practical considerations for lc/ms practical considerations for lc
TRANSCRIPT
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
©2015 Waters Corporation 3
and Alzheimer’s disease.
Practical Considerations for LC/MS Bioanalysis of Proteins via the Surrogate
P tid A hPeptide Approach
©2015 Waters Corporation 4
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?
©2015 Waters Corporation 5
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
©2015 Waters Corporation 6
Possible Protein Bioanalysis Workflowsfor Surrogate Peptide Approachg p pp
©2015 Waters Corporation 7
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
©2015 Waters Corporation 8
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)
©2015 Waters Corporation 9
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
©2015 Waters Corporation 10
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
©2015 Waters Corporation 11
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)
©2015 Waters Corporation 12
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
©2015 Waters Corporation 13
Topicsp
Challenges in Workflow Options and Typical Steps
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
©2015 Waters Corporation 14
What about intact antibody analysis?
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
©2015 Waters Corporation 15
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)
©2015 Waters Corporation 16
http://www.drugbank.ca/drugs/DB00072
Bevacuzimab (Avastin)
Bevacizumab light chainDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECBevacizumab heavy chainEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Unique Signature
Generic Signature
Drug Bank
Generic Signature
http://www.drugbank.ca/drugs/DB00065
Formula: C6538H10034N1716O2033S44Molecular Weight: ~ 149.0 kD
©2015 Waters Corporation 17
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
©2015 Waters Corporation 18
http://www.drugbank.ca/drugs/DB00051
Peptide Choice: Impact on Protocol for Unique Peptides
Eliminate reduction/alkylation?
%Area
3 step, normalized to 5 step protocol
©2015 Waters Corporation 19
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
©2015 Waters Corporation 20
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)
©2015 Waters Corporation 21
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
©2015 Waters Corporation 22
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
10014Aug2015_RemicadeSPE_ProteinA_01038 MRM of 11 Channels ES+
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
©2015 Waters Corporation 23
Workflow check Standard
Topicsp
Challenges in Workflow Options and Typical Steps
Antibodies and Choice of Representative Peptide
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
©2015 Waters Corporation 24
What about intact antibody analysis?
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
©2015 Waters Corporation 25
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
©2015 Waters Corporation 26
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
©2015 Waters Corporation 27
Protein A/G Cross Reactivity
Can be used as generic purification in discovery for certain pre-clinical species
©2015 Waters Corporation 28
Removal of Endogenous Background: Effect of Protein-Level Purification
Specific:Anti-human in Rat
Generic:Protein A in Human
None:Direct Human Plasma
©2015 Waters Corporation 29
Effect of Protein-Level Purification : Detection Limit Comparisonp
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 ~50 ng/mL 100 ng/mL
©2015 Waters Corporation 30
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
©2015 Waters Corporation 31
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
©2015 Waters Corporation 32
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
©2015 Waters Corporation 33
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
©2015 Waters Corporation 34
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
©2015 Waters Corporation 35
Topicsp
Challenges in Workflow Options and Typical Steps
Antibodies and Choice of Representative Peptide
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
©2015 Waters Corporation 36
What about intact antibody analysis?
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
©2015 Waters Corporation 37
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
©2015 Waters Corporation 38
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
©2015 Waters Corporation 39
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
22Dec2014_ProtA_nospe_1021 Sm (Mn, 3x3) MRM of 10 Channels ES+ 633.1 > 731.8 (TNO Remicade signatureDILLTQSPAILSVSPGER)
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
22Dec2014_ProtA_nospe_1012 Sm (Mn, 3x3) MRM of 10 Channels ES+ 633.1 > 731.8 (TNO Remicade signatureDILLTQSPAILSVSPGER)
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
22Dec2014_ProtA_nospe_1005 Sm (Mn, 5x5) MRM of 10 Channels ES+ 633.1 > 731.8 (TNO Remicade signatureDILLTQSPAILSVSPGER)
1.23e4Area
84
Blank plasma
©2015 Waters Corporation 40
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
22Dec2014_ProtA_nospe_1021 Sm (Mn, 2x3) MRM of 10 Channels ES+ 603.3 > 1110.6 (TNO Furlong Remicade signatureVVSVLTVLHQDWLNGK)
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
22Dec2014_ProtA_nospe_1012 Sm (Mn, 1x1) MRM of 10 Channels ES+ 603.3 > 1110.6 (TNO Furlong Remicade signatureVVSVLTVLHQDWLNGK)
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
22Dec2014_ProtA_nospe_1005 Sm (Mn, 1x1) MRM of 10 Channels ES+ 603.3 > 1110.6 (TNO Furlong Remicade signatureVVSVLTVLHQDWLNGK)
2.34e3Area
24Blank plasma
©2015 Waters Corporation 41
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
©2015 Waters Corporation 42
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
©2015 Waters Corporation 43
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
©2015 Waters Corporation 44
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 ‐ ‐ ‐ ‐
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 9.40 100.0350.00 353.43 4.85 1.37 101.0
QC Conc (ug/mL)Mean Cal. Conc
(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
QC Conc (ug/mL)Mean Cal. Conc
(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
Peptide QC Conc (ug/mL)Mean Cal. Conc
(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
©2015 Waters Corporation 45
350.00 347.27 13.57 3.91 99.2
QC Conc (ug/mL)Mean Cal. Conc
(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
©2015 Waters Corporation 46
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
©2015 Waters Corporation 47
50 334224 62240 49.4 ‐1.3
Standardized Protocol: Avastin(bevacizumab) QC Statistics(bevacizumab), QC Statistics
Peptide QC Conc (ug/mL)Mean Cal. Conc
(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
QC Conc (ug/mL)Mean Cal. Conc
(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
QC Conc (ug/mL)Mean Cal. Conc
(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
©2015 Waters Corporation 48
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
9Jan2015_ProtA_nospe_1031 Sm (Mn, 2x2) MRM of 10 Channels ES+ 751.88 > 836.47 (Merck DSTYSLSSTLTLSK)
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
9Jan2015_ProtA_nospe_1028 Sm (Mn, 2x2) MRM of 10 Channels ES+ 751.88 > 836.47 (Merck DSTYSLSSTLTLSK)
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
9Jan2015_ProtA_nospe_1021 Sm (Mn, 2x2) MRM of 10 Channels ES+ 751.88 > 836.47 (Merck DSTYSLSSTLTLSK)
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
9Jan2015_ProtA_nospe_1006 Sm (Mn, 2x2) MRM of 10 Channels ES+ 751.88 > 836.47 (Merck DSTYSLSSTLTLSK)
4.40e5Area
0.10 g/mL
Blank Plasma
©2015 Waters Corporation 49
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
14Aug2015_Dir_5step_SPE_mabs_HumPlsm_10p0_033 Sm (Mn, 2x3) MRM of 12 Channels ES+ 535.3 > 901.44 (Humira APYTFGQGTK LC)
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
14Aug2015_Dir_5step_SPE_mabs_HumPlsm_5p0_030 Sm (Mn, 2x3) MRM of 12 Channels ES+ 535.3 > 901.44 (Humira APYTFGQGTK LC)
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
©2015 Waters Corporation 50
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
14Aug2015_RemicadeSPE_ProteinA_01019 MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )
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
14Aug2015_RemicadeSPE_ProteinA_01028 MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )
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
10014Aug2015_RemicadeSPE_ProteinA_01035 MRM of 11 Channels ES+
469.6 > 603.8 (Remicade SINSATHYAESVK )2.05e7
Area
857961
14Aug2015_RemicadeSPE_ProteinA_01045 MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )
1.32e86321264
35.0 g/mL
©2015 Waters Corporation 51
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
©2015 Waters Corporation 52
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
©2015 Waters Corporation 53
Topicsp
Challenges in Workflow Options and Typical Steps
Antibodies and Choice of Representative Peptide
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
What about intact antibody analysis?
©2015 Waters Corporation 54
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
©2015 Waters Corporation 56
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
©2015 Waters Corporation 57
Data courtesy of Liuxi Chen
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
©2015 Waters Corporation 58
Data courtesy of Liuxi Chen
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
©2015 Waters Corporation 59
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
©2015 Waters Corporation 60
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
©2015 Waters Corporation 61
Data courtesy of Liuxi Chen
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
Company A ‐ Batch 2
H30 H43 H65 H76
0
2
4
L126 L145 L149 L169 L183 L188 L190 L207 H136 H216 H217
Relativ
e
20
Company A ‐ Batch 2
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
Data courtesy of Liuxi Chen
Topicsp
Challenges in Workflow Options and Typical Steps
Antibodies and Choice of Representative Peptide
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
What about intact antibody analysis?
©2015 Waters Corporation 63
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
©2015 Waters Corporation 64
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
©2015 Waters Corporation 65
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
©2015 Waters Corporation 66
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
©2015 Waters Corporation 67
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
©2015 Waters Corporation 68
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……………
©2015 Waters Corporation 69
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
©2015 Waters Corporation 70
Thank You for Attending!g
Post-Event Home Page: http://www.waters.com/Sep24
30% Introductory Offer on Protein, Peptide and30% Introductory Offer on Protein, Peptide and Glycan Separation Columns and Kits
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©2015 Waters Corporation 71