lc–ms/ms strategies for therapeutic proteins · lc–ms/ms strategies for therapeutic proteins...
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LC–MS/MS STRATEGIES FOR THERAPEUTIC PROTEINS
Johannes Stanta PhDScientific Manager, BioanalysisEBF Focus Workshop – June 2017
Copyright © 2017 Covance. All Rights Reserved
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Presentation Overview
► A brief introduction to LC-MS/MS quantification of proteins and the aims of our work
► Presentation of strategies for LC-MS/MS quantification of therapeutic proteins
► Case study: Affibody® technology► ADA effects on LC-MS assays
► Case study: Herceptin®
LC–MS/MS strategies for therapeutic Proteins2
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Considerations for LC-MS/MS Quantification of Proteins in Biological Samples
Most bioanalytical strategies are based on tryptic digestion… …. but beyond that everyone does it differently!
Considerations
What digestion approach:Guanidine/urea denaturation and overnight digestion?Detergent denaturation and facilitated digestion?Microwave or methanol assisted digestion?Immobilised Trypsin?
To reduce or not to reduce?Do we really need to reduce/alkylate?
Sample pre-treatment:Direct digestion of plasma?Protein precipitation or size exclusion?Protein A/G mediated extraction of antibodies?Bespoke ultra-selective immunoaffinity cleanup?
Post-digestion analysis:Additional cleanup after digestion?Conventional reversed phase LC-MS/MSNanoflow, ion-mobility, …Which tryptic peptide do we choose?
LC–MS/MS strategies for therapeutic Proteins3
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Introduction
► LC-MS/MS can provide complementary data to ligand binding assays as well as offering merits as a standalone bioanalytical approach
► We wanted an approach that was cost effective, sensitive, quick/easy to develop, no requirement for analyte-specific reagents, and high throughput for use with conventional UHPLC-MS/MS instrumentation
LC–MS/MS strategies for therapeutic Proteins4
AB Sciex API5000™ and Waters Acquity™
Image: Covance
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Affibody® molecule fusion protein
Affibody® technology
Two 6 kDa units linked by 5 kDa albumin binding domain (ABD)
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LC–MS/MS strategies for therapeutic Proteins Albumin
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MS infusion
Strategy One: Intact protein analysis
Copyright © 2017 Covance.
LC–MS/MS strategies for therapeutic Proteins
+Q1: 17 MCA scans from Sample 1 (TuneSampleID) of MT20150112131503.wiff (Turbo Spray) Max. 1.6e7 cps.
600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200m/z, Da
0.0
1.0e6
2.0e6
3.0e6
4.0e6
5.0e6
6.0e6
7.0e6
8.0e6
9.0e6
1.0e7
1.1e7
1.2e7
1.3e7
1.4e7
1.5e7
1.6e7
Inte
nsi
ty,
cps
1166.5
1244.11097.91037.0
1332.9
982.6
1865.5
910.9
1435.3933.4
1138.2
1554.8
889.01695.9
2072.3782.2
759.3 2079.5848.8 1043.1 1114.2 1193.5668.2 974.3
1878.1908.7838.8 1010.9717.8 811.3 1067.6 1188.0644.0 1285.51085.5874.4 1007.7 1560.0825.7 2093.41206.0749.8612.6 1707.41266.7 1392.6 1517.41291.4 2114.21408.9 1893.81908.41590.3 1789.4 1987.6
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XIC of +MRM (9 pairs): 1431.800/86.200 Da ID: Lysozyme A from Sample 18 (8314846 Chrom150116 BEH300 C18) of 8314846 Chrom1501... Max. 4.1e4 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
Inte
nsi
ty,
cps
3.10
3.07
LC–MS/MS strategies for therapeutic Proteins7
CSH C18 Column 300Å
Intact protein chromatography
1 µg/mL just about achievable
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Strategy Two: Plasma Digestion
15 µL plasma + 40 µL of 100 µg/mL chicken lysozyme
Load all into the SMART Digest™ tubes
Perform SPE (Waters Oasis® HLB)
LC-MS/MS (Sciex API 5000™, Waters Acquity®)Phenomenex Kinetex® XB-C18 1.7 µm column
Water/acetonitrile phases + formic acid
Total extraction time <5.5 hours per 96x sample run
Two hour digestion at 70ºC
Add 175 µL supplied digestion buffer and mix briefly
Eliminates salts and undigested proteins
Evaporative concentration and reconstitution
(Generic protein internal standard)
LC–MS/MS strategies for therapeutic Proteins8
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► Several peptides from variable region predicted
► After optimisation three of these gave desirable analytical performance
► Peptide with best signal/noise chosen
Affibody® molecule signature peptide method
LC–MS/MS strategies for therapeutic Proteins9
+Q1: 25 MCA scans from Sample 1 (TuneSampleID) of MT20150113124452.wiff (Turbo Spray) Max. 1.1e8 cps.
400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100m/z, Da
0.00
5.00e6
1.00e7
1.50e7
2.00e7
2.50e7
3.00e7
3.50e7
4.00e7
4.50e7
5.00e7
5.50e7
6.00e7
6.50e7
7.00e7
7.50e7
8.00e7
8.50e7
9.00e7
9.50e7
1.00e8
1.05e81.08e8
Inte
nsi
ty,
cps
592.6473.8
888.1
828.8
940.1
417.8
501.3
861.7753.6 941.0
613.3573.7 679.3488.6 553.2
646.6 919.2565.5437.2 745.7586.6471.7 627.3 662.5531.6 847.7 1018.3454.4 762.7426.6 971.1558.6 640.1495.3 951.1815.7523.8 872.8450.6 882.4784.6612.3 988.8680.6580.7 700.3407.0 983.3759.6540.5 1062.81009.8839.6
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Copyright © 2017 Covance.
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Method Validation in Monkey plasma (Whole plasma digestion, Affibody® molecule, 1 – 1000 µg/mL)4 precision and accuracy runs with selectivity and stability tests
► Six individual lots of matrix at LQC: <12.3% bias
► Haemolysed LQC and HQC samples: acceptable
► Stability samples acceptable:• 24 hour on ice• 2 hour at RT• 4x freeze-thaws• 102 hours for extracts
LC–MS/MS strategies for therapeutic Proteins10
LLOQ LQC MQC HQCMean Concentration (µg/mL) 1.01 3.13 53.1 839Inter-run RSD (%) 10.6 8.8 6.8 8.7Inter-run %Bias 1.0 4.3 6.2 4.9n 18 23 24 24
Data generated at Covance
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Method Validation in Monkey plasma (Whole plasma digestion, Affibody® molecule)
LLOQ (1 µg/mL)
Matrix blank(No peak)
Retention time 4.76 min
Retention time 4.33 min
Surrogate peptide of Lysozyme (ISTD)
LC–MS/MS strategies for therapeutic Proteins11
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SMART Digest™ Method Validation (Affibody® molecule, 20 – 10,000 ng/mL human serum, Lysozyme ISTD)
5 precision and accuracy runs with selectivity and stability tests
► Six individual lots of matrix at LoQC: <14.2% bias
► Haemolysed and lipidaemic LQC and HQC samples: acceptable► Stability samples acceptable:
• 19 hours room temp (plasma)• 2 hours room temp and refrigerated (blood)• 4x freeze-thaws
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LLOQ LQC MQC HQCMean Concentration (ng/mL) 20.3 59.8 710 8070Inter-run RSD (%) 17.0 8.6 8.2 7.0Inter-run %Bias 1.5 -0.3 1.4 0.9n 23 30 30 30Data generated at Covance
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Human Method Validation (SMART Digest™, Affibody® molecule)
LLOQ (20 ng/mL)
Lysozyme surrogate peptide (ISTD)
Retention time 4.51 min
Retention time 4.11 minutes
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LC–MS/MS strategies for therapeutic Proteins14
20 – 10,000 ng/mL
Clinical Assay for Ph1 and Ph2a
Ø Lysozyme Internal standard
Ø ~1700 samples analysed
Issues: Ø LLOQ robustness
Ø ChromatographyØ Response drift
SIL IS peptide for more robust methodØ synthetic peptide with Leucine-D10Ø 9 AA flanking on each side.
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Human Method Validation (SMART Digest™, Affibody® molecule)
LLOQ (20 ng/mL)
SIL IS peptide (ISTD)
Retention time 4.59 min
Retention time 4.71 min
LC–MS/MS strategies for therapeutic Proteins15
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Conclusions
LC–MS/MS strategies for therapeutic Proteins16
► Whole plasma/serum digestion can work very well when you have a selective peptide
► More robust assay with SIL IS
► Better selectivity/sensitivity could be achieved using immunoaffinity cleanup
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WHAT ABOUT ANTI-DRUG-ANTIBODY EFFECTS (ADA)?
Herceptin® case study
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►Common assumption that LC-MS/MS assays will always measure total (free, soluble target-bound and ADA-bound)
►How can we test this? ►Does it need to be validated?►Case Study
Anti-Herceptin® rabbit polyclonal antibody (pADA)Anti-Herceptin® human monoclonal antibody (mADA)
Was investigated for the following validated assays:(1) Ligand binding assay(2) Whole serum digestion LC-MS/MS method
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Impact on Ligand Binding Assay
What effect does these
levels of ADA have on our LC-MS/MS
assay?
LC–MS/MS strategies for therapeutic Proteins19
Ewles, M, Mannu R, Fox C, Stanta J et al., Bioanalysis (2016) 8(24), 2565–2579
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Copyright © 2017 Covance.
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Sample Preparation
LC–MS/MS strategies for therapeutic Proteins20
SampleHerceptin®
to ADA ratio
ADA typeLigandBinding Assay
Wholeserum digest and LC-MSMS
Whole serumdigestion (DMSO)
LQC (0.3 µg/mL)
1:0 Accurate Accurate Accurate
1:533 Polyclonal No response Inaccurate(-65%) Accurate
1:133 Monoclonal No response Accurate Accurate
HQC (80 µg/mL)
1:0 Accurate Accurate Accurate
1:2 Polyclonal Inaccurate (-20 – -40%) Accurate Accurate
2:1 Monoclonal Inaccurate (-20 – -40%) Accurate Accurate
Ewles, M, Mannu R, Fox C, Stanta J et al., Bioanalysis (2016) 8(24), 2565–2579
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Results Using LC-MS/MS Assays
Using WHOLE SERUM DIGESTION approach
► No impact of mADA on quantification► Where the polyclonal antibody is present at high molar excess there
is serious negative bias on the LC-MS/MS assay► Hence: LC-MS/MS assays can be affected by ADA effect!
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Clinical Relevance
► Polyclonal antibody used raised in rabbit to Fab fragment► Therefore significant amount of antibody unique to variable region
therefore could mimic a true ADA response in clinical samples► What might be going on here??? (hypothesis…)
YADA Target mAb (variable region
protected from trypsin)
Immobilised Trypsin
LC–MS/MS strategies for therapeutic Proteins22
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Strategies investigated to dissociate Herceptin® ADA prior to digestion
► Denaturation with DMSO – Solves the problem!• Impact of anti-Herceptin® polyclonal antibody eliminated• DMSO improved selectivity/sensitivity of whole plasma digestion• Work ongoing to assess ADA effects on protein G enrichment
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A Resolution to Ensure Total Therapeutic mAb Quantification
Conclusions
► We have shown that for LC-MS/MS, an anti-drug-antibody effect can result in an underestimation of the total drug even when digesting whole plasma/serum.
► However careful optimisation can overcome this
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Acknowledgements
►Matthew Ewles: Covance Bioanalytical, Harrogate ►David Firth, James Duffy, Len Bell, Sian Estdale:
Covance Biopharm CMC, Harrogate (research collaborators)
►Chris Fox, Graeme Evans: Covance Bioanalytical Services (Immunochemistry), Harrogate (ligand binding assay data)
►Joachim Feldwisch and everyone at Affibody
Covance Inc., headquartered in Princeton, NJ, USA, is the drug development business of Laboratory Corporation of America Holdings (LabCorp). COVANCE is a registered trademark and the marketing name for Covance Inc. and its subsidiaries around the world.
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