[PPT] - Horseradish Peroxidase Immunoassays Patrick A. Williams Ian M. PowerPoint Presentation

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Horseradish Peroxidase Immunoassays Patrick A. Williams Ian M. Klink Brandon D. Shaw

Chemical Engineering Senior Design Project

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Overview

I. Purpose II. Immunoassay III. Market IV. Characteristics of Our Immunoassay V. Design Options VI. Fabrication

VII. FDA Approval
VIII. Financial Risk

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Objectives & Goals

Produce a quality diagnostic test
Provide leading immunoassays for

bacterial detection

Target rural hospital market initially
Make Money

Purpose Purpose

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Product

Immunoassay Kit

Analytical test
Targets specific molecule
Results obtained by signal

generation

Purpose Purpose

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Why S. aureus immunoassay?

Staphylococcus aureus

Major cause of hospital infections
8 common infections
Can cause deadly complications
Hospital Infections 4th leading cause
f US deaths

www.ulb.ac.be/sciences/biodic/ ImBacterie2.html

Purpose Purpose

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Why S. aureus immunoassay?

Common Conditions

Description Infection

acute infection associated with tampon use during menstruation Toxic shock syndrome

nset after surgery

Osteomyelitis inflammation of the lungs Staphylococcal pneumonia A localized collection of pus in part of the body, formed by tissue disintegration and surrounded by an inflamed area Abscesses a contagious skin infection Impetigo Inflammation of one or more sebaceous glands of an eyelid Sties painful, pus-filled inflammation of the skin and subcutaneous tissue Boils inflammation of the follicles Folliculitis Purpose Purpose

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

S. aureus immunoassay
Early detection yields better

treatment

Sanitation control
Can be modeled and adapted to
ther immunoassays

Purpose Purpose

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Immunoassay

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Components

Antibody

Immune system vesicle

Antigen

Molecule being detected

Enzyme

Oxidizing agent using in signal generation

Substrate

Signal generator in conjunction with enzyme

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Background

Antibody
Chains
Regions
Types
Polyclonal
Monoclonal

Variable Region Constant Region Heavy Chain Light Chain

Immunoassay Immunoassay

Polyclonal

Same form of antibody with

varying binding specificities and affinities for the targeted antigen

Monoclonal

Arises from a single cell and have

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Background

Which antibody for our assay?
Immunoglobulin G (IgG)
Why IgG?
Widely available
Higher antigen affinity compared to
ther antibodies
Structure of IgG

Immunoassay Immunoassay

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Background

Antigen — Protein A
Source - Staphylococcus aureus

Immunoassay Immunoassay

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Background

Immunoassays
Immunometric
Enzyme linked immunosorbent assay

Wash to remove unbound labeled antibody Solid phase coated with antibody Enzyme labeled antibody Antigen

E E E E E E E E

Immunoassay Immunoassay

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Background

Immunoassays

E E E S S S S E E E S S S S

Enzyme Catalyzed Reaction to Produce Signal Immunoassay Immunoassay

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Immunoassay Operation

Equilibrium test
Approximately

3 hours

Working Range
Pico-molar

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Laboratory operation

96 Well Plate Wash

Sample

Wash

Conjugated HRP

Chromogenic Reagent

Stop Spectrophometer

λ=403 nm Immunoassay Immunoassay

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Market

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Market

Target market segment
Rural hospitals
Why rural hospitals?
Untapped market
Technology — Cost barrier
Automated throughput vs. manual
Prohibitive capital cost

Market Market

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Market

Rural Hospital Infection Rates
300,000 total infections annually in

rural hospitals

S. aureus responsible for

approximately 76,000 infections

300,000 assays required to test all

patients presenting symptoms

Market Market

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Market

Rural Hospital Trends
Recent growth in rural population
6% increase in hospital discharges between 1995 &

2000

11% growth in rural population between 1990 &

2000

Rural hospital discharges to increase
Discharges expected to increase 10% between

2005 & 2014

Market Market

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Market

Competition
Larger biotechnology companies
Remel, Sure-Vue, Murel
Our Advantages
Quantitative Detection
Shorter waiting time
Does not require sample culturing

Market Market

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Immunoassay Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Reagents & Components

What’s in the box?
Two 96 - well plates
HRP-IgG conjugate solution
Wash buffer
TMB Substrate solution
Stop Solution
Standard Antigen Solution
Standard Dose Response

Curve

Characteristics Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Reagents & Components

96 well plate
Standard for use with ELISA
Allows for multiple sample testing
Polystyrene
IgG-HRP conjugate solution
Signal generation component of assay

Characteristics Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Reagents & Components

Wash buffer
TRIS buffered saline with 0.1% Tween
Hydrogen peroxide
Oxidizing agent
TMB Substrate solution
3,3’,5,5’-tetramethylbenzidine
Produces strong signal
Lacks mutagenic qualities

Characteristics Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Reagents & Components

Assay buffer
TRIS buffered saline with 1% BSA

(Bovine Serum Albumin)

Standard antigen solution
Protein A antigen solution
4 concs. that span working range

Characteristics Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Reagents & Components

Stop solution
1M hydrochloric acid
Standard dose response curve
Signal vs. antigen conc.
Conc. as low as 800 pg/mL

Characteristics Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

HRP Kinetics

Enzyme Reaction

HRP undergoes three reactions to

generate signal

O H AH HRP AH HRP AH HRP AH HRP O H HRP O H HRP

k k k 2 2 2 2 2 2

2 2 1 1 +

+

→  + −

+

− →  + − + − →  +

γ β α

Color is generated from oxidized substrate radical

Characteristics Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

HRP Kinetics

Stop solution will be added to the

mix after 10 minutes.

Spectrophotometer is used to

measure the concentration of colored substrate in solution

λ=403 nm

Characteristics Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Kinetic Analysis

Antibody-antigen binding
Surface adsorption kinetics
Three binding reactions of interest
Antibody to surface
Antigen to antibody
Labeled antibody to antigen-

antibody surface complex

Ag Ab Ag Ab

k k

− +

   ← − →  1 1

Characteristics Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Kinetic Analysis

Evaluation of rate constants
How?
BIAcore s51

Characteristics Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Kinetic Analysis

Plate Antibody Binding

Binding reaction of antibody to surface
First order surface adsorption kinetics
Adsorption and Desorption reactions

considered

3 , 3 3 3 3 3 , 3 AD Ab v S DES SAb AD DES Ab v S SAb

Adsorption r k C C Desorption r k C k Equilibrium K k Netrate r r k C C k C

− − −

= = = = = = = − = −

3 3

k k

S Ab SAb

−

→ ←

+

Characteristics Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Kinetic Analysis

Binding of antigen to antibody

2 , 2 2 2 2 2 , 2 AD v SAb Ag DES SAb Ag AD DES v SAb Ag SAb Ag

Adsorption r k C C Desorption r k C k Equilibrium K k Net rate r r k C C k C

− − − − −

= = = = = = = − = −

2

Ag SAb Ag SAb

k k

− +

   ← −   →  2

Characteristics Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Kinetic Analysis

Surface complex binding

      − = = = = =

− = − = − − − − − − − − 5 , 5 5 5 5 5 , 5

K C C C k Netrate K k k m Equilibriu C k Desorption C C k Adsorption

HRP Ab Ag SAb Ag SAb v Ab HRP HRP Ab Ag SAb Ag SAb v Ab HRP

HRP Ab Ag SAb Ab HRP Ag SAb

k k

− − − − + −

   ← −   →  5 5

Characteristics Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Kinetic Analysis

Equilibrium interaction model

1 , 1 SAb Ag Ab HRP HRP Ab v SAb Ag

C r k C C K

− − − − −

= −

. 1 1 SAb Ag Ab HRP bind SAb HRP Ab Ag

C r k C C K

− − − − −

  = −    

      − =

− − 2 , 2

K C C C k r

Ag SAb SAb v Ag Ag SAb

Characteristics Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Kinetic Analysis

Assume Adsorption & Desorption rates are

equal to zero

Solve for unknown concentrations
Initial antigen concentration is known
Initial surface antibody conc. is known

( )

[ ]

SAb v Ag Ab HRP HRP Ab Ag SAb Ag SAb Ag Ag SAb Ag Ab HRP HRP Ab Ag SAb Ag SAb Ag Ag SAb v Ag Ag SAb

C C C C C K C C C C C C C C K C

, , 1 , , 1

) (

− − − − − − − − − − − − − −

− − − = − − − = =

Characteristics Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Kinetic Analysis

System of 3 equations
Solved using Polymath™
Conc. of surface antibody-

antigen complex critical variable

Strength of signal is defined by

this concentration

Characteristics Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Kinetic Analysis

Dose response curve
Indicates antigen concentration
Curve asymptotes as surface antibody

concentration is approached

Characteristics Characteristics

Dose Response Curve-varying CSA b(mol/cm2) 0.00E +00 2.00E

13

4.00E

13

6.00E

13

8.00E

13

1.00E

12

1.20E

12

1.40E

12

1.60E

12

1.80E

12

2.00E

12

550000 1100000 1650000 2200000 2750000 3300000 3850000 4400000 4950000 5500000 CAg, 0 (pg/mL) CSAbAgAbHRP (mol/cm^2 2.5e-10 CSAb (mol/cm^2) 5e-10 CSAb (mol/cm^2) 7.5e-10 CSAb (mol/cm^2) 1e-9 CSAb (mol/cm^2) 1.25e-9 CSAb (mol/cm^2)

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Sensitivity

Assay sensitivity
How low can we go?
Determined by surface complex

concentration

Minimum complex conc.=1.8x10-15 mol/cm2

Characteristics Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Sensitivity

Signal strength increases with surface

antibody concentration

Optimize cost of surface antibody

using this plot

Use minimum amount of surface

antibody that can generate signal

Characteristics Characteristics

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Sensitivity

Characteristics Characteristics

Dose Response Curve-varying CSA

b (mol/cm2)

0.00E +00 1.00E

15

2.00E

15

3.00E

15

4.00E

15

5.00E

15

6.00E

15

7.00E

15

8.00E

15

9.00E

15

1.00E

14

400 800 1200 1600 2000 2400 2800 3200 3600 4000 CAg, 0 (pg/mL) C

SAbAgAbHRP

(mol/cm^2)

2.5e-10 CSAb (mol/cm^2) 5e-10 CSAb (mol/cm^2) 7.5e-10 CSAb (mol/cm^2) 1e-9 CSAb (mol/cm^2) 1.25e-9 CSAb (mol/cm^2)

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Sensitivity

Dose response curve is only used

in linear range

Linear range= 800 pg/mL – 4000 pg/mL

Characteristics Characteristics

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Design Options

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Evaluated Processes

Horseradish Peroxidase (HRP) Extraction

2 Differential Extractors
Biphasic Salt Solution
Polyethylene Glycol (PEG) & KPO4
Reagent Cost≈$20,000
FCI≈$150,000

P-1 / DX-101 Differential Extraction PEG/ water Crude Extract P-2 / DX-102 Differential Extraction Extract 1 Salt Phase Raff KPO4 P-3 / V-101 Storage PEG RECYCLE PRODUCT HRP/BUFFER

Amount: 1.24kg Activity: 117U/mL

Modeled using SuperPro Designer 4.7

Design Options Design Options

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Evaluated Processes

Cost Comparison of Produced and

Purchased HRP

Purchase HRP
Annual requirement: 5 g/yr
Cost: $15,000/yr
Produce HRP
Approximate Labor cost: $35,000/yr

Design Options Design Options

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Evaluated Processes

HRP Extraction continued

Decided not to produce
Purchase HRP
Lyophilized
Small amounts needed
Efficient for small company
$266 per 100 mg (Sigma)

Design Options Design Options

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Fabrication

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Plate Preparation

Polystyrene Plate Steps

1. Fill wells with IgG coating solution 2. Incubate for 18 hours 3. Remove coating solution. Wash. 4. Fill wells with 2nd coating solution 5. Incubate for 30 minutes. 6. Remove coating solution. Wash, and dry for 24 hours.

Fabrication Fabrication

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Manufacturing

Equipment

3 major pieces
Sterilization
Bottling
Labeling

STS Series HEA 105 KFVG x212

Fabrication Fabrication

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Manufacturing

1

Assay Solution - Sterlization

0.5h 2

Standard Solution - Sterilization

0.5h 3

Wash Buffer - Sterilization

0.5h 4

Conjugated HRP - Sterilization

0.5h 5

Substrate (TMB) - Sterilzation

0.5h 6

Stop Solution - Sterilization

0.5h 7

Assay Solution - Filling

0.5h 8

Standard Solution - Filling

0.5h 9

Wash Buffer - Filling

1.0h 10 Conjugated HRP - Filling 0.5h 11 Substrate (TMB) - Filling 0.5h 12 Stop Solution - Filling 0.5h 13 Assay Solution - Labeling 1.0h 14 Standard Solution - Labeling 1.0h 15 Wash Buffer - Labeling 1.0h 16 Conjugated HRP - Labeling 1.0h 17 Substrate (TMB) - Labeling 1.0h 18 Stop Solution - Labeling 1.0h 5

Dail y Oper at ion

1 2 3 4 9 10 11 12 ID Task Name Duration 8

Gantt Chart: Components Gantt Chart: Components

$24,370 Packaging $490,169 Operating Labor $7,000,000 Raw Materials

Fabrication Fabrication

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Packaging

Shipping Box

6”x6”x4” Indestructo Mailing

Gray Convoluted Foam

8”x6”x2”

Yearly Packaging Costs $24,370

Fabrication Fabrication

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Quality Control

Random quality audits
2.5% of each assay batch
Verification of valid working range
Error greater than 5% fails audit
2.5% of tested assays fail - discard

batch

Fabrication Fabrication

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Quality Control

Apparatus
Xiril™ X100

combination liquid handler and plate reader

Higher test

throughput

Less chance of

human error

Fabrication Fabrication

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FDA Approval

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Why FDA Approval

Food & Drug Administration

Government organization
Protects Consumers
Promotes Public Health
Regulates Medical

Devices

Approval
Require for US sales
Quality product endorsement

FDA Approval FDA Approval

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

FDA Testing Phases

Phase 3 Scrap Phase 2 Scrap Phase 1 Scrap Phase 4 Scrap

Accuracy and Reproducibility Test Quality Control Human Serum Testing Chemical Inhibition Testing

FDA Approval FDA Approval

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

FDA Testing Decisions

How much pre FDA testing

should we do?

Should we perform many tests in

the same phase, or just a few?

After what phase should we enter

the FDA approval process?

To answer these questions we

developed a novel model of the FDA approval process.

FDA Approval FDA Approval

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Pertinent Steps

Phase 1: Accuracy and Reproducibility Tests: 500 Time: 4 weeks Cost: $28,000 Phase 2: Human Serum Testing Phase 3: Chemical Inhibition Testing Phase 4: Quality Control

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

FDA Approval Outline

Pre-Clinical Testing Business Establishment & Registration FDA Testing Phases

FDA Approval FDA Approval

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Financial Risk

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Stochastic Modeling

Financial Financial Risk Risk

Phase 3 Phase 1 & 2 1000 Phase 3 & 4 500

2

Phase 4 Phase 1 & 2 500 Phase 3 & 4 250

3

Phase 3 Phase 1 & 2 500 Phase 3 & 4 250

4

Phase 4 Phase 1 & 2 1000 Phase 3 & 4 500

1

FDA

Number of Tests

Option

Evaluated Design Variables

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Stochastic Modeling

Evaluate all possible pathways in

the FDA model for each design variable to determine:

Cost
Time
NPW

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Stochastic Modeling

500 1000 1500 2000 2500 3000 3500 4000

NPW ($1000) Frequency (6000) (5000) (4000) (3000) (2000) (1000) 1000

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Stochastic Modeling

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 (7,000) (6,000) (5,000) (4,000) (3,000) (2,000) (1,000) 1,000

NPW ($1000) Probability

Option 1 Option 2 Option 3 Option 4

Risk Assessment

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Stochastic Modeling

Option 1 gave the best results
Thorough testing through all phases

reduces the uncertainty in the FDA approval process

Estimated testing cost: $1.8 Million
Pre FDA Testing: $1 Million
FDA Testing: $800,000
Estimated time: 1.7 years

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Breakdown

Estimated Annual Sales
250,000 Immunoassay Kits
$125 per kit
Total Sales: $31 Million
Total Product Cost
$30 million
Total Capital Investment
$5.8 Million
Estimated NPW
-$2 Million

Financial Financial Risk Risk

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Purpose Purpose Immunoassay Immunoassay Characteristics Characteristics FDA Approval FDA Approval Financial Financial Risk Risk Design Options Design Options Fabrication Fabrication Market Market

Questions

???

Financial Financial Risk Risk