lc–ms/ms strategies for therapeutic proteins · public considerations for lc-ms/ms quantification...

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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?


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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


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)

Copyright © 2017 Covance.

LC–MS/MS strategies for therapeutic Proteins Albumin

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6

MS infusion

Strategy One: Intact protein analysis


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


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)


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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


+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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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


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


Surrogate peptide of Lysozyme (ISTD)




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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


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 4.11 minutes



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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)





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Conclusions


► 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?


Ewles, M, Mannu R, Fox C, Stanta J et al., Bioanalysis (2016) 8(24), 2565–2579



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Sample Preparation


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



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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


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.


lc–ms/ms strategies for therapeutic proteins · public considerations for lc-ms/ms quantification...

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