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Practical Considerations for LC/MS Practical Considerations for LC/MS BioanalysisBioanalysis of of Proteins via the Surrogate Peptide ApproachProteins via the Surrogate Peptide Approachg p ppg p pp

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Today’s Presenter y

Erin E. Chambers, Ph.D. Director, Technology Advancement P i i l A li ti Ch i tPrincipal Applications Chemist Waters Corporation, Milford, MA USA

Erin Chambers is the Director of the Technology Advancement group and a Principal Applications Chemist, providing pharmaceutical applications development and support for Waters Erin’s primary scientific roles are toWaters. Erin s primary scientific roles are to support regulated and discovery Bioanalysis, and develop bioanalytical methods for drug molecules, both large and small. She is responsible for sample preparation, mass

t t d LC th d d l tspectrometry, and LC method development, and also provides customer and in-house training on these topics. Her most recent focus has been on peptide and protein bioanalysis. Her primary research interests include diabetes


and Alzheimer’s disease.

Practical Considerations for LC/MS Bioanalysis of Proteins via the Surrogate

P tid A hPeptide Approach


Erin E Chambers, PhD

Topicsp

Challenges in Workflow Options and Typical Steps

Antibodies and Choice of Representative Peptide

Eff t f P t i d P tid l l l Effect of Protein and Peptide-level clean-up

Addressing the Challenges with a Flexible Kit-based Approachg g pp

ADC Quantification

What about intact antibody analysis?


Challenges for an LC/MS Approachg pp

Many possible workflow options– How do I choose?How do I choose?

– What steps should I include/omit for my protein?

Method DevelopmentM t t ti i– Many steps to optimize

Lack of standardization – approaches

– reagent choice/quality

– different labs, different results

ReproducibilityReproducibility– Long term lot-to-lot reproducibility/traceability

Sensitivity


Possible Protein Bioanalysis Workflowsfor Surrogate Peptide Approachg p pp


Sample Preparation Method Development Challenges: Digestion

Optimization of protein denaturationOptimization of protein denaturation

Optimization of protein reduction/alkylation: time, temp, Optimization of protein reduction/alkylation: time, temp, y , p,reagent; omit?

y , p,reagent; omit?

Finding the appropriate enzyme,Finding the appropriate enzyme,Finding the appropriate enzyme, protein : enzyme ratio, tempFinding the appropriate enzyme, protein : enzyme ratio, temp

Digestion time optimizationDigestion time optimization


Testing digestion reproducibilityTesting digestion reproducibility

Optimization Complexity: Enzyme

native peptide13C15N peptide

80

90

100

Digestion Efficiencynative peptide13C15N peptide

40

50

60

70

Increasing Digestion Time

20

30

10 20 30 50 100

Protein to trypsin ratioIncreasing Protein:Trypsin Ratio

Type A

Type B ($9.2/mg)

TypeA

($473/mg)

Type B ($820/mg)

Type A ($1030/mg)

yp($150/mg)

Type A ($0.3/mg)

Type C ($0.5/mg)

Type B ($0 2/mg)

Type C ($0.7/mg)

($0.2/mg)


Vendor #1 Vendor #2 Vendor #3 Vendor #4

Optimization Complexity: Reducing Agent Concentration There is a sweet spot for reducing agent concentration

– For a representative generic signature peptide (GPSV), area counts reached a peak in the middle of the concentration range

F th i i t tid f R i d t i d ith d i

g

– For the unique signature peptide of Remicade, area counts increased with decreasing concentrations of reduction reagent

%Area


Decreasing Concentration

Optimization Complexity: Reduction Temperature So many literature protocols

Different reagents, different optimal temperature

Reagent 1 prefers higher temperature

Reagent 2 prefers lower temperature

p

Reagent 2 prefers lower temperature

%AreaNormalized to 80C

%AreaNormalized to 80C

Reducing Agent 1 60°C Reducing Agent 2 60°C

One must carefully choose the specific


y preducing agent, the concentration of each reagent, and the time and temperature

4 variables to optimize!

Optimization Complexity: Reduction/Alkylation Time

Peptide area increases with decreased reduction time

Peptide area decreases with decreased alkylation time

y

p y

%Area%Area Reduction Time Alkylation Time

Decreasing time D i tiDecreasing time(normalized to longest time)

Decreasing time(normalized to longest time)


Sensitivity and Sample Prep: What is Enough and How do I Get There?g

Specific:Anti-human in Rat

Generic:Protein A in Human

None:Direct Human Plasma

100 ng/mL500 ng/mL 500 ng/mL

100 ng/mL100 ng/mL

50 ng/mL

50 ng/mL 50 ng/mL

Blank Plasma Blank Plasma Blank Plasma


Topicsp


Antibodies and Choice of Representative Peptide– Unique or generic signature peptide

o Impact on 3 vs 5-step protocol

Effect of Protein and Peptide-level clean-up Effect of Protein and Peptide level clean up

Addressing the challenges with a Flexible Kit-based Approach

ADC Quantification



Infliximab (Remicade)Remicade Light chain [2]:Remicade Light chain [2]: DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQSHSWPFTFGSGTNLEVKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Remicade Heavy chain [2]: EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMNSLRTEDTGVYYCSRNYYGSTYDYGQGTTLTVSXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

C d iUnique signature

Van Dongen et al. 61st ASMS, MP525 Minneapolis Minnesota, USA 9-13 June 2013.

Conserved region: bluevariable regions: redCDR regions: green

Generic signature

USA 9 13 June 2013.

Formula: C6428H9912N1694O1987S46Molecular Weight: ~ 149.1 kD


http://www.drugbank.ca/drugs/DB00065

Trastuzumab (Herceptin)

Conserved region Surrogate Peptides

Anti-HER2 Light chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP

Anti-HER2 Heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY

RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Q Q QADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Unique SignatureGeneric Signature

Formula: C6470H10012N1726O2013S42Molecular Weight: ~ 145.5 kDa(claims are 148 package insert)



Bevacuzimab (Avastin)

Bevacizumab light chainDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECBevacizumab heavy chainEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Unique Signature

Generic Signature

Drug Bank

Generic Signature


Formula: C6538H10034N1716O2033S44Molecular Weight: ~ 149.0 kD


Adalimumab (Humira)( )

Light chain:DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Heavy chain: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVS Q QSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Unique Signature

Generic Signature

Drug Bankhttp://www drugbank ca/drugs/DB00051

C6428H9912N1694O1987S46Protein average weight 144190.3000



Peptide Choice: Impact on Protocol for Unique Peptides

Eliminate reduction/alkylation?

%Area

3 step, normalized to 5 step protocol


For unique signature peptides from 4 monoclonal antibody drugs, 3 step protocol works well

Peptide Choice: Impact on Protocol for Generic Peptides

Eliminate reduction/alkylation?

3 step, normalized to 5 step protocol

%Area

For generic signature peptides from 4 monoclonal antibody drugs 5 step protocol is more efficient more often than not


drugs, 5 step protocol is more efficient, more often than not

Labeled Antibody Internal Standard: SILu™Mab

SILuMab Heavy ChainEVQLVESGGGLVQPGGSLRLSCVASGFTLNNYDMHWVRQGIGKGLEWVSKIGTAGDRYYAGSVKGRFTISRENAKDSLYLQMNSLRVGDAAVYYCARGAGRWAPLGAFDIWGQGTMVTVSS|ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFAPLGAFDIWGQGTMVTVSS|ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSQ Q QRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SILuMab Light ChainQSALTQPRSVSGSPGQSVTISCTGTSSDIGGYNFVSWYQQHPGKAPKLMIYQSALTQPRSVSGSPGQSVTISCTGTSSDIGGYNFVSWYQQHPGKAPKLMIYDATKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGDYTPGVVFGGGTKLTVL|GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

Labeled Peptides:DTLMISR Heavy Chain (IgG1, IgG2, IgG3, IgG4)FNWYVDGVEVHNAK Heavy Chain (IgG1)VVSVLTVLHQDWLNGK Heavy Chain (IgG1 IgG3 IgG4)


VVSVLTVLHQDWLNGK Heavy Chain (IgG1, IgG3, IgG4)NQVSLTCLVK Heavy Chain (IgG1, IgG2, IgG3, IgG4)GFYPSDIAVEWESNGQPENNYK Heavy Chain (IgG1, IgG4)AGVETTTPSK Light Chain (lambda)YAASSYLSLTPEQWK Light Chain (lambda)

Intact Murine mAb Mass Check Standard (Waters)( )

Murine mAb Light chain:DVLMTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECQ QMurine mAb Heavy chain:QVQLKESGPGLVAPSQSLSITCTVSGFSLLGYGVNWVRQPPGQGLEWLMGIWGDGSTDYNSALKSRISITKDNSKSQVFLKMNSLQTDDTAKYYCTRAPYGKQYFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTDSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAHTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG

Conserved region: blue Variable regions: in red Signature peptides: BOXED

Signature

Generic

Signature peptides: BOXED


Murine Monoclonal Antibody

prot A spe blk ISTD murine mab

10014Aug2015_RemicadeSPE_ProteinA_01004 MRM of 11 Channels ES+

983.95 > 397.21 (Murine 4)4.91e6

2

4

Murine mAbInternal Standard

Retention Time( i )

Murine mAb IS Peptides

%

1

2

3

Internal Standard (min)1 MNSLQTDDTAK 4.062 VNSAAFPAPIEK 5.073 NTQPIMDTDGSYFVYSK 5.374 SVSELPIMHQDWLNGK 5.66

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

0

prot a spe 50 ug/ml


633.1 > 731.8 ( Remicade DILLTQSPAILSVSPGER)8.35e6

6

7

8A tib d D U i

Retention Time(min)

5 SINSATHYAESVK* 4.22

mAb Peptides

Time

%

0

5

9

Antibody Drug Unique and Generic Signature Peptides

6 DSTYSLSSTLTLSK 5.437 GPSVFPLAPSSK 5.488 DILLTQSPAILSVSPGER* 6.029 VVSVLTVLHQDWLNGK 6.16

Generic Internal StandardW kfl h k St d d

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

0


Workflow check Standard

Topicsp



Effect of Protein and Peptide level clean up Effect of Protein and Peptide-level clean-up– Precipitation pre-treatment– Generic and Specific Affinity Capture of Protein– Peptide purification from digest– Peptide purification from digest

Addressing the challenges with a Flexible Kit-based Approach

ADC Quantification



Albumin Interferences: HSA peptides and Trastuzumab peptidep p

MEOH 1:1

%

10025Jan2015_Herceptin_dilution_MEOH_1002 MRM of 24 Channels ES+

485.2 > 608.3 (Herceptin-FTISADTSK HC)7.03e5

5.18

Trastuzumab (100ug/mL)

100

-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.000

4.59 5.90

25Jan2015_Herceptin_dilution_ACN_1014 MRM of 24 Channels ES+ TIC

2.22e88.696.07

5.566.33

7.14

0 00 1 00 2 00 3 00 4 00 5 00 6 00 7 00 8 00 9 00 10 00 11 00 12 00 13 00

%

0

4.48

4.24 4.94

6.657.84

8.12

Albumin Peptides

%

40.000

60.000

80.000

-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.0025Jan2015_Herceptin_dilution_MEOH_1002 % A

Range: 900.02

% Aqueous

Time-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

20.000


PPT Pretreatment: Albumin depletion and Herceptin Recoveryp y Normalized to Direct Digestion Incubation (100 uL Plasma)

Reduced recovery of trastuzumab (Herceptin) seen with organic solvents with greatest albumin removal

Solvent 4 in 1:1 ratio to plasma yields greatest balance of albumin removal and increased area counts for trastuzumab


Off-line Sample Preparation Options for Purification at the Protein Level

Off‐line Sample Preparation Technique

General Proteins

Monoclonal Antibodies Discovery

Development/Clinical Sensitivity

None x x x lowMW cutoff filters x x x x lowDepletion Plates x x x x lowProtein A/G x x x mediumKappa (anti‐human) x x highKappa (anti human) x x highSpecific Capture Reagent x x x high


Protein A/G Cross Reactivity

Can be used as generic purification in discovery for certain pre-clinical species


Removal of Endogenous Background: Effect of Protein-Level Purification





Effect of Protein-Level Purification : Detection Limit Comparisonp




100 ng/mL

100 ng/mL

LLOQ 50 ng/mL

LOD ~50 ng/mL 100 ng/mL


LOD >100 ng/mL

Conclusions: Comparison of Pre-fractionation Techniquesq

Absolute LLOQ/LOD for Kappa anti-human and Protein A similar– Kappa S:N much higher and integration much cleaner

– Protein A LLOQ may be 2-3X higher/worse due to interferences and ease of integration

LLOQ/LOD for whole digest 2-10X worse than Kappa or Protein AQ/ g pp

NSB may occur after very specific clean-up at protein level!– Too clean?

All t h i i ld li All techniques yield linear curves– Anti-human 3 orders magnitude

o Due to capacity

• When more beads are used, the linear range could be extended

o Will NSB impact be even greater using more beads?

– Protein A 4 orders magnitude


g

– Whole plasma digestion 3 – 4 orders magnitude

Matrix Effects at the Signature Peptide LevelPeptide Level

1 nM trastuzumab in ~1500 area countsas u u abSolvent A

~1500 area counts

human serum digest

1 nM trastuzumab in serum digest

~500-700 area counts= 2-3X lower!g


Addressing the problem with sample prep………

Peptide Clean-up: Mixed-mode Cation Exchange SPE

100

120

Oasis MCX

g

0

20

4060

80100

0

OasisWCX

6080

100120

Oasis WCX

Single SPE method recovers unique and generic signature

02040unique and generic signature

peptides with high efficiencyStrong cation exchange mixed-mode (Oasis® MCX) best overallµElution format eliminates dry


µElution format eliminates dry down and concentrates up to 15X

Example 1: Incremental Improvement in Signal Combining Clean-up Options

1001: MRM of 2 Channels ES+

485.2 > 721.4 (FTISADTSK HC)6.23e5

Area

5 00 5 50 6 00 6 50 7 00 7 50 8 00 8 50 9 00 9 50 10 00

%

0

7.093185

Direct plasma digestion: no clean-up

%

100

5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.001: MRM of 2 Channels ES+

485.2 > 721.4 (FTISADTSK HC)6.23e5

Area

7.1014439 Protein A plasma clean-up

5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

%

0

1: MRM of 2 Channels ES+ 485 2 > 721 4 (FTISADTSK HC)7 09

p p

%

100 485.2 > 721.4 (FTISADTSK HC)6.23e5

Area

7.0927490

Protein A plasma clean-up, Oasis MCX digest clean-up


Time5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.000

Example 2: Mixed-mode Cation Exchange Clean-up of Unique Herceptin Peptide

Before SPE

Summary

Strong cation exchange most

Before SPE

Strong cation exchange most universal– >90% recovery for unique and

generic signature peptidesgeneric signature peptides tested

Eliminates digest reagents

I iti itAfter SPE

Increases sensitivity

Eliminates phospholipids

Digest concentration without

After SPE

gevaporation

Increased system robustness


Topicsp



Effect of Protein and Peptide level clean up Effect of Protein and Peptide-level clean-up

Addressing the challenges with a Flexible Kit-based ApproachR d ibilit d St d di ti– Reproducibility and Standardization

– Broadly applicable protocol– Sensitivity

ADC Quantification



Solution to the Complexity:Waters Kits

Prototype Kits: A Few Key Attributes

Processing time for 3-step: ~2 hours for antibodies in plasma

Processing time for 5-step: ~3 hours for antibodies in plasma

Standardized protocols

Scalable, flexible format

Reduced variability Reduced variability

High Sensitivity

Eliminates Method Development for Discovery Studies

No capital investment required


Large Molecule Drugs

Anti-infectivePEGIntronPegasys

AvastinRitHe ceptin R i d

2009 sales ($Billion)

OncologyErbitux 5.7

AvastinRituxanHerceptin Remicade

Humira

Rheumatology

4.3Lantus Neulasta6.1

Enbrel

Levemir

Norditropin NovoMixEndocrine

CNS

NovoRapid/NovoLog

Avonex RebifTysabri

SimpleXx Humalog1.1

0.8

BloodNovoSeven KogenateProcrit/Eprex Monoclonal Ab

Other protein


Modified slide from McKinsey and CompanyData Source: Evaluate Pharma

201520142013201220112010 20202019201820172016

US Patent Expiration Date

2021

Reproducibility:Waters Prototype Kits Mean Area % CV’s

Intra-kit

Inter-kit


Standardized Protocol: Remicade(infliximab) Unique Signature Peptide(infliximab) Unique Signature Peptide

prot a no spe 350 ug/ml

%

10022Dec2014_ProtA_nospe_1042a Sm (Mn, 5x5) MRM of 10 Channels ES+

633.1 > 731.8 (TNO Remicade signatureDILLTQSPAILSVSPGER)1.37e7

Area

1080273

350.0 g/mL

%

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.500

22Dec2014_ProtA_nospe_1029 Sm (Mn, 5x5) MRM of 10 Channels ES+ 633.1 > 731.8 (TNO Remicade signatureDILLTQSPAILSVSPGER)

1.58e6Area

119196

35.0 g/mL

%

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.500


2.91e5Area

12417

g

3.5 g/mL

%

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.500


3.70e4Area

1398

0.35 g/mL

%

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.500


1.23e4Area

84

Blank plasma


Time2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

%

0

84

Standardized Protocol: Remicade(infliximab) Generic Signature Peptide( ) g p

prot a no spe 350 ug/ml

%

10022Dec2014_ProtA_nospe_1042a Sm (Mn, 5x5) MRM of 10 Channels ES+

603.3 > 1110.6 (TNO Furlong Remicade signatureVVSVLTVLHQDWLNGK)1.09e7

Area

867524

350.0 g/mL

%

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

%

0

22Dec2014_ProtA_nospe_1029 Sm (Mn, 3x3) MRM of 10 Channels ES+ 603.3 > 1110.6 (TNO Furlong Remicade signatureVVSVLTVLHQDWLNGK)

2.16e6Area

104779

35.0 g/mL

%

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

%

0


2.52e5Area

11082

g

3.5 g/mL

%

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

%

0


3.78e4Area

1272

0.35 g/mL

%

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

%

0


2.34e3Area

24Blank plasma


Time2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

%

0

Standardized Protocol: Remicade(infliximab) Unique Signature Peptide( ) q g p

Std. Curve Range Linear fit

Mean % Accuracy of all

PeptideRange (ug/mL) Weighting

Linear fit (r2)

of all points

DILLTQSPAILSVSPGER0.25‐500 1/x 0.998 100.01

DILLTQSPAILSVSPGER* 0.25‐500 1/x 0.995 101.26

Compound name: Remicade DILLTQSPAILSVSPGERCorrelation coefficient: r = 0.998949, r̂ 2 = 0.997898Calibration curve: 0.16721 * x + 0.0293672Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Otherwise quant using DTL Silumab peptide as IS* Quantified using SILUMAB‐VVSV (IS)

Conc

Res

idua

l

-10.0

0.0

10.0

Res

pons

e

25.0

50.0

75.0


Conc-0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

-0.0

Standardized Protocol: Remicade(infliximab) Unique Signature Peptide(infliximab) Unique Signature Peptide

Mean Cal ConcQC Conc (ug/mL)

Mean Cal. Conc (ug/mL) Std. Dev. %CV Mean Accuracy

DILLTQSPAILSVSPGER* 0.35 0.33 0.02 5.80 93.2SILUMAB‐DTL(IS) 3.50 3.79 0.02 0.49 108.2

35.00 39.58 0.17 0.44 113.1350 00 350 02 3 09 0 88 100 0350.00 350.02 3.09 0.88 100.0

QC Conc (ug/mL)Mean Cal. Conc

(ug/mL) Std. Dev. %CV Mean AccuracyDILLTQSPAILSVSPGER* 0.35 0.34 0.00 0.89 96.5SILUMAB‐VVSV (IS) 3.50 3.65 0.05 1.47 104.2

35 00 36 51 0 73 2 01 104 335.00 36.51 0.73 2.01 104.3350.00 350.80 3.90 1.11 100.2

* Signature


Standardized Protocol: Remicade(infliximab) Generic Signature Peptide( ) g p

PeptideStd. Curve Range (ug/mL) Weighting Linear fit (r2)

Mean % Accuracy of all points

GPSVFPLAPSSK 0.25‐250 1/x2 0.997 100.01GPSVFPLAPSSK 0.25 250 1/x 0.997 100.01

STSGGTAALGC[+57]LVK 0.25‐500 1/x2 0.986 99.01DSTYSLSSTLTLSK 0.25‐500 1/x 0.998 100.00DSTYSLSSTLTLSK* 0.25‐500 1/x 0.998 100.00VVSVLTVLHQDWLNGK 0.25‐500 1/x 0.999 100.00VVSVLTVLHQDWLNGK* 0 25‐500 1/x 0 998 100 00

Compound name: Merck DSTYSLSSTLTLSKCorrelation coefficient: r = 0.999151, r̂ 2 = 0.998303

All others are quant using DTL Silumab pepitde as ISCompound name: Furlong Remicade VVSVLTVLHQDWLNGKCorrelation coefficient: r = 0.999486, r̂ 2 = 0.998973Calibration curve: 0.139921 * x + 0.020816Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

VVSVLTVLHQDWLNGK 0.25‐500 1/x 0.998 100.00*Quantified using SILUMAB‐VVSV (IS)

Calibration curve: 0.430163 * x + 0.0341835Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Res

idua

l50

0.0

5.0

Res

idua

l

-10.0

0.0

10.0

pons

e

200

Conc

R

-10.0

-5.0

Res

pons

e

200

40.0

60.0

Conc


Conc-0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

Res

p

-0

100

Conc-0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520

-0.0

20.0

Standardized Protocol: Remicade(infliximab)Generic Signature Peptide(infliximab)Generic Signature Peptide

Peptide QC Conc (ug/mL)Mean Cal. Conc

(ug/mL) Std. Dev. %CV Mean AccuracyGPSVFPLAPSSK 0.35 0.38 0.00 1.05 109.7SILUMAB‐DTL(IS) 3.50 3.87 0.07 1.90 110.7( )

35.00 36.49 0.61 1.67 104.2350.00 ‐ ‐ ‐ ‐


(ug/mL) Std. Dev. %CV Mean AccuracySTSGGTAALGC[+57]LVK 0.35 0.38 0.01 3.39 109.4SILUMAB‐DTL(IS) 3.50 3.62 0.22 6.20 103.5

35.00 35.01 3.29 9.40 100.0350.00 353.43 4.85 1.37 101.0


(ug/mL) Std. Dev. %CV Mean AccuracyDSTYSLSSTLTLSK 0.35 0.37 0.00 0.27 105.7SILUMAB‐DTL(IS) 3.50 3.80 0.08 2.22 108.5

35.00 37.53 0.61 1.64 107.235.00 37.53 0.61 1.64 107.2350.00 347.51 2.50 0.72 99.3


(ug/mL) Std. Dev. %CV Mean AccuracyDSTYSLSSTLTLSK 0.35 0.37 0.00 0.95 105.5SILUMAB‐VVSV (IS) 3.50 3.69 0.15 4.17 105.6

35.00 35.03 0.55 1.56 100.1350.00 364.85 7.64 2.10 104.2


(ug/mL) Std. Dev. %CV Mean AccuracyVVSVLTVLHQDWLNGK 0.35 0.35 0.01 3.89 100.9SILUMAB‐DTL(IS) 3.50 3.91 0.04 0.95 111.6

35.00 37.03 1.59 4.28 105.8350 00 347 27 13 57 3 91 99 2


350.00 347.27 13.57 3.91 99.2


(ug/mL) Std. Dev. %CV Mean AccuracyVVSVLTVLHQDWLNGK 0.35 0.35 0.01 3.85 99.7SILUMAB‐VVSV (IS) 3.50 3.66 0.02 0.50 104.7

35.00 37.41 0.68 1.81 106.9350.00 351.28 3.01 0.86 100.4

Standardized Protocol: Herceptin(trastuzumab) Unique Signature Peptide

Compound name: FT peptideCorrelation coefficient: r = 0.999927, r̂ 2 = 0.999853Calibration curve: 0.523327 * x + 0.00974718Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )C t Li O i i E l d W i hti 1/ A i t N

Trastuzumab (Herceptin) Whole Plasma Digest: Linearity 100 ng/mL – 500 µg/mL

Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

se

200

Conc

Res

pons

-0

100 Std. Concug/mL Area IS Area Conc. %Dev

5321Conc-0 50 100 150 200 250 300 350 400 450 500

0 53210.05 57280.1 276 4576 0.1 ‐3.20.5 1526 5315 0.5 60.75 1957 4409 0.8 10.60.75 1957 4409 0.8 10.6

1 2279 4695 0.9 ‐9.15 9652 4078 4.5 ‐9.910 19661 3540 10.6 5.950 53272 2684 37.9 ‐24.2


250 329709 2533 248.7 ‐0.5500 694625 2648 501.1 0.2

Standardized Protocol: Herceptin(trastuzumab) Unique Signature Peptide( ) q g pCompound name: FT peptideCorrelation coefficient: r = 0.999068, r̂ 2 = 0.998137Calibration curve: 0.108766 * x + -0.000178401Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )C t Li O i i E l d W i hti 1/ A i t N

Trastuzumab (Herceptin) Kappa affinity: Linearity 50 ng/mL – 50 µg/mL

Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

4 00

Res

pons

e

2.00

4.00

Std. Conc Area IS Area Conc. %Dev61 87479 0

Conc-0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

-0.00 61 87479 00.05 419 76438 0.05 4.10.1 843 69846 0.1 12.60.5 3521 67483 0.5 ‐3.70.75 5140 72747 0.7 ‐13.2

1 7191 69310 1 ‐4.45 36193 69041 4.8 ‐3.610 69955 58730 11 9.5


50 334224 62240 49.4 ‐1.3

Standardized Protocol: Avastin(bevacizumab) QC Statistics(bevacizumab), QC Statistics


(ug/mL) Std. Dev. %CV Mean AccuracyGPSVFPLAPSSK 0.35 0.34 0.03 9.88 96.4SILUMAB‐DTL(IS) 3.50 3.84 0.06 1.67 109.8

35 00 37 88 1 49 3 94 108 235.00 37.88 1.49 3.94 108.2*350.00 368.26 1.29 0.35 105.2


(ug/mL) Std. Dev. %CV Mean AccuracySTSGGTAALGC[+57]LVK 0.35 0.35 0.02 6.80 100.6SILUMAB DTL(IS) 3 50 3 62 0 10 2 73 103 4SILUMAB‐DTL(IS) 3.50 3.62 0.10 2.73 103.4

35.00 38.20 1.52 3.99 109.1350.00 341.18 17.14 5.02 97.5


(ug/mL) Std. Dev. %CV Mean AccuracyDSTYSLSSTLTLSK 0 35 * * * *DSTYSLSSTLTLSK 0.35 * * * *SILUMAB‐DTL(IS) 3.50 3.36 0.17 5.06 96.0

35.00 37.60 1.50 3.99 107.4350.00 381.10 8.99 2.36 108.9

* Outside of curve dynamic range


Standardized Protocol: Avastin(bevacizumab)

prot a no spe 0.5 ug/ml

%

0

1009Jan2015_ProtA_nospe_1037 Sm (Mn, 2x2) MRM of 10 Channels ES+

751.88 > 836.47 (Merck DSTYSLSSTLTLSK)2.13e6

Area

71399

(bevacizumab)

10.0 g/mL

%

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

%

0

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.000

9Jan2015_ProtA_nospe_1035 Sm (Mn, 2x2) MRM of 10 Channels ES+ 751.88 > 836.47 (Merck DSTYSLSSTLTLSK)

1.15e6Area

35600


6.04e518766

5.0 g/mL

2.5 g/mL

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

%

0

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

%

0Area


5.91e5Area

8506

9Jan2015_ProtA_nospe_1026 Sm (Mn, 2x2) MRM of 10 Channels ES+

g

1.0 g/mL

2 00 2 50 3 00 3 50 4 00 4 50 5 00 5 50 6 00 6 50 7 00 7 50 8 00

%

0

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

%

0

100 751.88 > 836.47 (Merck DSTYSLSSTLTLSK)5.03e5

Area4764


5.46e5Area2509

0.5 g/mL

0.25 g/mL

%

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

%

0

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.009Jan2015_ProtA_nospe_1020 Sm (Mn, 2x2) MRM of 10 Channels ES+

751.88 > 836.47 (Merck DSTYSLSSTLTLSK)4.71e5

Area1374


4.40e5Area

0.10 g/mL

Blank Plasma


Time2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

%

0Area

548

Standardized Protocol: Humira(adalimumab) Unique Signature Peptide(adalimumab) Unique Signature Peptide

Peptide: APYTFGQGTK

1 500 / L

250 ug/mL

10014Aug2015_Dir_5step_SPE_mabs_HumPlsm_250p0_041 Sm (Mn, 2x3) MRM of 12 Channels ES+

535.3 > 901.44 (Humira APYTFGQGTK LC)4.93e4

Area

5.29;2528

250 0 g/mL

Compound name: HumiraCorrelation coefficient: r = 0.997626, r̂ 2 = 0.995257Calibration curve: 0.00300808 * x + -0.00157281Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

40.0

1-500 ug/mL

%

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

14Aug2015_Dir_5step_SPE_mabs_HumPlsm_50p0_038 Sm (Mn, 2x3) MRM of 12 Channels ES+ 535.3 > 901.44 (Humira APYTFGQGTK LC)

1.33e4Area

5.29;599

250.0 g/mL

50.0 g/mL

Conc

Res

idua

l

-20.0

-10.0

0.0

10.0

20.0

30.0

%

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.000


1.88e3Area

5.30;90

10.0 g/mL

Res

pons

e

0.50

0.75

1.00

1.25

1.50

Conc

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.000


966Area

5.29;53

5.0 g/mLConc

-0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500-0.00

0.25

050


Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

10014Aug2015_Dir_5step_SPE_mabs_HumPlsm_Blk_002 Sm (Mn, 2x3) MRM of 12 Channels ES+

535.3 > 901.44 (Humira APYTFGQGTK LC)1.65e3

Area

5.289

Blank plasma

Standardized Protocol: High Sensitivity Remicade (infliximab)

QC 0.035 ug/mL14Aug2015 RemicadeSPE ProteinA 01005 MRM of 11 Channels ES+

SINSATHYAESVK Unique Signature Peptide

100

-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

100g _ _ _

469.6 > 603.8 (Remicade SINSATHYAESVK )2.19e4

Area

14Aug2015_RemicadeSPE_ProteinA_01010 MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )

2.76e4Area

1143

Blank plasma

0 035 g/mL 35 ng/mL

%

100

-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

Area


2.40e5Area

10119

0.35 g/mL

0.035 g/mL g/

-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

100

-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.000


2.28e6Area

95867

14Aug2015 RemicadeSPE ProteinA 01035 MRM of 11 Channels ES+

3.5 g/mL

100

-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0


469.6 > 603.8 (Remicade SINSATHYAESVK )2.05e7

Area

857961


1.32e86321264

35.0 g/mL


Time-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

Area350.0 g/mL

Competitor vs. Waters: Digestion, No Reduction/AlkylationyArea for Unique Signature Peptides of 4 Monoclonal Antibodies,

Normalized to Waters Prototype Kit

100

120

60

80

Commerically Available C i Ki

20

40

Competitor Kit

Waters Prototype Kit

0

20


Both protocols were completed in comparable time

Competitor vs. Waters

Area for Generic Signature Peptides of 4 Monoclonal Antibodies, Normalized to Waters Prototype Kit

80

100

120

40

60

80

Commerically Available

0

20

Commerically Available Competitor Kit

Waters Prototype Kit


Topicsp



Effect of Protein and Peptide-level clean-up

Addressing the challenges with a Flexible Kit based Approach Addressing the challenges with a Flexible Kit-based Approach

ADC Quantification



Trastuzumab Emtansine (T-DM1)

Trastuzumab based

MCC (Linker)

90 lysines

SMCC linker

DM1 + MCC (D + Li k )

MCC (Linker)219.0895 Da

Drug Maytansinoid

Two-step conjugation

Hydrophobic DM1 + MCC (Drug + Linker)956.3644 Da ADCs from 3 sources

©2015 Waters Corporation 55Bioconjugate Chemistry 2014, 25, 1223-1232

Data courtesy of Liuxi Chen

Why Does Drug Location Matter ?

The potential effect of drug loading, location and distribution:

• Efficacy and Toxicity

• CDR Binding

• StabilityStability

• Pharmacokinetics

• ADC manufacturing process control

PK

E2 E4

E4 E8


Clin. Cancer Res. 10, 7063–7070.Chemistry & Biology 20, 161-167, 2013

E4 E8

ADC Conjugated Peptide Quantification StrategyQ gy

Trypsin/Lys C Asp N

• Control mAb• Lys-conjugated ADC

Peptide Mapping Accurate Mass

Identification Quantification Trypsin/Lys C Asp N

Peptide Mapping

(MSE & DDA)

Accurate Mass Screening

(MSE)

• Identifies conjugated peptides

• Confirms conjugation sites

• Generates accurate mass list of

• Quantifies conjugated peptides across samples

• Automatically combines different charge states and adducts ions

• Automatically combines intensity • Generates accurate mass list of identified conjugated peptides

uto at ca y co b es te s tyfrom the same peptide at different RT



MultiEnzymes for Lys ADC Quantification

Tr psin Asp N

Quantification

Trypsin Asp N

Unconjugated K Kj gpeptide

K K

Conjugatedpeptide K K

miscleavage

Quantitation Relative abundance of conjugated peptides

Relative site occupancy ratio



Control mAb vs T-DM1 - LC/MSE Chromatograms- LC/MS Chromatograms

T-DM1Data courtesy of Liuxi Chen

Trypsin

TmabConjugated peptide Elution window

T-DM1

Elution window

Asp N


Tmab

Control mAb vs T-DM1 - LC/MSE Chromatograms

10x

T-DM1

- LC/MS ChromatogramsData courtesy of Liuxi Chen

Trypsin

Tmab

10x

T-DM1

Asp N


Tmab

Representative Data: Relative Site Occupancy AspN ResultsOccupancy, AspN Results

20%

25%

30% Light Chain Heavy Chain FcHeavy Chain Fab

upan

cy

10%

15%

20%

ve Site

Occu

0%

5%

LC42 LC183 HC HC30 HC43 HC65 HC76 HC225 HC251 HC277 HC395

Relati

LC42 LC183 HC N‐term

HC30 HC43 HC65 HC76 HC225 HC251 HC277 HC395

Lys Site Number



Relative abundance of conjugated peptidesfrom different sources (T-DM1)

6

8

10

from different sources (T DM1)

tensity

AspN resultsTrypsin results

10

12

14

0

2

4

N‐term L42 L107 N‐term H30 H43 H65 H76 H124

Relativ

e Int

0

2

4

6

8

H30 H43 H65 H76

6

8

10

12N term L42 L107 N term H30 H43 H65 H76 H124

e Intensity

Company A ‐ Batch 1


H30 H43 H65 H76

0

2

4

L126 L145 L149 L169 L183 L188 L190 L207 H136 H216 H217

Relativ

e

20


Company B

Company C ‐ Process 1

Company C ‐ Process 2

tive Intensity

8

12

16

20

©2015 Waters Corporation 62Lys Residue Number

Relat

0

4

H225 H249 H251 H277 H291 H293 H323 H325 H329 H337 H343 H363 H395 H417 H442


Topicsp



Effect of Protein and Peptide-level clean-up

Addressing the challenges with a Flexible Kit based Approach Addressing the challenges with a Flexible Kit-based Approach

ADC Quantification



Improved Sensitivity Capability of ionKey for Deglycosylated mAb Analysisg y y y

0.1 ngPWHM: 8.5s %

100

%

100 2.90

• 40 pg LOD• 100 pg LOQ

1 ng

%

100

m/z2200 2400 2600 2800 3000 3200 3400

0

%

100

2.00 4.00 6.00 8.000

9.17

2.90

10 ng

m/z

%

0

100

m/z2200 2400 2600 2800 3000 3200 3400

0

%

0

100

2.00 4.00 6.00 8.000

2.90

man

25 ng

m/z2200 2400 2600 2800 3000 3200 3400

%0

100

m/z2200 2400 2600 2800 3000 3200 3400

0

2 00 4 00 6 00 8 00

%

0

100

2.00 4.00 6.00 8.000

2.88

f G

reg R

o

50 ng

m/z2200 2400 2600 2800 3000 3200 3400

%

0

100

2200 2400 2600 2800 3000 3200 3400

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

2.88

court

esy

o


100 ng

m/z2200 2400 2600 2800 3000 3200 3400

%

0

100

Time2.00 4.00 6.00 8.00

%

0

100 2.88

4.51

Dat

a c

Improved Sensitivity Capability of ionKey for Glycosylated mAb Analysisy y y y

• 0.4 ng LOD• 1 ng LOQ

man

f G

reg R

o

No Glycoform court

esy

o


No GlycoformCarryover

Dat

a c

Generic Affinity Capture of Humirafrom Rat Plasma

Humira LinearRange:25-500 g/mL

%

1005132015 Blank Rat Plasma_250pt0 ugmL 151 (2.947) 1: TOF MS ES+

2.00e52743.07842693.2227

2645.19122598.7388

2554.0410

2468 8767

2794.7969

2848.5042

2904.3540

2907.5510

2965.6665

%

1005132015 Blank Rat Plasma_250pt0 ugmL 151 (

2.00e52743.0784

2746.1213

250 g/mL

100

m/z2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 34000

2468.8767

2428.45852314.6531

2211.0151

3022.9172

3085.75783154.8474 3223.6106

3370.1011

5132015 Blank Rat Plasma_100pt0 ugmL 150 (2.930) 1: TOF MS ES+ 1.30e52794.82502743.0637

2693.22922848.5764

2904 3823

100

m/z2720 2740 2760

0

2739.3311

2716.3662

2748.4795

5132015 Blank Rat Plasma_100pt0 ugmL 150 (1.25e52743.0637

1005132015 Humira 100 ugmL_4 151 (2.947) 1: TOF MS ES+

7.53e5148078.0000

Deconvoluted Spectra

m/z2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400

%

0

2645.15582598.7661

2554.0063

2510.78322428.46562314.6995

2244.6521

2904.3823

2962.4216

3022.90093026.2302

3089.19563154.9912

3291.5718m/z

2720 2740 2760

%

0

2738.9309

2737.7749

2746.0850

2749.1294

2750.7810

100 g/mL

%

148222.0000

%

100

2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 34005132015 Blank Rat Plasma_50pt0 ugmL 150 (2.930) 1: TOF MS ES+

8.57e42794.81862743.0356

2645.1282

2598.7595

2553.99982510.6736

2428.47882314.6135

2848.5479

2904.3762

2907.5068

2965.64433022.8723

3026.9216

%

1005132015 Blank Rat Plasma_50pt0 ugmL 150 (2

8.44e42743.0356

2740.7654

2746.0999

2748.3723

50 g/mL1005132015 Humira 50 ugmL_3 147 (2.879) 1: TOF MS ES+

5.49e5148078.0000

mass148000 148250 148500 148750

0

148370.0000

148529.0000

100

m/z2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 34000

2200.60353026.9216

3155.0083

5132015 Blank Rat Plasma_25pt0 ugmL 151 (2.947) 1: TOF MS ES+ 4.26e42743.04222693.2717

2645.11382598.8704

2848.5107

2904.3823

2962.5107

100

m/z2720 2740 2760

0

2740.7654

2737.85402749.6160

2751.4180

5132015 Blank Rat Plasma_25pt0 ugmL 151 (24.26e42743.0422

2746.1492

% 148221.0000


m/z2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400

%

0

2554.0063

2468.88382351.42552314.69952204.6226

3022.8560

3026.2751

3089.1726

m/z2720 2740 2760

%

0

2742.1853

2716.86332748.8291

2749.8157

25 g/mL

mass148000 148250 148500 148750

0

148363.0000

148510.0000

Data courtesy of Greg Roman

Trastuzumab ADC (De-Glycosylated): IonKey/MS

%

1003252015 C4 Herceptin ADC 10000X Dilution_2 156 (3.051) Cm (144:169) 1: TOF MS ES+

6.08e32904.37552847.47022740.01492689.4590

2593.2161

2904.79442963.8701 3089.8511

3090.23750.25 ngTrastuzumab MW:

145165.1575 g/mol

100

m/z2400 2500 2600 2700 2800 2900 3000 3100 3200 3300

0

3252015 C4 Herceptin ADC 1000X Dilution_2 151 (2.947) Cm (143:161) 1: TOF MS ES+ 1.01e42847.47022792.9436

2792 83572904.24342904 7063

Linker and Drug: 958.5322 g/mol

1 Drug + 1 Antibody: 146123.6 g/mol

m/z2400 2500 2600 2700 2800 2900 3000 3100 3200 3300

%

0

2792.83572689.2893

2904.7063 3025.3850

3252015 C4 H ti ADC 100X Dil ti 2 151 (2 947) C (145 165) 1 TOF MS ES

2.5 ng%

1003252015 C4 Herceptin ADC 100X Dilution_2 151 (2.947) Cm (145:165) 1: TOF MS ES+

4.98e42848.01612794.3059

2724.76882644.5042

2884.98582904.7285

25.0 ng

100

m/z2400 2500 2600 2700 2800 2900 3000 3100 3200 3300

0

3252015 C4 Herceptin ADC 10X Dilution_2 150 (2.930) Cm (144:165) 1: TOF MS ES+ 2.68e52794.32762742.5422

2644.60942598.2388

2847.99412885.3372

2924.1621250 ng


m/z2400 2500 2600 2700 2800 2900 3000 3100 3200 3300

%

0

250 ng

Intact Mass Analysis on T-DM1

+Drug 2 +Drug 3

+Drug 1

+Drug 4

+Drug 5

+Drug 0 +Drug 6+Drug 7+Linker

+Linker

+Linker+Linker

+Linker +Linker

Antibody with 0-7 drugs observed

Linker-only peaks observed


Linker-only peaks observed

Conclusions

Protein BA workflows are complex– Optimization of multiple aspects of multiple steps is requiredOptimization of multiple aspects of multiple steps is required

– The number and nature of steps to include depends on many variables

Waters prototype kits provide standardized reproducible and Waters prototype kits provide standardized, reproducible, and fully flexible solutions to accommodate multiple workflows with fast, optimized protocols

C l d d b d f d d For ADC analysis, conjugated peptides can be identified and quantified using a 2-stage workflow, HRMS, and specifically designed software

Intact analysis is closer than you might think……………


Acknowledgementsg

Mary Lame

Hua Yang Hua Yang

Liuxi Chen

Henry Shion

Gregory Roman

James Murphy

Weibin Chen Weibin Chen

John Gebler

Sherri Naughton

Catalin Doneanu


Thank You for Attending!g

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