technology

– Peptide C18 Columns

– QC Tested with Digests

BEH Technology

– BEH130 C18 and BEH300 C18

– Outstanding Performance for Most Applications

– Two Pore Sizes

– Particle Sizes: 1.7 µm, 3.5 µm, 5 µm

– Analytical, Nano and Prep Columns

Now even more tools in the toolbox … CSH Technology – CSH130 C18

– Highest peak capacities in TFA and FA mobile phases.

– Unique selectivity

– Particle Sizes: 1.7 µm, 2.5 XP, 3.5 µm, 5 µm

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All Waters Peptide

All Waters Peptide SeparationColumnsSeparationColumns are Quality Control Tested with

are Quality Control Tested with TrypticTryptic Digest of

Digest of CytochromeCytochrome cc

CSH130 C18

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Fine Tuning Your Separation Fine Tuning Your Separation

Parameters that Influence Selectivity

– Ion Pairing Reagent (TFA, HFBA, etc.) and Concentration

– Organic Eluent (MeCN, MeOH, IPA)

– Column Temperature

– Gradient Slope/Column Length

Peak Tracking

– Ideally using LC-MS can expedite separation optimization

– Make several incremental changes

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% * * Rate of Change 0.75%/ col. vol. Method Optimization: Method Optimization: Gradient Slope Gradient Slope % 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 1 * Rate of Change 1.5%/ col. vol. * * *

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Why Does This Switch in Elution Why Does This Switch in Elution Order Occur? Order Occur? L o g k Elution at Higher % MeCN w/ Steeper Gradient ©2013 Waters Corporation 38 % MeCN L o g k Elution at Lower % MeCN w/ Shallower Gradient

Adapted from: Spicer, V., Grigoryan, M., Gotfrid, A., Standing, K. G., & Krokhin, O. V. (2010). Predicting retention time shifts associated with variation of the gradient slope in peptide RP-HPLC. Analytical chemistry, 82(23), 9678-9685.

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Gradient Slope and Segmented Gradients Gradient Slope and Segmented Gradients

Changes in gradient slope should occur in regions of separation where

there are no peaks of interest

Potential selectivity differences should be tracked

Approach could also be used to generate a focused gradient if only specific

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CSH130 C18 CSH130 C18

Useful Current Literature/Resources Useful Current Literature/Resources

http://pubs.acs.org/doi/abs/10.1021/ac401481z

Previously recorded webinar available:

http://www.waters.com/waters/promotionDetail.htm?id=134727909

Increasing Peak Capacity in Reversed Phase Peptide Separations with Charged Surface Hybrid (CSH) C18 Columns M.A. Lauber, S.M. Koza, K.J. Fountain

Waters Application Note 720004568EN 2013

Peptide Mapping and Small Protein Separations with Charged Surface Hybrid (CSH) C18 and TFA-Free Mobile Phases M.A. Lauber, S.M. Koza, K.J. Fountain

Waters Application Note 720004571EN2013

High Mass Loading of Peptides with Hybrid Particle C18 Columns and Acetic Acid Mobile Phases M.A. Lauber, S.M. Koza, K.J. Fountain

Waters Application Note 720004674EN2013 Recent Application Notes

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Part 2 - Gaining Efficiency:

Instrumentation and Informatics Platforms for Peptide Mapping

Asish Chakraborty, Ph.D

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A History of Relieving the Pressure on A History of Relieving the Pressure on Analysts …

Analysts …

Sample

Generation Preparation Sample Acquisition AnalysisData Generation Report

Sample Generation Data Analysis _GenerationReport

Chemistries Instrumentation and

Automation Informatics

Now It Becomes Routine

Sample Generation _GenerationReport

Sample

Generation

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Performance and Usability

through Engineered Simplicity

Automatically ensuring the system is ready to run

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Automating batch processing, annotatation, and comparison tools in BiopharmaLynxTM _increases

productivity

First shown at

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Biopharmaceutical Platform Solution Biopharmaceutical Platform Solution with UNIFI 1.7 with UNIFI 1.7 Biopharmaceutical Platform Solution Intact Protein

Mass MappingPeptide

DDA (Peptide & Glycan)

Xevo G2-S QTof

An analytical system for biotherapeutic analysis integrating UPLC/UV and UPLC/MS

Glycan GU + Mass

DDA (Peptide & Glycan)

Intact Protein: TUV, MS

Peptide Mapping: TUV, MSE_{, MS/MS}

Released Glycan: FLR (+MS, NIBRT Library), MS/MS Bioseparations: TUV, FLR

Workstation or Workgroup (Compliance)

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Discovery Discovery

Deploy high resolution analytics across Deploy high resolution analytics across a

a biotherapeuticbiotherapeutic organizationorganization

Few

compliance issues GxP Labs Regulatory Compliance

Discovery Development Production Post-Approval QC/QA Discovery Development Production Post-Approval QC/QA

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Office PC

The Biopharmaceutical Workgroup The Biopharmaceutical Workgroup

Office PC Office PC Lab Network Device (LND) Lab PC LND Lab PC UPLC-FLR-Xevo G2-S UPLC-TUV-Xevo G2-S

Intact Mass, Peptide Mapping

Intact Mass, Peptide Mapping Released Glycan AnalysisReleased Glycan Analysis

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UNIFI™ Meets the Biopharmaceutical UNIFI™ Meets the Biopharmaceutical industry’s Global reach

industry’s Global reach

A Scientific management system for the global nature of the Biopharmaceutical business

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Peptide Mapping in UNIFITM

Advanced Reporting Capabilities in a GxP-ready Environment

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Experimental Setup for Peptide Mapping Experimental Setup for Peptide Mapping

Therapeutic Proteins

Denature & Alkylate

Trypsin Digest LC/MSE Non-Reduced Peptide Map Reduction & Alkylation LC/MSE Reduced Peptide Map Peptide Map UNIFI Scientific Information System

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UPLC/MS

UPLC/MSE E _{Comprehensively Analyzes}_{Comprehensively Analyzes} Complex Samples

Complex Samples

UPLC/MSE is a simple method of unbiased data acquisition that

comprehensively analyzes all components in a single analysis.

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Surveying Chromatography and

Complexity in Peptide Mapping Data

Chromatogram with Peak Assignments

3D Chromatogram

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Peptide Mapping Data

Annotated

Chromatograms _{Fragment ions}

Spectrum Assignments

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Peptide Mapping Data

Data Table (linked to Coverage Map) Coverage Map Fragment ions Spectra Assignments

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Access to both raw and processed data

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Case Study 1 Case Study 1 Case Study 1 Case Study 1

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Experimental Setup for Peptide Mapping Experimental Setup for Peptide Mapping

Denature & Alkylate

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Equivalent protein coverage was obtained for innovator and biosimilar

Innovator HC Innovator Biosimilar HC Biosimilar LC Innovator LC BEH, C18, 1.7 µm, 130, 2.1x 100 mm, Biosimilar

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Asp

Asp IsomerizationIsomerization of Peptide T24 of Peptide T24 (FNWYVDGVEVHNAK) (FNWYVDGVEVHNAK) XIC Innovator Iso ASP

Isomerization: Asp to iso-Asp (no mass difference). isoAsp is not a natural amino acid and can potentially

Innovator

Biosimilar

isoAsp is not a natural amino acid and can potentially be immunogenic.

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Oxidation of HC Peptide T42 Oxidation of HC Peptide T42 I Biosimilar Batch Innovator % O xi d at io n I Batch

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Peptide Maps

Peptide Maps ReportReport: Unifi enables researchers to : Unifi enables researchers to focus on critical attributes of a molecule

focus on critical attributes of a molecule

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Experimental setup for disulfide Experimental setup for disulfide bond mapping

bond mapping

Denature & Alkylate

Trypsin Digest LC/MSE Non-Reduced Peptide Map Reduction & Alkylation LC/MSE Reduced Peptide Map UPLC BEH300 C18, 1.7 µm, 2.1 x 150 mm UNIFI Scientific Information System

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IgG1 mAb contains 16 S-S bonds (12

intra, and 4 inter)

Digestion Enzyme: Trypsin

Symmetry of IgG1 molecule provides

redundancy in mass-based search

Heavy chain V H C H1 VL Light chain S – -– S S – -– S S – -– S S – -– S S – -– S S – -– S S – -– S S – -– S –S -S_– S-S– –S-S –

Light Chain Light Chain

Expected disulfide bonds in IgG1 Antibody Expected disulfide bonds in IgG1 Antibody Trypsin

Trypsin Digest Digest

redundancy in mass-based search

8 unique S-S bonded peptides

LC: 2 Intra, HC: 4 Intra, HC-HC(Hinge): 1 inter HC-LC:1 inter CHO CHO CL C H₃ C H₂ – S-S _– – –S-S S S S S S S S S –S-S – -S-S-Humanized IgG Light Chain (1) Light Chain (4) Heavy Chain (2) Heavy Chain (3) K K

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Disulfide Containing Peptides

2:T21-3:T21

Nonreduced

Nonreduced peptide mapping enabled ID peptide mapping enabled ID

of all canonical S

of all canonical S--S peptidesS peptides

A simple filter to only display disulfide containing peptides

2:T21-3:T21 2:T21-3:T21 2:T21-3:T21 MSE Fragment Ions 2:T21-3:T21 Additional studies show there are no scrambled disulfide presence

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Disulfide Bonds

Disulfide Bonds ReportReport: Unifi enables researchers : Unifi enables researchers to focus on critical attributes of a molecule

to focus on critical attributes of a molecule

Component Plot for S-S peptides Analysis Information

KK

Component Summary

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Automated Processing and Reporting with Automated Processing and Reporting with UNIFI™: Intact Protein Analysis

UNIFI™: Intact Protein Analysis

INTACT PROTEIN ANALYSIS

Innovator Biosimilar 1 G0F/G1F G0F/G0F G1F/G2F G2F/G2F G0/G0F G1F/G1F G0F/G2F Innovator Biosimilar 2 G0F/G1F G0F/G0F G1F/G1F G0F/G2F G1F/G2F G2F/G2F G0/G0F

MaxEnt1 deconvoluted mass spectra in compare mode

UNIFI™ workflow automatically acquires, processes and reports the intact mass

– Deconvolution with MaxEnt

– Reporting with Flexible templates

Ivleva et al Poster - ASMS 2012;

http://www.waters.com/webassets/cms/library/docs/2012asms_ivleva_rituximab.pdf

Discrepancy needs to be explained

Biosimilar 2 glycoforms have a systematic mass

shift of 56 Da compared to innovator mAb Biosimilar 1 glycoforms broadly match the innovator

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Reduced Protein Analysis

Reduced Protein Analysis –– LCs LCs

identical identical + Reduction Innovator Biosimilar 1 Biosimilar 2 LC LC w/ PyQ _HC

Mass Analysis of the Light Chain Mass Analysis of the Light Chain

Innovator Biosimilar 1 Innovator Biosimilar 2 Light Chain Automated Processing and Reporting

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Reduced Protein Analysis of Heavy Reduced Protein Analysis of Heavy Chain Chain Innovator Biosimilar 1 G0F G1F G2F G0F+K G1F+K G0 G0 Quantification of C-terminal Lys Variation 0.00E+00 1.00E+06 2.00E+06 3.00E+06 4.00E+06 5.00E+06 6.00E+06 7.00E+06 1 2 3 4 5 6 7 8 9 MS Response Innovator Biosimilar 1 Biosimilar 2 Summary plots Based ©2011 Waters Corporation 70

Detailed Information automatically reported

in UNIFI™

Multiple aspects available from the dataset Response for each batch of each protein

measured and compared Automated Processing and Reporting MS Response 0.00E+00 2.00E+06 4.00E+06 6.00E+06 8.00E+06 1.00E+07 1.20E+07 1 2 3 4 5 6 7 8 9 Innovator Biosimilar 1 Biosimilar 2 Based on UNIFI results Distribution of G0 Glycoform G0F Innovator Biosimilar 2 G0F G1F G0F+K G2F G1F+K G0 G0 G0F G1F ∆m = 28 Da

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LC/MS

LC/MSEE _Tryptic_{Tryptic peptide mapping to locate}_{peptide mapping to locate} the sequence variance

the sequence variance

Tryptic Digest comparison between Innovator and Biosimilar 2 does not show

sequence differences in Light Chain

Light Chain - Innovator Compare

mode Biosimilar 2 Automated Reporting Coverage Map

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LC/MS

LC/MSE E _Tryptic_{Tryptic peptide mapping to locate}_{peptide mapping to locate} the sequence variance

Heavy Chain - Innovator

Compare mode

HC - Biosimilar 2 Biosimilar Coverage _{Map: shows section}

where no sequence match is made

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LC/MS

LC/MSE E _Tryptic_{Tryptic peptide mapping to locate}_{peptide mapping to locate} the sequence variance

Heavy Chain - Innovator

Compare mode

Biosimilar 2

Coverage Map

Alternative Enzyme to Trypsin needed to ascertain if there is a different sequence here in Biosimilar 2

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LC/MS

LC/MSE E _Chymotryptic_{Chymotryptic peptide mapping}_{peptide mapping}

analysis of Biosimilar 2 analysis of Biosimilar 2 Innovator Biosimilar 2 ∆m = 28 Da Innovator Biosimilar 2 BPI Peptide Map

Chymotryptic Digest used to reveal differences

– Peptides are highlighted in the coverage map as each is selected by the user Amino Acid Substitution can be identified

HC Coverage Map

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UNIFI™ Peptide Map Workflow:

UNIFI™ Peptide Map Workflow: MSMSEE _data_data

confirming the sequence Variant confirming the sequence Variant

K₂₁₈ → R₂₁₈ Automated fragment information from MSE data MSE Spectrum of chymotryptic digest confirms amino acid substitution

– K for R at position

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Summary of the Structural Analysis Summary of the Structural Analysis of Rituximab by LC/MS Approach

of Rituximab by LC/MS Approach

The differences between Innovator and Biosimilars Rituximab candidates are:

©2011 Waters Corporation 76 – Biosimilar 1 vs. Innovator o Same AA Sequences o Higher percentage of C-terminal variation o Increased G0 glycoform o Different percentage of pyroglutamation at the N-termini of both LC and HC

– Biosimilar 2 vs. Innovator

o Sequence Variant in HC, K₂₁₈ > R₂₁₈ o Lower percentage of C-terminal Lys

variation

o Much higher percentage of G0 o Different percentage of

pyroglutamation at the N-termini of both LC and HC

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Summary Summary Reduced LC Mass Analysis Intact Mass Analysis Reduced HC Reduced Peptide Mapping Aggregate Analysis Glycan Analysis Non-Reduced Peptide Mapping Charge variant Reduced HC

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2013 Waters Biopharmaceutical 2013 Waters Biopharmaceutical Application Notebook

Application Notebook

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Thank You!

Questions?

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