Transcript corporate overview - Frontage Laboratories

BIOMARKER DEVELOPMENT AND
VALIDATION
PRACTICES & EXPERIENCES
Shawn Li, M.D., Ph.D.
October 1, 2012
CORPORATE OVERVIEW
PRESENTATION OUTLINE
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Introduction
Frontage Capabilities and Approaches
Case Studies
Challenges and Solutions:
 Measurement of Analyte in Presence of Endogenous
Protein
 Qualified Assays
CORPORATE OVERVIEW
BIOMARKER DEFINITION
A characteristic that is objectively measured and evaluated as an
indicator of normal biologic processes, pathogenic processes, or
pharmacologic responses to a therapeutic intervention. [Clinical
Pharmacology & Therapeutics (2001) 69, 89–95]
 Disease-related Biomarkers
 Drug-related biomarkers
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Diagnostic Biomarker
Safety Biomarker
PD Biomarker
Efficacy Biomarker
Surrogate Biomarker
Prognostic Biomarker
Predictive Biomarker
CORPORATE OVERVIEW
 Classification:
 Type 0 - Natural history
markers
 Type 1 - Drug activity
markers
 Type 2 - Surrogate
markers
ROLE OF BIOMARKER IN DRUG DEVELOPMENT
Pharmacology markers for
•pharmacodynamic performance
•pharmacokinetic correlation
Disease biomarkers for
•diagnosis & prognosis
•predisposition assessment
•early detection of “toxicity”
Safety & efficacy biomarkers for
•clinical response monitoring
•surrogate endpoints
•response prediction
Compound selection
Dose selection
Dose monitoring
Therapeutical
performance
Patient selection
Utility of biomarkers to support decision making in drug development is
directly related to quality of data
CORPORATE OVERVIEW
BIOMARKER ASSAY CATEGORIES
Continuous Analytical Response
Definitive
Quantitative
Relative
Quantitative
Biomarker levels determined
using a ‘reference standard’
(i.e., calibration curve)
–Reference standard needs to be
representative of analyte
CORPORATE OVERVIEW
Categorical Response
QuasiQuantitative
Qualitative
Biomarker levels are determined
without using a reference standard
(i.e., calibrators)–Reference material is
not available, or–Not representative of
test samples
BIOMARKER ASSAY CATEGORIES AND PURPOSE
The fit-for-purpose approach to biomarker method validation tailors the burden of proof
required to validate an assay to take account of both the nature of technology utilized
and position of the biomarker in the spectrum between research tool and clinical end
point. Ultimately, fit-for-purpose requires an assessment of the technical ability of the
assay to deliver against the predefined purpose
IHC=immunohistochemistry; LBA=ligand binding assay; MS=mass spectrometry;
PD=pharmacodynamic; POM=proof of mechanism; POC=proof of concept.
CORPORATE OVERVIEW
BIOANALYTICAL VS. BIOMARKER ASSAYS
CORPORATE OVERVIEW
SUMMARY OF VALIDATION PARAMETERS APPLICABLE TO EACH CATEGORY OF
BIOMARKER ASSAY
Validation
Parameters
Definitive
quantitative
Relative
Quasi-quantitative Qualitative
quantitative
Sample stability
X
X
X
X
Reagent stability
X
X
X
X
Assay range
X
X
Parallelism
X
X
Dilution linearity
X
X
Accuracy
X
X
Precision
X
X
X
Sensitivity
X
X
X
X
Specificity
X
X
X
X
Example Assays
Mass
ELISAs
spectrometry
CORPORATE OVERVIEW
Immunogenicity
immunoassays
Immunohistochemistry
BIOMARKER CAPABILITIES AT FRONTAGE LABS
DNA
 Genotyping, Mutation, Haplotype determination, SNP
 Epigenetic, Methylation, Accetylation
RNA
 Gene expression (real time PCR), In situ hybridization
Protein/Peptide
 Single and multiplex Ligand binding assays
 ELISA, ECL, SDS-PAGE, Western Blot
 LC/MS/MS
Cytology / Histopathology
 Tissue array
 Immunohistochemistry / Immunocytochemistry
 Flowcytometry
Immunogenicity
 Screening/Confirmation/Titer determination
 Cell based assay, Neutralization antibody assay
CORPORATE OVERVIEW
VALIDATION APPROACHES AT FRONTAGE LABS
Biomarker validations
– Fit-for-purpose (FFP)
- Validation parameters differ depending on the purpose of study
and method categories
- Follow bioanalytical method validation guideline as close as possible
– Biomarker validation SOP and validation protocol implementation
-Design of the experiments
-Standard and QC preparations
-Acceptance criteria
-Data reporting
CORPORATE OVERVIEW
CASE STUDY 1: GENOTYPING OF FCGRIIA
Biomarker Category: Qualitative
FcgRIIa can have either histidine (H131) or arginine (R131) at
amino acid position 131 located in the IgG-binding site
H131
H131/R131
R131
CRP Binding
NO
YES
YES
CRP Activation of Fibrocytes
NO
YES
TBD
SAP Binding
YES
YES
YES
SAP Inhibition of Fibrocytes
YES
YES
YES
CRP cross-inhibit SAP
NO
TBD
TBD
1
H R H
2
3
Patient No.
R H R
FcgRIIA
CORPORATE OVERVIEW
Patients with different genotype response differently
to anti-inflammatory drugs
CASE STUDY 2: FISH DETECTION OF mRNA
Biomarker category: Qualitative/semi-quantitative
In situ hybridization of target biomarker mRNA in cancer tissue
Drug treated
CORPORATE OVERVIEW
Vehicle injected
CASE STUDY 3: WESTERN BLOT SEMI-QUANTIFICATION OF BIOMARKERS
Biomarker category: Quasi-quantitative
Target protein A
CORPORATE OVERVIEW
CASE STUDY 4: RT-PCR QUANTIFICATION
Biomarker category: Quasi-quantitative or Quantitative
Real-time RT-PCR quantification of BDNF mRNA in brain tissue
β-Actin
CORPORATE OVERVIEW
Drug x
Dosage 3
Drug x
Dosage 2
Drug x
Dosage 1
Positive
control
BDNF RT-PCR Amplification Plot
Vehicle
ΒDNF
CASE STUDY 5: MSD Multiplex Assay
TH1/TH2 (10-Plex)
CORPORATE OVERVIEW
Biomarker category: Quantitative
LLOD≠ LLOQ, Matrix effect
1 curve fail ≠ all curve fail
CASE STUDY 6: ANTI-DRUG ANTIBODY ASSAY
Immunogenicity: Unwanted immune response in the patient to
biologic drugs--Development of Anti-Drug Antibodies(ADA)
Neutralizing antibodies: Prevent drug from binding to the target
molecule either by binding directly to epitopes in active site or by
steric hindrance: abolish effect of the drug
 Hypersensitivity reactions
 Neutralize the activity of an endogenous equivalent,
resulting in a deficiency syndrome.
 Efficacy
 Altered drug PK profile due to change in clearance
Non-neutralizing antibodies: Bind to sites on the drug molecule
without affecting target binding and efficacy
CORPORATE OVERVIEW
CASE STUDY 6: ANTI-DRUG ANTIBODY ASSAY
Biomarker Category: Quasi-quantitative
Key parameters for
Points to consider for assay
development
validation
Titer-based assay
Screening cut point
PCs, prefer pAbs
Specificity/confirmation
cut point
Detect low and high
affinity
Sensitivity
Sensitivity/w drug
System suitability
controls(QCs) acceptance
• Preclinical: 500-1000
criteria
ng/mL
Selectivity/Interference
• Clinical: 250-500
ng/mL
• Matrix components
Screening cut point, 5% FP
• Drug
Confirmation and titration
Precision
steps are needed
Robustness
Stability
CORPORATE OVERVIEW
CASE STUDY 6: ANTI-DRUG ANTIBODY ASSAY
Method development and validation of anti-X123 antibody assay
Evaluation
Analytical Method
Matrix
Positive Control
Drug
Screening Assay Cut Point (S/N)
Confirmation Cut-Point
High Positive Control
Low Positive Control
Negative Control
Titration
Hook Effect
Drug Tolerance
Interference by Hemolysis and Lipemia
Immuno-depleted Control (LPC)
Immuno-depleted Control
Method Relative Sensitivity
Method Selectivity
Bench Top Stability
Refrigerated Stability
Freeze Thaw Stability
CORPORATE OVERVIEW
Summary
ELISA
Monkey Serum
anti-X123
X123
1.44
22.08%
Intra-Assay %CV: ≤ 12.6%
Inter-Assay %CV: 13.9%
Intra-Assay %CV: ≤ 14.5%
Inter-Assay %CV: 17.5%
Intra-Assay %CV: ≤ 16.5%
Inter-Assay %CV: 21.5%
The average assay titer was 1:55 for TPC
No Hook effect up to 100000 ng/mL
4.13µg/mL at 500ng/mL of ADA
No Interference observed
Average: 34.88%
Average: 92.66%
167.75 ng/
90% of the spiked and unspiked samples
were within 75%125% of the respective
controls.
Stable up to 24 hrs
Stable up to 3 days
Stable up to 6 cycles
CHALLENGES IN ELISA-BASED BIOMARKER ASSAY
Measurement of analyte in presence of endogenous protein:
Endogenous compound can exist in multiple isoforms or clipped forms in
matrix
Multiple configurations of LBA: options to measure different compound forms
– Appropriate choice of binding reagent, incubation times, buffers, sample
dilution etc
– Analog contains specific epitopes (characterization required)
– Specific reagent development may needed
Unexpected challenges encountered with most commercial assay kits
CORPORATE OVERVIEW
CHALLENGES IN ELISA BIOMARKER ASSAY
How to create STDs/QC when endogenous levels present in matrix
– Use of matrix with low endogenous levels
– Use of substituted matrix
– Prepared in buffer
Subtract basal level
– Analysis of blank sample (zero spike)
– Endogenous amount subtracted, nominal amount of added spike determined
Endogenous and therapeutic act in similar manner: correction factor applied
– If endogenous and therapeutic NOT linear, correction factor cannot be applied:
total measured concentration reported
Matrix can be stripped (charcoal):
– Not typically recommended
– Incomplete removal?
– Expensive,
CORPORATE
OVERVIEW time-consuming
CHALLENGES IN ELISA BIOMARKER ASSAY – MRD DETERMINATION
MRD: The smallest dilution to which a sample must be diluted in buffer
to optimize accuracy and precision in an assay run by reducing the
signal to noise ratio
CORPORATE OVERVIEW
CHALLENGES IN ELISA BIOMARKER ASSAY – SELECTIVITY EVALUATION
Subtract basal level
Human Plasma Lot Blank Result
#
(pg/mL)
BRH600992
208.892
BRH600993
BQL
BRH600999
BQL
BRH601000
BQL
BRH601002
265.448
BRH601003
BQL
BRH601004
BQL
BRH601005
BQL
BRH601006
BQL
BRH591695
330.846
BRH591696
BQL
BRH591706
236.192
BRH591709
362.267
BRH591714
556.763
BRH591716
368.536
BRH601001
859.823
Corrected
Spiked Sample Concentratio
Result (pg/mL)
n (pg/mL)
BQL
NA
303.002
NA
315.437
NA
249.797
NA
430.442
164.994
347.240
NA
341.945
NA
235.449
NA
401.342
NA
496.075
165.229
394.732
NA
415.751
179.559
518.714
156.447
786.363
229.600
699.857
331.321
1099.457
239.634
RE%
NA
NA
NA
NA
-17.5
NA
NA
NA
NA
-17.4
NA
-10.2
-21.8
14.8
65.7
19.8
Pass/Fail
Fail
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Fail
Pass
Method LLOQ = 200 pg/mL
Acceptance Criteria: If the measured concentration in the blank is ≥ LLOQ, the endogenous
level will be subtracted. If the measured concentration in the blank is < LLOQ, the spiked
concentration should be between 150-450 pg/mL (blank range + LLOQ range)
CORPORATE OVERVIEW
LC/MS/MS METHOD VALIDATION OF ENDOGENOUS COMPOUNDS
Case Study 1
A Highly Sensitive and Selective Method for the Determination of
Leukotriene B4 (LTB4) in Ex-vivo Stimulated Human Plasma by Ultra
Fast Liquid Chromatography–Tandem Mass Spectrometry
Case Study 2
Determination of an Endogenous Biomarker - 4β-Hydroxycholesterol
in K2EDTA Human Plasma by LC-MS/MS
CORPORATE OVERVIEW
QUALIFIED ASSAYS (COMPLETE BIOMARKER LIST AVAILABLE)
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Human IFNg-plasma-MSD
Human IL6-plasma-MSD
Human IL1-b-plasma-MSD
Human TNF-α-MSD
Human PSA-serum-Spectramax
Human Testosterone serumSpectramax
Human Complement C3a-plasmaSpectramax
Human Complement Bb-plasmaSpectramax
Podocin in human urine -Spectramax
Nephrin in human urine –Spectramax
Creatinine in human UrineSpectramax
NPY Human Plasma/serum spectramax
CORPORATE OVERVIEW
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Fibronectin in rat urine-Spectramax
MCP-1 in rat urine-MSD
Collagen IV in rat urine-Spectramax
sGAG in rat urine-Spectramax
Mouse IFNg-plasma-MSD
Mouse IL6-plasma-MSD
Mouse IL1-b-plasma-MSD
Mouse TNF-a-plasma-MSD
Mouse PSA-serum-Spectramax
Mouse Testosterone-serumSpectramax
Mouse Complement C3-plasmaSpectramax
Mouse Complement C5a-plasmaSpectramax
THANK YOU!
Shawn Li, M.D., Ph.D.
Director, Biologics Services
Frontage Laboratories, Inc.
700 Pennsylvania Drive
Exton, PA 19341
Tel: 484-348-4860 / Fax: 610.232.0101
Email: [email protected]
www.frontagelab.com
CORPORATE OVERVIEW