Peptide Mapping 101: Essential Tools for Effective Development

Peptide Mapping 101: Essential Tools for Effective

Development and Characterization

Part 1:Introduction to Peptide Mapping

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©2013 Waters Corporation 1

Stephan M. Koza, Ph. D.Principal Applications Chemist

Waters Technologies Corporation

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� Product specific information

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AgendaAgenda

�What is Peptide Mapping and Why Do It?

�Protein Digestion


�Peptide Separations

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 prepared in a side-by-side experiment.

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


– 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

Biopharmaceutical Classes That Use Biopharmaceutical Classes That Use Peptide Mapping MethodsPeptide Mapping Methods

� Peptides/Proteins derived through recombinant DNA-based processes– Insulin Diabetes– Erythropoietin Cancer– Monoclonal antibodies derived by recombinant DNAprocesses, and their derivatives

Herceptin Cancer


o Herceptin Cancer� Protein Conjugates– ADC– PEGylated proteins

� Synthetic peptides

� Oligonucleotides/siRNA� Vaccines� Gene therapy� Cells/Stem cells

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 mapSelective 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.

1. Structural Characterization and Confirmation (6.1.1)

AgendaAgenda





� On to Part II

Enzymes and Chemistries forEnzymes and Chemistries forProtein DigestionProtein Digestion


EUROPEAN PHARMACOPOEIA 5.0, 2.2.55. PEPTIDE MAPPING

Enzymes and Chemistries forEnzymes and Chemistries forProtein DigestionProtein 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 reproducible peptide maps.

In In silicosilico DigestionDigestion Tools for Tools for Selecting Selecting an Enzyme (or Chemical):an Enzyme (or Chemical):MassLynxMassLynxProtein/Peptide EditorProtein/Peptide Editor


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 this example

Flow Chart of Peptide MappingFlow Chart of Peptide Mapping

Protein (e.g. antibody)

Denaturation, Disulfide Reduction/Alkylation,

Buffer Exchange


Enzymatic Digestion (e.g. Trypsin)

Peptide Map Analysis

UPLC/ UV UPLC/ MS UPLC/ MS/MS UPLC/ MSE

What’s RapiGest™ SF

� Anionic detergent that improves solubility and digestion of


� 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 not interfere with LC/MS or MALDI MS analysis.

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 troubleshooting purposes

AgendaAgenda





� On to Part II

Peptide SeparationsPeptide Separations

� Column Selection– Ethylene Bridged Hybrid (BEH) Particle Technology

– UPLC vs HPLC

– Charged Surface Hybrid Technology


� Fine Tuning Your Separation

Bridged Ethanes

In Silica Matrix

U.S. Patent No. 6,686,035 B2and others patent pending

Organo Silica Hybrid Particles

Ethylene Bridged Hybrid - BEH Technology™

©2013 Waters Corporation 18Anal. Chem. 2003, 75, 6781-6788

Tetraethoxysilane Bis(triethoxysilyl)ethane

+4

Polyethoxysilane

Si

EtO

EtO OEtEtO

Si

EtO

EtOCH2

EtO

CH2Si

OEt

OEt

OEtSi

EtO

O

CH2 CH2

SiO

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

– Basis of Peptide Separation Technology

Small Particle SizeSmall Particle Size

Porous

Particle

Peptides

Mobile Phase

1500 Da Peptide

23.5 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 (mm)

1.7 µm

2.1 mm ID40

µL/min400

µL/min

Why UPLCWhy UPLC®® for peptide mapping for peptide mapping More resolution even using a shorter gradientMore resolution even using a shorter gradient

AU

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


Time20.00 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

AU

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

Time30.00 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 minUPLC 2.1 x 150 mm, 1.7 µ

Charged Surface Hybrid (CSH) TechnologyCharged 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. WalterWaters 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

Positive Surface Charge

100%

Peak CapacityPeak 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


0%

50%

Peak Height

tgradient

wh

2.35σ

4σw4σ13.4%

9 peakscould resolve ~300-400

A A Novel Novel Column Column Chemistry: Chemistry: CSH130 C18 (0.1% TFA) CSH130 C18 (0.1% TFA)

UV absorban

ce (214 nm)

BEH130 C18Porous (130Å) 1.7 µm

2.1 x 150 mm

Competitor’s “Industry Standard” C18

Porous (300Å) 5 µm2.1 x 250 mm


1 2

Time (min)10 50

10 20 30 40 5010 50

Time (min)

10 50Time (min)

10 50Time (min)

UV absorban

ce (214 nm)

CSH130 C18Porous (130Å) 1.7 µm

2.1 x 150 mm

Competitor’s Superficially Porous

“Peptide” C18SPP (100Å) 1.7 µm

2.1 x 150 mm

Peak Capacity Peak Capacity -- FA FA vsvs TFATFA

220

270

320

370

c,4σ


20

70

120

170

220

0.00 0.05 0.10

P c,4

Percent TFA0.050.05

0.100.00

0.000.10

% TFA% FA

Competitor’s “Industry Standard” Silica C18 5 µm2.1 x 250 mm

FA TFA


220

270

320

370

c,4σ

BEH130 C18 1.7 µm2.1 x 150 mm


20

70

120

170

220

0.00 0.05 0.10

P c,4

Percent TFA


0.050.05

0.100.00

0.000.10

% TFA% FA

FA TFA

220

270

320

370

c,4σ


BEH130 C18 1.7 µm2.1 x 150 mm

Competitor’s SPP “Peptide” C18 1.7 µm2.1 x 150 mm


20

70

120

170

220

0.00 0.05 0.10

P c,4

Percent TFA


0.050.05

0.100.00

0.000.10

% TFA% FA

FA TFA


220

270

320

370

c,4σ

Competitor’s SPP “Peptide” C18 1.7 µm2.1 x 150 mm

BEH130 C18 1.7 µm2.1 x 150 mm

CSH130 C18 1.7 µm2.1 x 150 mm

20%

90%


20

70

120

170

220

0.00 0.05 0.10

P c,4

Percent TFA


0.050.05

0.100.00

0.000.10

% TFA% FA

FA TFA

350

400

450High Mass Load

CSH C18

BEH C18

A 6 µg of mixture

LoadabilityLoadability

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

CSH130 C18 1.7 µm

Typical Mass Load6 µg of mixture (Equivalent to ~ 45 µg of a mAb)

350

400

450Low Mass Load

CSH C18

BEH C18

BCSH C18

BEH C18

0.6 µg of mixture

BEH130 C18 1.7 µm

CSH130 C18 1.7 µm

Low Mass Load0.6 µg of mixture(Equivalent to ~ 4.5 µg of a mAb)


Pc,4σ

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,4σ

Percent TFA0.050.05

0.000.10

0.100.00

% 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,4σ

Percent TFA

0.100.100.00

0.050.05

0.000.10

0.100.00

BEH130 C18 1.7 µm

*Previously shown

0.050.05

0.100.00

0.000.10

% TFA% FA

FA TFA

0.050.05

0.100.00

0.000.10

% TFA% FA

FA TFA

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

Inte

nsity

BEH C18 1.7 µmPc,4σ= 399

BEH130 C18 1.7 µmPc,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

Inte

nsity

Time(min)

CSH C18 1.7 µmPc,4σ= 532

CSH130 C18 1.7 µmPc,4σ= 532

0E+0

1E+6

5.5 6.5 7.5 8.5 9.5 10.5 11.5 12.5

Time(min)

LCLC--MS MS Retention and SelectivityRetention and Selectivity

BEH130 C18

T10T3

T5/T12 T19

T40

T3 SVYDSRT5 GVFRT12 ANIDVKT19 HLADSKT10 GVLHAVKT40 IATAIEK

More positive charge


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 T3T19 T40

T5/T12

UPLC UPLC andand HPLCHPLC

0.2

0.4

0.6

0.8

1.0

A214

0.2

0.4

0.6

0.8

1.0

A214

0.1 % FA

1.7 µm

CSH130 C182.1 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

A214

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

A214

Time (min)

2.5 µm XP

~3000 psi

Longer Run Time

LowerPressure

Method Transfer

UPLC UPLC andand HPLCHPLC

0.2

0.4

0.6

0.8

1.0

A214

0.2

0.4

0.6

0.8

1.0

A214

0.1 % FA

1.7 µm

CSH130 C182.1 x150 mm

0.1 % TFA

~8000 psi

High peak capacity separations not limited to UPLC

CSH130 C18 Peptide Separation Technology Columns

Available Now: Upcoming:


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

A214

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

A214

Time (min)

2.5 µm XP

~3000 psi

Longer Run Time

LowerPressure

Method Transfer

Available Now: Upcoming:Analytical Columns Nano (75, 150, 300 µm ID)1.7 µm2.5 µm XP3.5 µmPrep Columns (5 µm)

Three Outstanding Three Outstanding Peptide Separation Technology ColumnsPeptide Separation Technology Columns

0

0.2

0.4

0.6

0.8

1

1.2

10 15 20 25 30 35 40 45 50

A214

Time (min)1.2

CSH130 C18 1.7 µm

BEH130 C18 1.7 µm

2

34

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 Insulin(Bovine) 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

A21

4

Time (min)

0

0.2

0.4

0.6

0.8

1

1.2

10 15 20 25 30 35 40 45 50

A214

Time (min)

BEH300 C18 1.7 µm

BEH130 C18 1.7 µm

23

4

5

61

23

5

61

4

300 ÅACQUITY UPLC H-Class BioUV @ 214 nm / Xevo G2 QTOF 1 µg each component

2.1 x 150 mm columns2% ACN for 1 min,then to 50% ACN over 60 min0.3 mL/min40°C

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


– 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– Analytical and Prep Columns (Nano in development)

All Waters Peptide All Waters Peptide SeparationColumnsSeparationColumnsare Quality Control Tested with are Quality Control Tested with TrypticTrypticDigest of Digest of CytochromeCytochrome cc

CSH130 C180.1 % Formic Acid


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

– Peak areas and A280/A214 UV absorbance ratios

%

*

*

Rate of Change0.75%/ col. vol.

Method Optimization:Method Optimization:Gradient SlopeGradient Slope


Time10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00

%

1

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.001

*

Rate of Change1.5%/ col. vol.

*

*

*

Why Does This Switch in Elution Why Does This Switch in Elution Order Occur?Order Occur?

Log k

Elution at Higher % MeCNw/ Steeper Gradient


% MeCN

Log k

Elution at Lower % MeCNw/ 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.

Gradient Slope and Segmented GradientsGradient 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 peptides are of interest

CSH130 C18CSH130 C18Useful Current Literature/ResourcesUseful 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 ColumnsM.A. Lauber, S.M. Koza, K.J. FountainWaters Application Note 720004568EN 2013

Peptide Mapping and Small Protein Separations with Charged Surface Hybrid (CSH) C18 and TFA-Free Mobile PhasesM.A. Lauber, S.M. Koza, K.J. FountainWaters Application Note 720004571EN 2013

High Mass Loading of Peptides with Hybrid Particle C18 Columns and Acetic Acid Mobile PhasesM.A. Lauber, S.M. Koza, K.J. FountainWaters Application Note 720004674EN 2013

Recent Application Notes


End of Part 1

Part 2 - Gaining Efficiency:

Instrumentation and Informatics Platforms

for Peptide Mapping


Asish Chakraborty, Ph.D

[email protected]

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

Sample Generation

Sample Preparation Acquisition Data

AnalysisReport

Generation

Sample Generation Data Analysis Report Generation

Chemistries Instrumentation and Automation

Informatics


Now It Becomes Routine

Sample Generation Report Generation

Sample Generation

Performance and Usability through Engineered Simplicity

Automatically ensuring the system is ready to run


Automating batch processing, annotatation, and comparison tools in BiopharmaLynxTM increases productivity


First shown at WCBP 2007 Meeting

Biopharmaceutical Platform Solution Biopharmaceutical Platform Solution with UNIFI 1.7with UNIFI 1.7

Biopharmaceutical Platform Solution

Intact Protein Mass

Peptide Mapping

DDA (Peptide & Glycan)

Xevo G2-S QTof

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

ACQUITY UPLC H-Class & H-class BIO


BioseparationsSize Exclusion (UV)

ReleasedGlycan

GU + Mass

DDA (Peptide & Glycan)

Intact Protein: TUV, MSPeptide Mapping: TUV, MSE, MS/MSReleased Glycan: FLR (+MS, NIBRT Library), MS/MSBioseparations: TUV, FLR

Workstation or Workgroup (Compliance)

H-class BIO

DiscoveryDiscovery

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

ReleaseMonitoringCharacterization

Office PC

The Biopharmaceutical WorkgroupThe Biopharmaceutical Workgroup

Office PCOffice PC

Lab NetworkDevice (LND)

Lab PC

LND

Lab PC UPLC-FLR-Xevo G2-S

UPLC-TUV-Xevo G2-S

Intact Mass, Peptide MappingIntact Mass, Peptide Mapping Released Glycan AnalysisReleased Glycan Analysis


Server

Data Processing&

Database Storage

UPLC-TUV

Lab PC LND

UPLC-FLR

Lab PC LND

LABORATORY NETWORK

BioseparationsBioseparations

UPLC-TUV UPLC-FLR

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

A Scientific management system for the global nature of

the Biopharmaceutical business


Peptide Mapping in UNIFITM

Advanced Reporting Capabilities in a

GxP-ready Environment


Experimental Setup for Peptide MappingExperimental Setup for Peptide Mapping

Therapeutic Proteins

Denature & Alkylate

Trypsin DigestLC/MSE

Non-ReducedPeptide Map

Reduction &

Alkylation

LC/MSE

Reduced Peptide Map


UPLC BEH300 C18, 1.7 µm, 2.1 x 100 mm

Peptide Map

UNIFI Scientific Information System

UPLC/MSUPLC/MSE E Comprehensively Analyzes Comprehensively Analyzes Complex SamplesComplex Samples

� UPLC/MSE is a simple method of unbiased data acquisition that comprehensively analyzes all components in a single analysis.


Surveying Chromatography and Complexity in Peptide Mapping Data


Chromatogram with Peak

Assignments

3D Chromatogram

Peptide Mapping Data


AnnotatedChromatograms Fragment ions

Spectrum

Assignments

Peptide Mapping Data

Data Table (linked to Coverage Map)


Coverage Map

Fragment ionsSpectra

Assignments

Access to both raw and processed data


Case Study 1Case Study 1


Case Study 1Case Study 1

Experimental Setup for Peptide MappingExperimental Setup for Peptide Mapping


Denature & Alkylate



Reduction &

Alkylation LC/MSE

Reduced Peptide Map


UPLC BEH300 C18, 1.7 µm, 2.1 x 100 mm


Equivalent protein coverage was obtained for innovator and biosimilar

Innovator HCInnovator


Biosimilar HC

Biosimilar LC

Innovator LC

BEH, C18, 1.7 µm, 130, 2.1x 100 mm,Gradient 1 to 35% ACN, 0.05%TFA, 60 min

Biosimilar

Asp Asp IsomerizationIsomerization of Peptide T24 of Peptide T24 (FNWYVDGVEVHNAK)(FNWYVDGVEVHNAK)

XIC

� Innovator

IsoASP

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.

Oxidation of HC Peptide T42Oxidation of HC Peptide T42

I

Biosimilar

Batch

Innovator


% Oxidation

Sample Injections

I

Batch

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

Analysis Information

LC Coverage


HC Coverage

LC Coverage

% Oxidation I

Biosimilar

Batch

I

Innovator

Batch

Experimental setup for disulfide Experimental setup for disulfide bond mappingbond mapping


Denature & Alkylate



Reduction &

Alkylation

LC/MSE

Reduced Peptide Map


UPLC BEH300 C18, 1.7 µm, 2.1 x 150 mm


� 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

VH

CH1

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

TrypsinTrypsin 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 interCHOCHO

C L

CH 3

CH 2

– –

S-S––S-S

SS

SS

SS

SS

–S-S –-S-S-

Humanized IgG

Light Chain (1)

Light Chain (4)

Heavy Chain (2)

Heavy Chain (3)

K K

Disulfide Containing Peptides

2:T21-3:T21

NonreducedNonreduced peptide mapping enabled ID peptide mapping enabled ID

of all canonical Sof 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 Ions2:T21-3:T21

UNIFI enables researchers to focus on critical attributes of a molecule

Additional studies show there are no scrambled disulfide presence

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

Component Plot for S-S peptides

Analysis Information

KK


ComponentSummary

Disulfide containing peptides identified in both innovator and biosimilar mAb samples


Case Study 2

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

INTACT PROTEIN ANALYSIS

Innovator

Biosimilar 1

G0F/G1F

G0F/G0F

G1F/G2F

G2F/G2FG0/G0F

G1F/G1F

G0F/G2F Innovator

Biosimilar 2

G0F/G1F

G0F/G0F

G1F/G1F

G0F/G2F

G1F/G2F

G2F/G2FG0/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 mAbBiosimilar 1 glycoforms broadly match the innovator

∆m = 56 Da

Reduced Protein Analysis Reduced Protein Analysis –– LCs LCs identicalidentical

+Reduction

Innovator

Biosimilar 1

Biosimilar 2

LC

LC w/ PyQ HC

Mass Analysis of the Light ChainMass Analysis of the Light Chain


Innovator

Biosimilar 1

Innovator

Biosimilar 2

� Light chain masses are identical for Innovator, Biosimilar 1 and Biosimilar 2

Light Chain

Automated Processing and Reporting

MaxEnt1 deconvoluted mass spectra in compare mode Light Chain

Reduced Protein Analysis of Heavy Reduced Protein Analysis of Heavy ChainChain

Innovator

Biosimilar 1

G0FG1F

G2FG0F+KG1F+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

Summaryplots

Based


� 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

Basedon

UNIFIresults

Distribution of G0

Glycoform

G0F

Innovator

Biosimilar2

G0F G1F

G0F+K G2FG1F+K

G0

G0

G0F

G1F

∆m = 28 Da

LC/MSLC/MSEE TrypticTryptic peptide mapping to locate peptide mapping to locate the sequence variancethe 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

LC/MSLC/MSE E TrypticTryptic peptide mapping to locate peptide mapping to locate the sequence variancethe sequence variance

Heavy Chain - Innovator

Compare mode


HC - Biosimilar 2Biosimilar Coverage Map: shows section where no sequence match is made

LC/MSLC/MSE E TrypticTryptic peptide mapping to locate peptide mapping to locate the sequence variancethe 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

LC/MSLC/MSE E ChymotrypticChymotryptic peptide mapping peptide mapping analysis of Biosimilar 2analysis of Biosimilar 2

Innovator

Biosimilar 2

∆m = 28 Da

Innovator

Biosimilar 2BPI 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

UNIFI™ Peptide Map Workflow: UNIFI™ Peptide Map Workflow: MSMSEE data data confirming the sequence Variantconfirming 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 218

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

The differences between Innovator and Biosimilars Rituximab candidates are:


– 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

The differences between Innovator and Biosimilars Rituximab candidates are:

SummarySummary

Reduced LCMass Analysis

IntactMass Analysis

Reduced HC

ReducedPeptide Mapping

Aggregate Analysis

Glycan Analysis

Non-ReducedPeptide Mapping

Charge variant


Reduced HCMass Analysis

Charge variantAnalysis

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©2013 Waters Corporation 78www.waters.com/ biopharmbook

Thank You! Thank You!

� Questions?

� Landing Page…http://www.waters.com/AUG20

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Documents

Peptide Mapping 101: Essential Tools for Effective Development