Biological Assay Development and Qualification: Determine ... · Expected RP Measured RP CV%...
Transcript of Biological Assay Development and Qualification: Determine ... · Expected RP Measured RP CV%...
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Biological Assay
� Measuring the response of a living system to a biological substance
� Quality (RP, IU, pfu/mL) issue but not quantity issue (ng/mL, vp/mL)- Same quantity but different quality; e.g. Degraded product
� Activity and/or dynamic of the response
� Less precise (5-40% or higher)
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Biological Assay Formats
� In Vivo bioassay
� Cell based assay
� Organ or primary cell culture
� Stable cell line culture
- Receptor binding/activation assay
- Reporter gene
- Cellular Response
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In Vivo Bioassay
� Quality can only be demonstrated in vivo
� Labor intensive
� Larger N needed to overcome variability of the assay to demonstrate statistic significant.
� Expensive
� Variability: Inter- animal variability
� e.g. Colony formation assay
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Primary Cell Based assay
� Tissue/Organ: Tissue micro-environment
� Primary cell culture: specific cell population is needed.
� Purity and donor-to-donor variability
� e.g., Angiogenesis/Huvec (tube formation), Cartilage excretion/Chondrocyte.
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Stable Cell Line Based Assay
� Single homogenous source of cells
� Offer greater precision and accuracy
� Easy to use and analyze
� Easy to transfer from Lab to Lab: Assay transfer
� Cheaper
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Cell Based Assay1. Receptor Binding/Activation -Binding, KIRA
3. Cellular Response- Growth- Cytokine secretions
2. Reporter Gene Assay
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Receptor Binding/Activation
• Rapid: 5-10 minutes
• Specificity
• Reproducible
• Upstream response only
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Reporter Gene Assay
� Specificity
� Quick and robust: hours
� Engineer promoter of target gene with reporter gene
� Multiple genes may involved in the biological end point
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Cellular Response
� Provide whole picture of biological response
� Select relevant biological end point when possible� Cytokine secretion
� Proliferation
� Cellular markers
� Cytotoxicity
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Cellular Response
� Longer incubation time
� Affected by extraneous influences
� Less accurate and precise
� Lack of specificity
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Assay Dev. and Qua. FlowFeasibility Study
MCBWCB
Produce reference materialOr standard, control
Development
Qualification
Data analysis model
Validation
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Feasibility
� Development and qualification plan
� Test response & range
� Test multiple lots if possible
� If bridging to a new assay, correlate the response to old assay
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Biological Assay Development
� Design to fit:� Phase of the study and intended use
� Capability of receiving Lab.� Simple is the best
� Define assay configuration
� Understand and control the variable
� Data analysis model
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Phases of Study
� Time and Resource
� Research: Yes or No
� Phase I and II
� Phase III: lot-to-lot, validation
� Stability study: model decay curve
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Capability of Receiving Lab.
� Simple is the best
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Assay Configuration
� Nested Assay
� Biological Response
� End point Read out: May need to determine both quantity and quality (Two in one)
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Understand and Control the Variable
� Cell culture
� Reagents
� Reference material.
� Data Analysis
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Cell Culture
� Cell banks
� Establish MCB and WCB: demonstrate comparable performance
� Characterization
� Program to qualify a new bank.
� Variability within a banks: beginning, middle and end of freeze- time before freeze.
� Stability of banks.
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Cell Culture
� Passage # vs. response:� Continuous culture:
� One vial for several assays� Great if assay is performed often� Performance may drift as passage increase� May be affected extraneous factors
� Fixed Passage: � One vial for one assay� Fixed passage � Great if assay is not perform as often� May need a big WCB
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Cell Culture
� Pre-assay culture condition: � Reagent used for cell culture, e.g. FBS� Post-thaw culture time for recovery
� Culture condition: seeding and feeding schedule.
� Assay culture condition� Assay incubation time for cellular response
� Seeding density
� Edge effect
� Dose response range: upper, lower asymptote, at least 4 or 5 points for linear portion of curve.
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Reagents
� FBS
� Media
� Other supplement reagents
� Stability
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Reference Material
� As a reference to
� Demonstrate product consistency or process characterization.
� Monitor assay performance
� Assign value to test sample
� Representative product if possible
� Monitor the performance: set up criteria
� Program to qualify a new reference material
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Data Analysis
� ED50
� Relative potency
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Point Determination
� ED50
� Curve may shift and change shape depending on the condition of culture and sample quality.
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ED50Test Sample
C= ED50
Resp
on
se
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ED50
C= ED50
Test Sample A
Resp
on
se
Test Sample B
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Relative Potency
� Compare test sample to reference product dose response curve
� Full Curve Analysis
� Measure activity and kinetic of stimulation
� RP can be translated to IU if international standard exist.
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Parallel Line Data Analyses (Finney)
M10R and =
−
=
b
YYM
RT
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ED50 vs. Parallel Line Analysis
� ED50� Simple data output
� Easy to understand
� May be misleading
� Parallel line analysis� Full curve analysis
� Complicate calculation
� May not easy to understand
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Assay Qualification
� Assay pre-validation or characterization
� Decision to proceed
� The assay is as it is and the assay performance is as it is.
� Q2R for pre-validation.
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Q2R
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Assay Qualification
� Specificity
� Only the target analyte can induce the activity
� Often difficult for biological assay in a complex sample matrix
� Test degrade product: peptide
� Test sample matrix if possible.
� Product from mock process
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Accuracy and Range
� Reference material is concentrated or diluted to simulate test samples with different activities.
Expected RP Measured RP CV% Accuracy
400% 356.21% 7.32% 89.05%
350% 334.55% 21.11% 95.59%
300% 312.24% 15.63% 104.08%
200% 210.39% 17.66% 105.20%
150% 171.00% 12.44% 114.00%
100% 97.66% 8.23% 97.66%
80% 85.34% 12.41% 106.68%
50% 55.44% 7.32% 110.88%
30% 33.16% 14.56% 110.53%
10% NA NA
Dilution Linearity
0%
100%
200%
300%
400%
500%
0% 100% 200% 300% 400% 500%
Expected RP%
Measu
red
RP
%
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Precision
� Intra-assay precision� Flask-to-flask
� Plate-to-Plate
� Position effects
� Inter-assay precision� Day-to-day
� Operator-to-operator
� Lab-to-Lab
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Robustness
� Pre-assay culture time
� Assay culture time (time for cellular response)
� Seeding density
� Freeze-and-Thaw samples for endpoint readout
� Not intended to make SOP flexible
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Assay Acceptance Criteria
� Reference Product: test assay performance
- Upper and lower asymptote
- Slope
- Intercept
- RMSE; Curve fit
� Parallelism: test kinetic of stimulation of test sample� F-test
� Tolerance range for slope difference between two vials of reference material.
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Q and A