Biological Assay Development and

Qualification:
Determine Potency

Pocheng Liu, Ph.D.

pocheng.liu@google.com
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)
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
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
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.
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
 Cell Based Assay
1. Receptor
Binding/Activation -
Binding, KIRA

2. Reporter Gene Assay

3. Cellular Response
- Growth
- Cytokine secretions
Receptor Binding/Activation
• Rapid: 5-10 minutes
• Specificity
• Reproducible
• Upstream response only
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
Cellular Response

Provide whole picture of biological
response

Select relevant biological end point
when possible
 Cytokine secretion
 Proliferation
 Cellular markers
 Cytotoxicity
Cellular Response

Longer incubation time

Affected by extraneous influences

Less accurate and precise

Lack of specificity
 Assay Dev. and Qua. Flow
Feasibility Study

MCB Produce reference material

WCB Or standard, control
Development

Data analysis model

Qualification

Validation
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
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
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

Capability of Receiving Lab.

Simple is the best
Assay Configuration

Nested Assay

Biological Response

End point Read out: May need to
determine both quantity and quality (Two
in one)
Understand and Control the Variable

Cell culture

Reagents

Reference material.

Data Analysis
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.
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
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.
Reagents

FBS

Media

Other supplement reagents

Stability
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
Data Analysis

ED50

Relative potency
Point Determination
 ED50
 Curve may shift and change shape
depending on the condition of culture
and sample quality.
 ED50
Test Sample
Response

C= ED50
 ED50

Test Sample A
Response

Test Sample B

C= ED50
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.
Parallel Line Data Analyses
(Finney)

YT − YR
M = and R = 10 M
b
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
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.
Q2R
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
Accuracy and Range

Reference material is concentrated or diluted to
simulate test samples with different activities.

Dilution Linearity
Expected RP Measured RP CV% Accuracy
500%
400% 356.21% 7.32% 89.05%
350% 334.55% 21.11% 95.59%
400%
300% 312.24% 15.63% 104.08%

Measured RP%
200% 210.39% 17.66% 105.20%
300%
150% 171.00% 12.44% 114.00%
100% 97.66% 8.23% 97.66% 200%
80% 85.34% 12.41% 106.68%
50% 55.44% 7.32% 110.88% 100%
30% 33.16% 14.56% 110.53%
10% NA NA 0%
0% 100% 200% 300% 400% 500%
Expected RP%
Precision
 Intra-assay precision
 Flask-to-flask
 Plate-to-Plate
 Position effects
 Inter-assay precision
 Day-to-day
 Operator-to-operator
 Lab-to-Lab
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
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.
Q and A