lba and lc-ms: advantages of incorporating both platforms ...€¦ · overview iq consortium large...
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© 2016, Genentech Confidential 2016_10_10_World_ADC_San_Diego_Saad,O Genentech proprietary information – Please do not copy, distribute or use without prior written consent
LBA and LC-MS: Advantages of Incorporating Both Platforms in Large Molecule Drug Development
Surinder Kaur, Ph. D. Director, ADC Programs & MS BioAnalytical Sciences, Genentech S. San Francisco, California
EBF Focus-Workshop
Bioanalytical Strategies for Large Molecules in Modern Drug Development: LBA and LC-MS United
21st June, 2017
Overview
IQ Consortium Large Molecule LCMS Working Group
Ligand binding, LC-MS/MS and hybrid IA-LC-MS/MS strategies
Advantages of having diverse large molecule platforms
Case study: Unusual clinical PK investigation
Summary and acknowledgements
S. Kaur, EBF Workshop, Lisbon, 6.21.17 2
IQ Consortium Large Molecule LCMS Working Group
• Kickoff 2016 • Mission: Best use of LC-MS for biotherapeutic bioanalysis and biotransformations • Upcoming: Joint IQ/AAPS LCMS/LBA Workshop at AAPS, San Diego, 11/2017
AbbVie: Jens Sydor Amgen: Hongyan Li Bayer: Erik Haaf BMS: Jianing Zeng Celgene: Ma=hew Myers Daiichi Sankyo: Joseph Pav Eli Lilly: Mike Berna Genentech: Surinder Kaur GSK: Jack Kellie Merck: Kevin Bateman NovarJs: Jim Glick NovarJs: Carsten Krantz Pfizer: Hendrik Neubert
Pierre Fabre: Stéphane Couffin Roche: Faye Vazvaei Sanofi: Olivier Pasquier Takeda: Dhiman Ghosh Vertex: Zhenmin Liang BI: Lin-‐zhi Chen Sea=le GeneJcs: Shawna Hengel UCB: Mark Jairaj UCB: Ludovicus Staelens
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New Product formats for Biotherapeutics: Drive Need for Complex Bioanalysis
Cyclic Peptides
Antibody-drug conjugates
Bispecific antibodies
Fusion proteins and novel macromolecule conjugates
PEGylated molecules
Small protein formats and novel protein scaffolds
More complicated biotherapeutics
Analytes are mixtures and have various catabolic products
Different in vivo PK properties
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A Role of Bioanalysis in Drug Discovery & Development: Pharmacokinetic Assays for Exposure Assessment
Exploratory PK Assays: Exposure in efficacy and Tox species to understand Therapeutic Window
Preclinical Development
Clinical Development Filing/Approval Discovery
Molecule Selection
IND Enabling Studies
Phase I Phase II Phase III Post Marketing
Validated PK Assays: Allow exposure assessment in Tox species to determine first in human dose
* IND
Validated PK Assays: Allow exposure response understanding to determine optimum dose and dose regimen
Quantitative PK assays play a key role in drug discovery & development
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Monoclonal Antibody
Growth FactorDigoxin
Analysis of Large and Small Molecule Drugs: Past Trends Molecular Structure was a Major Driver
6
Ligand Binding Assays: Fundamental Characteristics
Ligand Binding Assays: E.g. ELISA
Antigen or Anti-Hu IgG
Anti-Hu IgG HRP
Therapeutic Antibody
Protein Calibration Curve
unknown
Reagents • One or two high quality binding reagents required
for performance
Sensitivity
• New micro-fluidic platforms for super sensitivity:
Singulex, Quanterix
Selectivity
• Binding specificity (2 epitopes) • Total or free format • Potential for interference • Sample dilution typical • “Free” or “total” format
Multiplexing
• Some multiplexing possible
Assay Training
• Established industry expertize • Trained analyst pool available
Outsourcing • Ready CRO capacity • Low capital investment • Assay maintenance
requirements S. Kaur, EBF Workshop, Lisbon, 6.21.17 7
Direct Digestion LC-MS/MS Assays: Fundamental Characteristics
Enzymatic digestion
Peptide Digest
Direct Digestion LC-MS/MS Assays
Protein Calibration Curve
Protein Concentration
Pept
ide
/ IS
ratio
unknown
MRM
LC-MS/MS
Stable isotope-labeled IS
Serum/Plasma Proteome
Reagents • No binding reagents required • Stable labeled peptide/protein
Sensitivity/ Identity
• Limited sensitivity with direct digestion (1 µg/ml for mAb)
• Catabolite ID information
Selectivity
• Based on (i) retention time, (ii) peptide mass, (iii) peptide fragmentation
• Low potential for interference • No dilution (except Cmax) • Total Format
Multiplexing
• Highly multiplexed
Assay Training
• New industry expertise • Limited trained analyst pool
Outsourcing • Few expert CROs • Building CRO capacity a slow
process • Initial capital investment • Robust assay transfer & facile
assay maintenance S. Kaur, EBF Workshop, Lisbon, 6.21.17 8
Enzymatic digestion
Peptide Digest
Immunoaffinity-LC-MS/MS Assays
Protein Calibration Curve
Protein Concentration
Pept
ide
/ IS
ratio
unknown
Immuno-Enrichment
MRM
LC-MS/MS
Magnet
Protein A, Anti-Hu IgG or Antigen Capture Stable isotope-labeled IS
Combining Ligand Binding Approach with LC-MS/MS: Hybrid Immunoaffinity (IA)-LC-MS/MS
Bio$n-‐An$ Id or (ECD)
Magnet
Streptavidin Coated Paramagne$c Bead
Generic Capture
Specific Capture
Therapeu$c mAb
Magnet
Protein A, G or (xhuIgG) Bead
Endogenous Ab
Therapeu$c mAb
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Platform Reagents Sensitivity/ Identity
Selectivity/ Multiplexing
Training/Outsourcing
LBA
• One or two high quality binding reagents
for performance
• New micro-fluidic platforms for super sensitivity:
Singulex, Quanterix
• Binding specificity • Total or free format • Potential for interference • Sample dilution typical • Multiplexed possible
• Established analysts Ready CRO capacity
• Low capital investment
• Assay maintenance requirements
Direct Digest LC-MS/MS
• No binding reagents required
• Stable labeled peptide/protein
• Limited sensitivity (1 µg/mL for mAb) • Catabolite ID
• Based on (i) retention time, (ii) peptide mass, (iii) peptide fragmentation
• Low potential for interference
• No dilution (except Cmax) • Total Format • Highly multiplexed
• Few expert CROs • Building CRO
capacity a slow process
• Initial capital investment
• Robust assay transfer
Hybrid IA-LC-MS/MS
• One reagent used to enrich analyte
• Otherwise, as above
• pg/mL sensitivity with anti-peptide reagents
• Otherwise, as above
• Binding specificity • Total or free format • Otherwise, as above
• New industry expertise
• Limited analyst pool • Otherwise, as above
Differences and Similarities for LBA, Direct Digestion LC-MS/MS and Immunoaffinity LC-MS/MS
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Considerations for choice of assay platform: LBA, LC-MS/MS, Hybrid IA-LC-MS/MS
“Free/Total” or “Generic/Specific” measurement required?
Assay sensitivity requirements
What binding reagents are available?
Exploratory or validated assay?
Is there a need to compare to historical platform data?
Resources/time available for assay development
Will the assay be outsourced?
Overall implementation and Regulatory strategy
Mix of technologies, collaboration, cross-training important
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K. Peng*, K. Xu* (*contributed equally), L. Liu, R. Hendricks, R. Delarosa, R. Erickson, N. Buda, M. Leabman, A. Song, S. Kaur, and S.K. Fischer, “Critical role of bioanalytical strategies in investigation of clinical pharmacokinetics, a Phase I case study”, MAbs 6 (6) 1500-08 (2014). doi: 10.4161/mabs.36208
Case Study: Investigating Unusual Phase I Clinical PK using ELISA and Hybrid LC-MS/MS
• Fully human RG7652 mAb targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) for treatment of hypercholesterolemia
• Target binding ELISA designed to quantify total RG7652
• Phase I showed unexpected nonlinear dose normalized exposure
• Is this an assay artifact or biology?
• Nonlinear PK results confirmed using hybrid LC-MS/MS
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ELISA Showed Unexpected Nonlinear Phase I PK
SA-coated plate
biotin-target
Mu anti-drug mAb
HRP anti-mu IgG
Clinical ELISA format: target-‐binding
Assay sensitivity: 0.1 ug/mL • Non-dose proportional increase in AUC
• Could PK non-linearity be due to assay artifacts?
Therapeutic mAb A
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Rapidly Develop Orthogonal LC-MS/MS Assay for Quantification of mAb RG7652 in Human Serum
• Signature Peptides selected from the CDRs or variable domains
L. Liu, K. Xu, S. Kaur
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Assay Performance Using Affinity Enrichment
Protein A Capture An$-‐Id Capture 0.05 ug/mL
Human serum background
2.0 3.0 4.0 5.0 Time, min
0
2000
4000
6000
8000
9500
1.0
Human serum background
1.5 ug/mL
1.0 2.0 3.0 4.0 5.0 0.0
5000.0
1.0e4
1.5e4
2.0e4
2.4e4
Time, min
0.05 – 125 ug/mL 1.5 – 125 ug/mL
Calibration range
Direct Diges$on
Inte
nsity
1.0 2.0 3.0 4.0 5.0 Time, min
0.0
5000
1.0e4
1.5e4
1.9e4
3.0 ug/mL
Human serum background
3.0 – 100 ug/mL
Trypsin
All forms of mAb
Protein A AnJ-‐Id mAb
Affinity Enrichment
Signature peptide
FNWYVDGVEVHNAK
y9
Trypsin
Increasing Sensitivity of Total LC-MS/MS PK Assay by Affinity Enrichment of Signature Peptide
Other immunoglobulins
+ Rb anti-peptide pAb
Signature peptide
L. Liu, K. Xu, S. Kaur
• Direct serum digestion ensures measuring the total Ab conc. • Immuno-enrichment by anti-peptide antibody enhances assay sensitivity
LC-MS/MS with Anti-ID Capture Allows Detection of Total Concentrations of mAb RG7652 in Presence of Soluble Antigen at Clinically Relevant Levels
Endogenous antigen levels in patients range from 0.13 ug/mL to 0.59 ug/mL
Good Correlation between LC-MS/MS and ELISA PK Data Rapidly Confirmed Unusual Nonlinear PK
Summary: Based on LC-MS/MS & ELISA comparable data unusual nonlinear clinical PK results were readily confirmed
Summary
• LBA, micro-fluidic LBA, LC-MS/MS and and IA-LC-MS/MS provide complimentary methods for protein quantification
• Unusual large molecule PK results can be rapidly investigated using a combination of diverse platforms
• Multiple platforms provide advantages during all stages of discovery and drug development
BioAnalytical Sciences/ ADC Group Mass Spectrometry: Keyang Xu, Luna Liu, Carl Ng, Dian Su, Jintang He Mass Spectrometry: Ola Saad, Neelima Koppada, Violet Lee, Suk-Joon Hyung Immunoassays: Randy Dere Montse Carrasco, Helen Davis, Connie Mahood, Kyu Hong, Collaborator Groups and Teams Administrative Assistant Cassie Duenas Dev Sci Management An Song, Patty Siguenza, Sara Kenkare
Acknowledgements
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