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 Assay1. Receptor Binding/Activation -Binding, KIRA

3. Cellular Response- Growth- Cytokine secretions

2. Reporter Gene Assay

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. FlowFeasibility Study

MCBWCB

Produce reference materialOr standard, control

Development

Qualification

Data analysis model

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.

ED50Test Sample

C= ED50

Resp

on

se

ED50

C= ED50

Test Sample A

Resp

on

se

Test Sample B

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)

M10R and =

−

=

b

YYM

RT

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.

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

%

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