practical considerations for lc/ms biliioanalysisofiihf ... · µelution format eliminates dry down...

Practical Considerations for LC/MS i l i f i i hBioanalysis of Proteins via the Surrogate

Peptide Approach

E i E Ch b PhD

©2015 Waters Corporation 1

Erin E Chambers, PhD

TopicsTopics

Ch ll i W kfl O ti d T i l St Challenges in Workflow Options and Typical Steps

I St d di d A h P ibl ? Is a Standardized Approach Possible?

ADC Quantification

What about intact antibody analysis?


Possible Protein Bioanalysis Workflowsfor Surrogate Peptide Approachfor Surrogate Peptide Approach


Infliximab (Remicade)( )Remicade Light chain [2]: DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQSHSWPFTFGSGTNLEVKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Remicade Heavy chain [2]: EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMNSLRTEDTGVYYCSRNYYGSTYDYGQGTTLTVSXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Conserved region: blueUnique signature

Generic signature

Van Dongen et al. 61st ASMS, MP525 Minneapolis Minnesota,

gvariable regions: redCDR regions: green

Generic signature

g , p ,USA 9-13 June 2013.

Formula: C6428H9912N1694O1987S46

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

Molecular Weight: ~ 149.1 kD



Trastuzumab (Herceptin)Trastuzumab (Herceptin)

Anti-HER2 Light chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Anti-HER2 Heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

THQGLSSPVTKSFNRGEC

ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Unique Signature

Formula: C6470H10012N1726O2013S42M l l W i ht 145 5 kD

Generic Signature

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



Bevacuzimab (Avastin)

Bevacizumab light chain

( )

gDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECBevacizumab heavy chainEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKQ Q QSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEQ Q QQALHNHYTQKSLSLSPGK

Unique Signature

Generic Signature

Formula: C6538H10034N1716O2033S44Molecular Weight: ~ 149 0 kD

Drug Bank


Molecular Weight: 149.0 kD


Adalimumab (Humira)Adalimumab (Humira)

Light chain:gDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Heavy chain: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Unique Signature

Generic Signature

C6428H9912N1694O1987S46Protein average weight 144190 3000

Generic Signature

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

Protein average weight 144190.3000


Peptide Choice: Impact on Protocol for Unique Peptides

Eliminate reduction/alkylation?Unique Peptides

%Area

3 step, normalized to 5 step protocol

For unique signature peptides from 4 monoclonal


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

Peptide Choice: Impact on Protocol for Generic PeptidesGeneric Peptides

Eliminate reduction/alkylation? / y

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


Labeled Antibody Internal Standard: SILu™MabSILu™MabSILuMab Heavy ChainEVQLVESGGGLVQPGGSLRLSCVASGFTLNNYDMHWVRQGIGKGLEWVSKIEVQLVESGGGLVQPGGSLRLSCVASGFTLNNYDMHWVRQGIGKGLEWVSKIGTAGDRYYAGSVKGRFTISRENAKD SLYLQMNSLRVGDAAVYYCARGAGR WAPLGAFDIWGQGTMVTVSS|ASTKG PSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG

SILuMab Light ChaingQSALTQPRSVSGSPGQSVTISCTGT SSDIGGYNFVSWYQQHPGKAPKLMI YDATKRPSGVPDRFSGSKSGNTASLT ISGLQAEDEADYYCCSYAGDYTPGV VFGGGTKLTVL|GQPKAAPSVTLFP PSSEELQANKATLVCLISDFYPGAV TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

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


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

Murine Monoclonal Antibody I t l St d dInternal Standard

prot A spe blk ISTD murine mab

10014Aug2015_RemicadeSPE_ProteinA_01004 MRM of 11 Channels ES+

983.95 > 397.21 (Murine 4)4 91e6

4

%

4.91e6

2Murine mAbInternal Standard

Retention Time(min)

1 MNSLQTDDTAK 4.06

Murine mAb IS Peptides

Time1 00 2 00 3 00 4 00 5 00 6 00 7 00 8 00 9 00

0

1 32 VNSAAFPAPIEK 5.073 NTQPIMDTDGSYFVYSK 5.374 SVSELPIMHQDWLNGK 5.66

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

prot a spe 50 ug/ml

10014Aug2015_RemicadeSPE_ProteinA_01038 MRM of 11 Channels ES+

633.1 > 731.8 ( Remicade DILLTQSPAILSVSPGER)8.35e6

6

7

8

Retention Time(min)

5 SINSATHYAESVK* 4 22

mAb Peptides

%

5

6 8Antibody Drug Unique and Generic Signature Peptides

5 SINSATHYAESVK 4.226 DSTYSLSSTLTLSK 5.437 GPSVFPLAPSSK 5.488 DILLTQSPAILSVSPGER* 6.029 VVSVLTVLHQDWLNGK 6.16

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

0

9

Generic Internal StandardWorkflow check Standard


Effect of Protein-Level Purification : Detection Limit ComparisonDetection Limit Comparison

Specific: Generic: None:

100 ng/mL

Specific:Anti-human in Rat

Generic:Protein A in Human

None:Direct Human Plasma

100 ng/mL

100 ng/mL

LLOQ ≤50 ng/mL

LOD ~10-50 ng/mL 100 ng/mL


LOD >100 ng/mL

Challenges for an LC/MS ApproachChallenges for an LC/MS Approach

Many possible workflow options– How do I choose?– What steps should I include/omit for my protein?M h d D l Method Development– Many steps to optimize

Lack of standardization Lack of standardization – approaches – reagent choice/qualityreagent choice/quality– different labs, different results

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

Sensitivity

Is it possible to have a standardized approach for discovery t di ?


studies?

ProteinWorks™ eXpress Digest Kit: Inter and Intra Kit Reproducibility Inter and Intra-Kit Reproducibility for Direct Digest of Whole Plasma

©2015 Waters Corporation 145 Lots trypsin, 4 prep days, 3 analysts

Peptide Clean-up: ProteinWorks™ µElution SPE

Oasis MCX

µElution SPE

6080

100120

Oasis MCX

0204060

Single SPE method recovers Single SPE method recovers unique and generic signature peptides with high efficiencyStrong cation exchange mixed-Strong cation exchange mixed-mode (Oasis® MCX) best overallµElution format eliminates dry down and concentrates up to


down and concentrates up to 15X

Standardized Protocol: Remicade(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)1080273

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

100 633.1 > 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 9.5022Dec2014_ProtA_nospe_1029 Sm (Mn, 5x5) MRM of 10 Channels ES+

633.1 > 731.8 (TNO Remicade signatureDILLTQSPAILSVSPGER)1.58e6

Area

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

22Dec2014_ProtA_nospe_1021 Sm (Mn, 3x3) MRM of 10 Channels ES+ 633.1 > 731.8 (TNO Remicade signatureDILLTQSPAILSVSPGER)

2.91e5Area

12417

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

22Dec2014_ProtA_nospe_1012 Sm (Mn, 3x3) MRM of 10 Channels ES+ 633.1 > 731.8 (TNO Remicade signatureDILLTQSPAILSVSPGER)1398

3.5 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 8.50 9.00 9.50

%

0

003.70e4

Area0.35 g/mL

%

10022Dec2014_ProtA_nospe_1005 Sm (Mn, 5x5) MRM of 10 Channels ES+

633.1 > 731.8 (TNO Remicade signatureDILLTQSPAILSVSPGER)1.23e4

Area

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

Standardized Protocol: Remicade(infli imab) Uniq e Signat e Peptide(infliximab) Unique Signature Peptide

Peptide

Std. Curve Range (ug/mL) Weighting

Linear fit (r2)

Mean % Accuracy of all points

0 25QC Conc (ug/mL)

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

DILLTQSPAILSVSPGER0.25‐500 1/x 0.998 100.01

DILLTQSPAILSVSPGER* 0.25‐500 1/x 0.995 101.26

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 0.88 100.0

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)QC Conc (ug/mL)


DILLTQSPAILSVSPGER* 0.35 0.34 0.00 0.89 96.5SILUMAB‐VVSV (IS) 3.50 3.65 0.05 1.47 104.2

Res

idua

l

0.0

10.035.00 36.51 0.73 2.01 104.3350.00 350.80 3.90 1.11 100.2

* Signature

75.0

Conc

-10.0

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

Res

pons

e

-0.0

25.0

50.0


Standardized Protocol + Affinity: High S iti it R i d (i fli i b)Sensitivity Remicade (infliximab)

Blank rat plasma digest, no IS, after protein A, SPE, 1

100092315_WAA678_CD_006a Sm (Mn, 2x3) MRM of 11 Channels ES+

469.6 > 603.8 (Remicade SINSATHYAESVK )2.20e4

Area

%

4.1536110 ng/mL

2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.400

09231 WAA6 8 CD 004 S (M 2 3) MRM f 11 Ch l ES100

092315_WAA678_CD_004a Sm (Mn, 2x3) MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )

2.20e4Area

Blank Plasma

%

Blank Plasma

Time2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40

0


Standardized Protocol: Remicade(infli imab) Gene ic Signat e Peptide(infliximab) Generic Signature Peptide

prot a no spe 350 ug/ml

22Dec2014 ProtA nospe 1042a Sm (Mn, 5x5) MRM of 10 Channels ES+

%

0

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 9.500

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 9.500

22Dec2014_ProtA_nospe_1021 Sm (Mn, 2x3) MRM of 10 Channels ES+ 603.3 > 1110.6 (TNO Furlong Remicade signatureVVSVLTVLHQDWLNGK)

2.52e5Area

11082

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

22Dec2014_ProtA_nospe_1012 Sm (Mn, 1x1) MRM of 10 Channels ES+ 603.3 > 1110.6 (TNO Furlong Remicade signatureVVSVLTVLHQDWLNGK)1272

3.5 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 8.50 9.00 9.50

%

0

100 ( g g )3.78e4

Area0.35 g/mL

%

10022Dec2014_ProtA_nospe_1005 Sm (Mn, 1x1) MRM of 10 Channels ES+

603.3 > 1110.6 (TNO Furlong Remicade signatureVVSVLTVLHQDWLNGK)2.34e3

Area24

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 9.500

Standardized Protocol: Remicade(infli imab) Gene ic Signat e Peptide(infliximab) Generic Signature Peptide

Std Curve Range Mean % AccuracyMean Cal. Conc

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

Mean % Accuracy of all points

GPSVFPLAPSSK 0.25‐250 1/x2 0.997 100.01

STSGGTAALGC[+57]LVK 0.25‐500 1/x2 0.986 99.01

Peptide QC Conc (ug/mL) (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 ‐ ‐ ‐ ‐

DSTYSLSSTLTLSK 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

QC Conc (ug/mL)Mean Cal. Conc

(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 9.40 100.0

Compound name: Merck DSTYSLSSTLTLSK

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 )

*Quantified using SILUMAB‐VVSV (IS) 350.00 353.43 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 2.22 108.5p

Correlation coefficient: r = 0.999151, r̂ 2 = 0.998303Calibration 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

50

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

sidu

al

0.0

10.0

35.00 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 0.95 105.5

ConcR

esid

ual

-10.0

-5.0

0.0

5.0

Conc

Re

-10.0

0.0SILUMAB‐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

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

(ug/mL) Std. Dev. %CV Mean AccuracyR

espo

nse

100

200

C

Res

pons

e

00

20.0

40.0

60.0 VVSVLTVLHQDWLNGK 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

Mean Cal. 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

-0

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

-0.0QC Conc (ug/mL) (ug/mL) Std. Dev. %CV Mean Accuracy

VVSVLTVLHQDWLNGK 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(trastuzumab) Unique Signature PeptideCompound name: FT peptideCorrelation coefficient: r = 0.999927, r̂ 2 = 0.999853

Trastuzumab (Herceptin) Whole Plasma Digest: i i 00 / 00 /

Co eat o coe ce t 09999 , 0 999853Calibration curve: 0.523327 * x + 0.00974718Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Linearity 100 ng/mL – 500 µg/mL

nse 200

Res

pon

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

Conc-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 60.5 1526 5315 0.5 60.75 1957 4409 0.8 10.6

1 2279 4695 0.9 ‐9.15 9652 4078 4 5 9 95 9652 4078 4.5 ‐9.910 19661 3540 10.6 5.950 53272 2684 37.9 ‐24.2250 329709 2533 248 7 ‐0 5


250 329709 2533 248.7 0.5500 694625 2648 501.1 0.2

Standardized Protocol: Herceptin(trastuzumab) Unique Signature Peptide(trastuzumab) Unique Signature PeptideCompound name: FT peptideCorrelation coefficient: r = 0.999068, r̂ 2 = 0.998137

Trastuzumab (Herceptin) Kappa affinity: Linearity 50 ng/mL 50 µg/mLCalibration curve: 0.108766 * x + -0.000178401

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

Linearity 50 ng/mL – 50 µg/mL

se

4.00

Res

pons

2.00Std. Conc Area IS Area Conc. %Dev

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 9.550 334224 62240 49.4 ‐1.3


Standardized Protocol: Avastin(bevacizumab), QC Statistics

Mean Cal. Conc Peptide QC Conc (ug/mL) (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 3.94 108.2*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 6.80 100.6[ ]SILUMAB‐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

Mean Cal. ConcQC Conc (ug/mL)


DSTYSLSSTLTLSK 0.35 * * * *SILUMAB‐DTL(IS) 3.50 3.36 0.17 5.06 96.0

35 00 37 60 1 50 3 99 107 435.00 37.60 1.50 3.99 107.4350.00 381.10 8.99 2.36 108.9

* Outside of curve dynamic range


Standardized Protocol: Humira( d li b) i Si id(adalimumab) Unique Signature Peptide

Peptide: APYTFGQGTK 1-500 ug/mL

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

300

40.0

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

50.0 g/mL

Res

idua

l

-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 10.00

%

0


1.88e3Area

5.30;90

10 0 / Le 1.00

1.25

1.50

Conc-20.0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0


9665.29;53

10.0 g/mL

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

Res

pons

e

-0.00

0.25

0.50

0.75

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

Area

14Aug2015_Dir_5step_SPE_mabs_HumPlsm_Blk_002 Sm (Mn, 2x3) MRM of 12 Channels ES+ 535.3 > 901.44 (Humira APYTFGQGTK LC)

1 65e3

5.0 g/mL


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

%

0

1.65e3Area

5.289

Blank plasma

Competitor vs. ProteinWorks eXpress:Direct Digestion of Whole PlasmaDirect Digestion of Whole Plasma

Competitor vs Waters 3 and 5‐Step*% Area Unique Signature Peptides

150

% Area Unique Signature Peptides

100

125

75Area

25

503‐Step

5‐Step

Competitor

0


Both protocols were completed in comparable time* Normalized to Waters 5‐Step

Competitor vs. ProteinWorksp

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

WatersWaters 3 and 5‐Step vs. Competitor *

125

Waters 3 and 5 Step vs. Competitor % Area Generic Signature Peptides

75

100

50

Area

Waters 3‐Step

Waters 5‐Step

0

25

Generic VVSVLTVLHQDWLNGK Generic GPSVFPLAPSSK Generic DSTYSLSSTLTLSK

Competitor

Generic VVSVLTVLHQDWLNGK Generic GPSVFPLAPSSK Generic DSTYSLSSTLTLSK

* Normalized to Waters 3‐Step 35 uL Plasma


TopicsTopics


I St d di d A h P ibl ? Is a Standardized Approach Possible?

ADC Quantification



Trastuzumab Emtansine (T-DM1) Trastuzumab Emtansine (T DM1)

Trastuzumab based

90 lysines

SMCC linkerMCC (Linker)219.0895 Da

Drug Maytansinoid

Two-step conjugation

DM1 + MCC (Drug + Linker)956.3644 Da

Drug Maytansinoid Hydrophobic payload

©2015 Waters Corporation 28Bioconjugate Chemistry 2014, 25, 1223-1232

Data courtesy of Liuxi Chen

Lysine Conjugated ADC Q tifi tiQuantification

Trypsin Asp N

Unconjugatedtid

K Kpeptide

Conjugatedpeptide K K

miscleavage

Non-lysine containing peptide for best Relative site

miscleavage

Quantificationp p

quant, others may be okay depending on site-occupancy %

Relative site occupancy ratio


Data courtesy of Liuxi Chen

ADC Analysis: Total Antibody Q tifi ti U i P t i W k ™ Quantification Using ProteinWorks™ eXpress Digest Kit

TrastuzumabStandard Curve

T-DM1Standard Curve

Trastuzumab T-DM1QC Samples QC Samples


T-DM1 Summary Standard Curve Statistics: Direct Plasma Digest, 35 µL

Peptide Std. curve

Weighting

Linear fit (r2)

Mean % Accuracy

f ll range (ug/ml)

of all points,

duplicate curvescurves

IYPTNGYTR (T-DM1/trastuzumab

0.25 -500

1/x 0.998 100.00

Signature peptide)GPSVFPLAPSSK

(Generic mAb peptide)0.1 - 500 1/x 0.998 100.00

( p p )FTISADTSK

(Typical tratuzumabSignature peptide T-DM1

0.25 -500

1/x 0.999 100.00

Signature peptide, T DM1 with no drug)

S l d ith P t i W k ™ X Di t Di t kit


Samples prepared with ProteinWorks™ eXpress Direct Digest kit and standardized protocol

T-DM1 and Trastuzumab Quantification Using Trastuzumab standard curve: Using Trastuzumab standard curve: direct digest, 35 µL plasma

Peptide

TrastuzumabSample QCs

conc. (ug/ml)

Mean Cal. Conc. (ug/ml)

Std. Dev. %CV Mean %Accuracy

# of QCs passed

(ug/ml)0.65 0.64 0.03 4.58% 99.77% 3 out of 33.5 3.25 0.19 5.96% 92.90% 3 out of 3

IYPTNGYTR 6.5 6.83 0.16 2.29% 105.13% 3 out of 335 36.41 0.42 1.16% 104.03% 3 out of 365 63.31 2.18 3.44% 97.40% 3 out of 3350 345.64 18.66 5.40% 98.73% 3 out of 3

Peptide

T‐DM1 SampleQCs conc.



# of QCs passed

(ug/ml) (ug/ml)

0.65 0.65 0.05 6.94% 100.50% 3 out of 33.5 3.36 0.24 7.10% 95.87% 3 out of 36 5 7 10 0 05 0 66% 109 20% 2 out of 36.5 7.10 0.05 0.66% 109.20% 2 out of 3

IYPTNGYTR 35 34.51 1.09 3.17% 98.57% 3 out of 365 59.74 3.72 6.22% 91.90% 3 out of 3350 324.72 17.06 5.25% 92.80% 3 out of 3


MRM transition: 542.77 > 808.40

U i T t b t d d Trastuzumab Mean Cal. Mean # of QCs

Using Trastuzumab standard curve

Peptide QC conc. (ug/ml)

Conc. (ug/ml)


# of QCs passed

3.5 3.47 0.17 4.89% 99.20% 3 out of 36 5 6 70 0 11 1 69% 103 07% 3 t f 36.5 6.70 0.11 1.69% 103.07% 3 out of 3

FTISADTSK 35 38.30 0.28 0.73% 109.47% 3 out of 365 64.12 1.68 2.63% 98.67% 3 out of 3350 357.47 9.65 2.70% 102.13% 3 out of 3350 357.47 9.65 2.70% 102.13% 3 out of 3

T‐DM1 Mean Cal. Mean # of QCsPeptide QC conc. (ug/ml)

Conc. (ug/ml)


# of QCs passed

3.5 3.03 0.06 1.87% 86.60% 3 out of 36 5 6 91 0 30 4 37% 106 30% 2 t f 36.5 6.91 0.30 4.37% 106.30% 2 out of 3

FTISADTSK 35 33.35 0.65 1.94% 95.27% 3 out of 365 58.70 2.93 4.99% 90.30% 3 out of 3350 322.02 7.51 2.33% 92.00% 3 out of 3350 322.02 7.51 2.33% 92.00% 3 out of 3



Using Trastuzumab standard curveUsing Trastuzumab standard curve

Trastuzumab Mean Cal. M # f QCPeptideTrastuzumabQC conc. (ug/ml)



# of QCs passed

0.65 0.66 0.05 7.97% 100.87% 3 out of 33.5 3.04 0.07 2.29% 86.90% 3 out of 3

GPSVFPLAPSSK 6.5 6.27 0.09 1.41% 96.50% 3 out of 335 35.50 1.62 4.55% 101.43% 3 out of 365 71 38 3 04 4 26% 109 83% 3 out of 365 71.38 3.04 4.26% 109.83% 3 out of 3350 379.79 21.64 5.70% 108.50% 3 out of 3

PeptideT‐DM1 QC conc. (ug/ml)



# of QCs passed

0 65 0 67 0 02 2 84% 103 30% 2 f 30.65 0.67 0.02 2.84% 103.30% 2 out of 33.5 3.10 0.06 1.80% 88.47% 3 out of 3

GPSVFPLAPSSK 6.5 6.19 0.31 4.98% 95.15% 2 out of 335 33.55 1.44 4.29% 95.87% 3 out of 335 33.55 1.44 4.29% 95.87% 3 out of 365 63.08 4.04 6.40% 97.03% 3 out of 3350 336.36 15.35 4.56% 96.10% 3 out of 3



Monitoring miscleaved peptideFTISADTSKNTAYLQMNSLR

ADC QC 350 ug/ml Direct 5 step no SPE 2

FTISADTSKNTAYLQMNSLR with the small molecule drug attached

ADC, QC, 350 ug/ml, Direct 5 step, no SPE, 2110415_WAA678_CD_070b Sm (SG, 2x3) MRM of 17 Channels ES+

1073.167 > 485.22 (Kadcyla miscleavage FTISADTSKNTAYLQMNSLR 2)1.93e5

Area13.38;7282

13.27;9539

%

1073.167 > 485.22

11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.501

110415 WAA678 CD 070b S (SG 2 3) MRM f 17 Ch l ES+

1073.167 > 485.22

110415_WAA678_CD_070b Sm (SG, 2x3) MRM of 17 Channels ES+ 1073.167 > 547.2 (Kadcyla miscleavage FTISADTSKNTAYLQMNSLR 1)

5.82e5Area13.39;2496613.27;29901

%

1073 167 > 547 20

Time11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50

0

1073.167 > 547.20


The peptide elutes in pairs (Diastereomers). Two MRM transitions confirm that the correct peptide is being monitored.

Monitoring miscleaved peptideFTISADTSKNTAYLQMNSLR FTISADTSKNTAYLQMNSLR with the small molecule drug attached

%MRM of 17 Channels ES+


12.21

13.9312.88 14.46

11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50

%

1

11.8511.56

12.55 13.1713.67

14.09 14.88

15.28

15.12 15.46

MRM f 17 Ch l ES

Rat plasma blank digest

Trastuzumab digest 350 ug/ml%

MRM of 17 Channels ES+ 1073.167 > 547.2 (Kadcyla miscleavage FTISADTSKNTAYLQMNSLR 1)

7.54e412.21

14.60

13.9412.88 14.41 350 ug/ml

11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.501

11.82

12.01 12.55 13.3213.56

14.1014.86 15.30

MRM of 17 Channels ES+

ADC digest350 ug/ml

%


Area

350 ug/ml

Time11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50

0


The peptide can only be found in ADC digest.

Monitoring miscleaved peptideFTISADTSKNTAYLQMNSLR FTISADTSKNTAYLQMNSLR with the small molecule drug attached

MRM of 17 Channels ES+%


7.47e412.21

11 97

13.9312.88

12 56 13.3213 6

14.4114.09 14.88Rat plasma blank digest

13 27;3690

12.00 12.50 13.00 13.50 14.00 14.50 15.001

11.97 12.56 13.3213.67 15.12


7.95e4Area13.39;2867

35 ug/ml13.27;3690

12.00 12.50 13.00 13.50 14.00 14.50 15.00

%

1

Area

65 ug/ml13.27;5300 13.39;4409

%


9.03e4Area

65 ug/ml

12.00 12.50 13.00 13.50 14.00 14.50 15.001


5.82e5

350 ug/ml

13.27;29901 13.39;24966

Time12 00 12 50 13 00 13 50 14 00 14 50 15 00

%

0

Area


The signal is concentration dependent.

12.00 12.50 13.00 13.50 14.00 14.50 15.00

TopicsTopics


I St d di d A h P ibl Is a Standardized Approach Possible

ADC Quantification



Intact mAb analysis using ionKey/MS Xevo G2 XS QTof system

ACQUITY UPLC M-Class Xevo G2-XS QTof

ionKey/MS Xevo G2-XS QTof system

ESI Voltage: 3.5 kV Sample Cone: 190 V Source Offset: 150 V

MPA: Water, 0.1% Formic Acid MPB: Acetonitrile, 0.1% Formic Acid Flow rate: 5 µL/min

Q Q

Source Offset: 150 V Source Temperature: 150C Cone Gas: 50 L/h Nano Flow Gas: 0.10 Bar Scan: 500 4000 Da 1 second

Flow rate: 5 µL/min Gradient: 1 min gradient delay, 3% B

to 98% B in 3.5 min., hold for 4.5 min at 98% B, equilibrate at 3% for 3 min.

Column Temp: 80⁰C Scan: 500 – 4000 Da, 1 second scan

Column Temp: 80 C 1 uL Loop (m-class) BEH C4, 300Å, 1.7 µm x 5 cm


Gregory Roman; et al: High Sensitivity Intact Mass Analysis of Antibodies using ionKey/MS: Waters Application Note P/N: 720005378EN

Improved Sensitivity Capability of ionKey for Deglycosylated Analysisfor Deglycosylated Analysis

100 2.90

• 40 pg LOD• 100 pg LOQ

0.1 ngPWHM: 8.5s

m/z2200 2400 2600 2800 3000 3200 3400

%

0

100

2.00 4.00 6.00 8.00

%

0

100

9.17

• 100 pg LOQ

1 ng

m/z

%

0

100

2 00 4 00 6 00 8 00

%

0

100 2.90

10 ng

%

100

m/z2200 2400 2600 2800 3000 3200 3400

%

100

2.00 4.00 6.00 8.00

2.90

25 ng

%

100

m/z2200 2400 2600 2800 3000 3200 3400

0

%

100

2.00 4.00 6.00 8.000

2.88

50 ng100

m/z2200 2400 2600 2800 3000 3200 3400

%

0

100

2.00 4.00 6.00 8.000

2.88

50 ng

100

m/z2200 2400 2600 2800 3000 3200 3400

%

0

100

2.00 4.00 6.00 8.00

%

0

2.88


100 ng

m/z2200 2400 2600 2800 3000 3200 3400

%

0

100

Time2.00 4.00 6.00 8.00

%

04.51

Improved Sensitivity Capability of ionKey for Glycosylated mAb AnalysisionKey for Glycosylated mAb Analysis

• 0.4 ng LOD• 1 ng LOQ• 1 ng LOQ

No Glycoform


Carryover

Waters mAb Standard, Deconvoluted Spectra ExamplesDeconvoluted Spectra Examples

1 ng 50 ng GOF/GOF 148221 4 148221 6 0 2GOF/GOF 148221.4 148221.6 0.2GOF/G1F 148383.5 148383.2 0.3G1F/G1F 148544.1 148544 0.1G1F/G2F 148708.1 148708 0.1G2F/G2F 148869 2 148869 5 0 3G2F/G2F 148869.2 148869.5 0.3

N-Acetylglucosamine (203Da)Mannose (162Da)Galactose (162Da)Fucose (146Da)Fucose (146Da)


Trastuzumab ADC (De-Glycosylated) i h i i

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

6.08e32904.37552847.47022740 01492689 4590 2904 7944

High Sensitivty CSD Spectra

m/z

%

0

2740.01492689.4590

2593.2161

2904.79442963.8701 3089.8511

3090.23750.25 ngTrastuzumab MW:

145165.1575 g/mol

Linker and Drug: 958 5322 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

2792.83572689 2893

2904.2434

2904.7063 3025.3850 2 5 ng

958.5322 g/mol

1 Drug + 1 Antibody: 146123.6 g/mol

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

%

0

2689.2893 3025.3850 2.5 ng%

100

2400 2500 2600 2700 2800 2900 3000 3100 3200 33003252015 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

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+

g

%

1003252015 C4 Herceptin ADC 10X Dilution_2 150 (2.930) Cm (144:165) 1: TOF MS ES+

2.68e52794.32762742.54222644.6094

2598.2388

2847.99412885.3372

2924.1621 250 ng


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

0

Trastuzumab ADC (De-Glycosylated) Trastuzumab ADC (De Glycosylated) Deconvolution on ionKey/MS

3252015 C4 Herceptin ADC 100X Dilution 2 151 (2 947) M1 [Ev 337277 It12] (Gs 1 500 1955:3634 11003252015 C4 Herceptin ADC 100X Dilution_2 151 (2.947) M1 [Ev-337277,It12] (Gs,1.500,1955:3634,1

1.88e5147079.0000

146122.0000145166 0000

148036.0000

148994.0000

Trastuzumab MW: 145165.1575 g/mol

Linker and Drug: 25.0 ng (on-column)

mass

%

0

145166.0000

144300.0000

149953.0000150902.0000 151866.0000

g958.5322 g/mol

1 Drug + 1 Antibody: 146123.6 g/mol

144000 146000 148000 150000

Trastuzumab MW: 145165.1575 g/mol

Linker and Drug:

+Linker and Drug: 958.5322 g/mol


ConclusionsConclusions

Standardized approaches to protein quantification can eliminate Standardized approaches to protein quantification can eliminate method development for discovery studies

Single digit accuracy and precision is achieved for multiple Single digit accuracy and precision is achieved for multiple mAbs and an ADC using a standardized protocol and kit

For ADC analysis total mAb concentrations can be determined For ADC analysis, total mAb concentrations can be determined using an optimized, yet generic standardized protocol and kit designed for protein quantificationg p q

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


Ackno ledgementsAcknowledgements

Mary Lame Mary Lame Hua Yang Jay Johnson Jay Johnson Liuxi Chen

J M h James Murphy Henry Shion

G R Gregory Roman Weibin Chen John Gebler Sherri Naughton Catalin Doneanu


practical considerations for lc/ms biliioanalysisofiihf ... · µelution format eliminates dry down...

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