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Complete, creative solutions for developers

and manufacturers of rapid assays

Complete, creative solutions for developers and manufacturers of rapid assays

Seminar on Business and Innovation in BiotechnologyINOVATEC

Belo Horizonte, Brazil. October 5th, 2010

• Brief introduction to DCN

• State of the art in applied rapid assay technology– Standard formats for standard applications– More advanced formats for quantification, high

sensitivity and digital interpretation

Agenda


• Market Opportunities and Market Demands

• Market Driven Innovation: Meeting market demand– Materials and reagents– Cassette and sample handler design– Reader development

• System development case studies

Key Points

1. Rapid testing is a dynamically growing, readily accessible market with opportunities available in numerous market segments

2. Assays can be developed to handle almost any reasonable market condition, based on existing technology– Simple, low cost lateral flow assays for simple, qualitative low

resolution applications– More complex, more expensive lateral flow systems for high


– More complex, more expensive lateral flow systems for high sensitivity, more quantitative, difficult applications

– These two types of assay are very different in how they are developed and manufactured

– Other approaches to high performance rapid testing are possible, but with a much longer outlook.

3. All of the elements needed to manufacture high performance tests are available today, so while technological innovation is ongoing, we can also choose to innovate in terms of where we apply these technologies

Introduction to DCN

• Based in Carlsbad, California

• Founded in 2003 with the purpose of assisting companies in

developing, manufacturing and commercializing rapid tests for

any application.

• Our primary platform technologies are lateral flow, flow


through, microfluidic, dipstick (chemistry) and rapid ELISA,

using visual, fluorescent or paramagnetic detection systems

• We work in any market segment where these technologies can

be applied

Primary Services and Products

Rapid assay development and manufacturingFrom Feasibility and Prototyping to full Development and Manufacturing Transfer

Education and training courses in rapid assay technologyLearn how to develop and manufacture rapid tests

Custom conjugation servicesprotein – particle, protein-protein, small molecule etc


Consulting on any aspect of rapid assay development and commercialization

ProductsMaterials starter kitsGold conjugation kitsLatex conjugation kitsColloidal goldAll related lateral flow materialsLateral flow reader systems: Fluorescent and visual

• Fully equipped for the manufacture of lateral flow, flow through,

and other rapid assay formats

• 8000 square feet, containing 3000 square feet of wet lab, housed in

a class 10,000 capable cleanroom, and an associated controlled

humidity room

• ISO 9001:2008 compliant, with EN 13485 by end 2010

Facility and Personnel


• ISO 9001:2008 compliant, with EN 13485 by end 2010

• Development staff of 14 industrially experienced assay development

scientists

Reagent Prep Area

Main Lab (Class 10,000 capable cleanroom)

Component Processing Area (Temperature / Humidity Controlled)

Our Markets

Medical Diagnostics

Veterinary Diagnostics

Agricultural / Environmental

Food Testing

OTC/Consumer

Our market is

international and

spread over multiple

application areas.


OTC/Consumer

Pharmaceutical

Biowarfare

Law enforcement / Forensics

Microbiology

Nucleic Acid Testing

Industrial Health and Safety

Approximately 40% of

our assay development

programs are US-

based, 40% are

European and 20% are

spread among Asia and

Latin America

Representative Clients

• Johnson and Johnson (UK)

• Novozymes (Denmark)

• Amic (Sweden)

• ICP Bio (Australia)

• Rockeby (Australia)

• Government of Thailand NIH (Thailand)

• Pacific Biotech (Thailand)

• BioRad (US/EU)

• Chiron Corporation (US)

• Amgen (US)

• Abbott (US)

• Genzyme Diagnostics (US)

• Becton Dickinson (US)

• Church and Dwight (US)


• Pacific Biotech (Thailand)

• Span Diagnostics (India)

• Daiichi Fine Chemical (Japan)

• Elixir Biosciences (China)

• MARS/Masterfoods (UK)

• Laboratorio Silanes (Mexico)

• Mintek (South Africa)

• Biosensia (Ireland)

• Church and Dwight (US)

• 3M (US)

• Whatman /GE (US / Europe)

• Aspen BioPharma (US)

• Genetic Testing Institute (GTI) (US)

• Cibavision (US)

• CDC (US)

• NIH (US)

Simple Devices for Rapid TestingStandard Lateral Flow Strip


Sample addition

Conjugate solubilization

Capillary flow;antibody binding

Membrane clearing;optical reading

Step 1

Step 2

Step 3

Step 4

Shown here are drawings of these simple devices. Both are based on the binding of the test substance (the analyte) by antibodies immobilized on a porous membrane. In a flow-through immunoassay, the role of the membrane is primarily to concentrate the reactants. This immunoconcentration brings reactants into the matrix of the membrane, where the diffusional distances are short enough so that the kinetics of binding closely approach the kinetics of binding in a solution. To get a high capacity for immobilizing the immunoreactants, the membrane should have a large surface area and therefore a small-pore size structure. As a consequence, the sensitivity may be higher than that of large pore membranes. While flow-through immunoassays are generally believed to be less sensitive than lateral flow immunoassays, this difference can be offset by the fact that much larger volumes of sample can be be applied to a flow-through device.

Simple Devices for Rapid TestingStandard Flow – through device


Sample addition

Optional wash

Conjugate addition; antibody binding

Final wash; membrane clearing;optical reading

In a flow-through immunoassay the sample is applied to the surface of a membrane containing immobilized antibodies. These can be in an area defined as a simple spot, or a more elaborate design, such as a plus sign. The sample flows through the membrane. Liquid is absorbed by a porous pad in contact with the opposite side of the membrane. If desired, an intermediate wash step can be employed. After the sample is added a detector is added. In the simplest embodiment, this detector is a solution of visible particles which are conjugated to antibodies to the test substance. These detectors bind to any analyte previously captured on the membrane by the immobilized antibodies and are observed visually. A final wash to remove background may or may not be necessary, depending on the specific nature and design of the test.

Evolution in Point of Care AssaysDrivers for Innovation and Market Growth

• Early POC devices were designed for qualitative applications

• Earliest applications in the 1980s were pregnancy tests,

growing in the 1990’s to include drugs of abuse and

infectious disease tests

• Market has grown steadily at a minimum CAGR of 10% since


the late 1990’s

• Market demand for tests in non-traditional areas has

expanded to include quantitative, high sensitivity

applications using difficult matrices, driving development

• Intellectual property issues have driven technological

innovation strongly in the past decade

Innovative Applications


Why lateral flow assays dominate the (non glucose) POC market

• Ease of use: Minimal operator dependent steps and interpretation

• Can handle small volumes of multiple sample types

• Can have high sensitivity, specificity, good stability

• Good Manufacturability and Scalability


• Good Manufacturability and Scalability

• Minimal market and user education required

• Market presence and acceptance

• Short development cycle – once reagents are developed, the timeline to manufacturing transfer and approval is short relative to other biotech products

• Sensitivity

• Control of user interpretation

• Reproducibility (CV)/ Quantification

• Multiplexing is difficult

Typical performance challenges in standard rapid assays


Application Requirements are Driving the Technology Forward

•The requirements that the major rapid test markets are demanding

include:

• Speed to result: 2-10 minutes

• Sensitivity: high picogram/ml to low nanogram/ml

• Ability to handle difficult sample matrices

• Quantification


• Quantification

•Multiplexing

• Removal of interpretation issues

• Control of data

Evolution in Rapid Test Technology: An Exercise in Market Driven Innovation

Clients provide use inspired basic research

Pasteur's Quadrant describes scientific research that both seeks fundamental understanding of scientific problems, and, at the same time, seeks to be eventually useful.


Development in rapid diagnostic testing incorporates elements of use inspired basic research (application development) and pure applied research (technology development)

DCN and partners provide the applied element

Bridging the Concept Gap

DCN


• Product Requirements Development

• Concept Refinement

• Feasibility and prototyping of all system elements

• Product development and system integration

• Transfer to manufacturing

• Commercialization

Veterinary9

Food and Beverage1

Pharmceuticals0.6

Envionmental0.2

Water0.2

Medical Diagnostics

Veterinary

Food and Beverage

High Performance Allows for New Applications Rapid Test Market Penetration and Opportunities


Medical Diagnostics89

Percent of Market by Application Total Market 2007: USD $2.3 billion Total Market 2012: USD $$3.7 billion

Pharmceuticals

Envionmental

Water

Major Rapid Test Market Opportunities1. Medical Diagnostics

35%

20%

21%

13%7% 4%

% market 2007Pregnancy Infectious diseases Cardiac markers

Cholesterol/lipds Drugs of abuse Other

•Growth opportunities exist in all medical segments

•Growth will be driven by reimbursement in US and EU markets


20%

24%

27%26%

15%

5% 3%

predicted market 2012Pregnancy Infectious diseases Cardiac markers

Cholesterol/lipds Drugs of abuse Other

•Requires performance improvements in quantification, sensitivity, reproducibility and multiplexing

Major Rapid Test Market Opportunities2. Veterinary Diagnostics

•Market was $205 million (USD) in 2007

• 9% CAGR to 2012 predicts a $310 million market by 2012

• Highly fragmented, price sensitive market, split among small animals and livestock

•Major segments are infectious disease testing, metabolic testing (cancer), diabetes and fertility/pregnancy


•Small animal tests tend to be paid for by owners and are non-regulated for the most part. Large animal testing tends to be government mandated and is regulated

•Growth driven by acceptance in large animal testing for infectious disease and fertility, and control of antibiotic use and in small animals by cancer and infectious disease testing


Major Rapid Test Market Opportunities3. Food and beverage testing

•Worldwide market of about $30 million in 2007

•Expected CAGR of 15% predicts $60 million by 2012

•Market is under-penetrated by rapid tests due to the preponderance of microbiological testing (slow, traditional technology largely used)


•Combinations of molecular and immunological testing may be important to this market

•Growth will be driven by increased awareness of rapid formats coupled with sampling and performance improvements that can generate appropriate sensitivity


Major Rapid Test Market Opportunities4. Water Testing and Environmental Remediation

•Worldwide market of about $5 million in 2007.

• Expected CAGR of 4% predicts $6 million by 2012.

• Highly regulated markets, with local or governmental agencies setting requirements for testing, often in association with AOAC (Association of Official Analytical Chemists International)

•Most testing is performed in certified laboratories by approved methods


methods

• Rapid tests are typically performed as spot checks between mandated test periods

• Also used for household and industrial testing of water quality in certain markets

• Growth will be driven by increased awareness of rapid formats coupled with performance improvements


• Option 1: Develop completely new platform technologies

based on microfluidics and alternative detection technologies

e.g. lab on a chip,

Market – Driven InnovationMaking test performance meet market demands


• Option 2: Improve the existing technology platform

A System Approach

• Rapid lateral flow and flow through assays can meet many of the market’s requirements, but a different approach to the assay is needed

• High performing rapid assays need to be approached in the same way as high performance laboratory based systems

• Key system elements:


• Key system elements:1) A properly developed assay made using

1) High quality materials2) High quality reagents3) Highly sensitive label for sensitive analyte detection

2) A well designed sample device and housing for the test3) A controlled method for result interpretation4) A well designed and controlled manufacturing process

1. AssayMaterials

• Analytical procedures usually require multiple steps

• Fluidics minimize number of steps in a test and increase ease of use

• In most POC devices these steps are performed using treated porous

matrices

• Fluid flow in POC devices usually relies on capillary flow

• Fluidic elements are usually designed to be disposable, and are


• Fluidic elements are usually designed to be disposable, and are

therefore made from low cost materials

• Suppliers must be of the highest quality. Lowest cost materials will not

support higher specified applications

Test Line

Control Line

Particle Conjugateon conjugate pad

WickSample Pad

Nitrocellulose MembraneBacking

1. Assay Reagents

• Recognition is generally Ab-Ag or Ab-Ab based

• Different characteristics are required for high performance in a lateral flow system than in an ELISA, for instance, due to the mechanics of the system:

– The method chosen for screening is important


• Important Parameters:

– Specificity

– Dissociation Constant (Kd)

– High Affinity (High Binding Rate Constant (kon))

– Antibody Class (subtype: IgG1, IgG2A, IgG3, etc.)

– Monoclonals typically preferred, but high purity polyclonals can also be used effectively

7777

Overall assay performance is affected by a number of variables including antibody quality. This includes such obvious parameters as antigen specificity and affinity (as defined by the dissociation constant). However, more important may be things like the binding rate constant, which is typically not measured during antibody screening and selection, and antibody classes. Some subtypes are easier to conjugate or fragment, and can pose special challenges with respect to purification, solubility and long-term storage. IgG3 for example, is not bound very well by Protein A, and tends to precipitate during freezing. Of course the quality of the membrane materials and assembly are critical, as is the often overlooked contribution of the plastic housing to assay performance. Oh, and did I mention antibody quality?

1. AssayLabels

• Colloidal gold– Typically 20-40nm– Low cost, difficult to control passive conjugations, one color,

relatively sensitive. Most common in qualitative applications

• Latex: Colored, Fluorescent, Paramagnetic– Typically 100-300nm– Low cost, covalent conjugations, variety of chemistries, well


– Low cost, covalent conjugations, variety of chemistries, well controlled, stable, variety of particles and labels. Most common for high sensitivity, quantitative applications (fluorescent, paramagnetic)

• Others– Upconverting phosphors– Colloidal carbon– Direct labeling using fluorophores and enhancers

Commonly used fluorescent labels in POC Applications

• We love fluorescence for high end applications!

• DCN’s Experience:

– Dark Red Labels (660-680nm) give good results due to lack of

background fluorescence in biological fluids in that spectral area

– Europium: Highly sensitive and commonly used due to broad

Stokes’ shift and stability (300 – 625nm)


Stokes’ shift and stability (300 – 625nm)

– Direct labeling of antibodies using fluorescent dyes is also an

option – sensitivity an issue – requires customization of system

components.

2. ReadersThe use of readers in lateral flow assay systems

Detection of the signal generated

by the reagents, translation into a

numerical or alphanumerical

output, and delivery of that

output

Use of readers allows for:Negative Positive

100% Visually Negative

100% Visually Positive

Operator Dependent Negative or Positive


Use of readers allows for:

• Improved sensitivity

• Reduction in user interpretation

issues

• Data capture, analysis and

storage

• Connectivity

• Quantification

• New applications

Operator Dependent Negative or Positive

Critical features:

• Hardware

• Algorithms for data processing,

• Signal to noise reduction,

•Measurement and data distribution

• Calibration methods

Reader Supply Partners

• Off-the-shelf readers are available that can be readily modified for any development program

• DCN represents two suppliers: LRE and Qiagen Lake Constance (formerly ESE) – Provide a base platform from which to start– Have a history of development of this type of product


– Have experience of regulatory and technical requirements– Can advise on important technical design considerations– May be faster to market– Generally have dedicated manufacturing capability

• Readers can be custom designed by experienced designer such as Source Scientific, Polygenesis, Omnica (all DCN partners)– Slower and more expensive route in return for a

custom solution

• Sensitive

• Affordable

• Simple to operate

• Small, Mobile and Robust

• Documented Results

• Objective Results

• Quantitative Results

ESE Quant

DCN’s Reader Partners: Handheld and Benchtop Readers


• Quantitative Results

DCN distributes both ESE and LRE readersDCN acts as the assay integration partner for both suppliers for

customers worldwide

LRE POC Reader

0

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1400

0 200 400 600 800 1000 1200 1400Data points

Intensity [mV]

Scan1

Scan2

Scan3

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Control

Test

Comparison of gold and fluorescent labelsLF Tuberculosis Fluorescent Assay

• Based on a dark red dye (latex particle based)

• Monoclonal sandwich assay

• Compared directly to a colloidal gold method

• Results read digitally using the ESE-Quant reader


Dark Red Dye (Latex)ex 660 em 680

0

20

40

60

80

100

120

500 550 600 650 700 750

Wavelength (nm)

Rel. Intensity

Excitation

Emission

LF Tuberculosis Fluorescent AssayGold vs. Fluorescent Latex

6

8

10

12

300

400

500

600

700

Peak Height (pixel units)

Peak Height (mV)

Sensitivity in TB Protein Assay:Gold vs Dark Red Flourescent Latex


0

2

4

0

100

200

0 0.1 0.5 0.75 1 1.25 5

Peak Height (pixel units)

Peak Height (mV)

Concentration (ng/ml) Flourescent Latex Gold

• Sensitivity greater than 0.2 ng/mL.

• Using same antibody / antigen combination, the assay

developed using a Dark Red fluorescent particle is >10 fold

more sensitive than the assay developed using gold.

Anthrax Protective Antigen Assay

• Assay for a recombinant

Anthrax protein

• Quantitative &

qualitative results


234567891011

Signal / Noise Ratio

Gold vs. Latex Comparison - Lateral FlowAnthrax Protective Antigen Assay

Gold

Fluorescent

Anthrax Protective Antigen Results


012

1 10 100 1000 10000

Ng/mL rPA

• Fluorescent assay sensitive to

2 ng/mL. Approximately 60-

fold better sensitivity than

comparable gold assay

• Visual or reader based

results

3. Device DesignThe importance of device design in test performance

• Cassette design plays an integral role in the

performance of the assay

• Early integration of cassettes with sample delivery

elements and with strips is a critical element of

the design process


the design process

• If a reader is to be used, design device with this

in mind – off the shelf cassettes will rarely work

• Novelty in approach can lead to major benefits

e.g. separation of sample handling and conjugate mixing from the

remainder of the device can significantly improve CV’s

The SymbioSysTm Program

• Rapid diagnostics have historically had less emphasis on engineering than other industries. (ie. Medical Device)

• Typical approach: Develop the strip, then find or try to design a cassette that works with it

– highly inefficient and counter-productive.

• With current demands for ease of use, precision, quantification,


• With current demands for ease of use, precision, quantification, low cost of goods and reduced time to market, this method is no longer acceptable.

• Concurrent engineering, assay development and user needs development is required to achieve this level of performance.

• DCN and Symbient developed the SymbioSys program to address this demand.

DCN’s Design Partner: Symbient Product Development

• Established in 2004.• Focus on development of single use diagnostic devices.• Product Types:

– Lateral Flow Cartridges– Fluidic Cartridges with Integrated Functions– Sample Delivery Systems - pipettes, lancet devices,

urine cups.


urine cups.– Instrument Driven Systems

Services Provided

• Mechanical Product Design• Industrial Design• 3D Solid Modeling (Solidworks)• Program Management• Precision Prototype Machining• Rapid Prototype Tooling and Molding• LIM Silicone Molding• Product Testing and Test Method Development.

Complete, creative solutions for developers and manufacturers of rapid assaysCONFIDENTIAL

• Product Testing and Test Method Development.• Failure Modes and Effects Analysis (FMEA)• Process Development • Manufacturing Transition

Sample Handling Device and Cartridge Design

• DCN and Symbient have

• DCN and Symbient have developed various diagnostic cartridges including single test strip, multiple test strip and specialty housings.


• DCN and Symbient have designed and developed several devices for collecting and processing samples for diagnostic testing.

• Sample types include saliva, whole blood, fecal matter, urine, mucous and surfaces.

• Methods include integrated buffers, mixing, filtering and metering.

Device

Optical Reader

Example: Sample handling in a quantitative agricultural test

• Vertical flow assay

• Rapid and quantifiable

• Increased sensitivity with

integrated pre-incubation

• Particulate samples do not cause


• Particulate samples do not cause

flow problems

1. PRE-INCUBATION

2. FLOW THROUGH

3. RESULT

Alpha Amylase Detection in Wheat

Hagberg Falling Numbers are determined by dropping a ball through a tube of ground wheat. The time it takes in seconds for it to reach the bottom correlates inversely to the amount of alpha-amylase in the wheat. High falling numbers indicate low concentrations of alpha-amylase, and low falling numbers indicate a high concentration of alpha-amylase.

Falling Number


050100150200250300350400

srw170 srw138 srw139 cbh2.7 cbh2.8

Falling Number

Wheat Sample

Comparison of Values from Device and True Falling Number Values

True FN

DiagnostIQ Avg FN

DiagnostIQ Avg [Amylase]

Falling Number

True FN

avg amyl conc.

St Dev %CV

304 1.54 6.4 1.77%

244 21.82 3.56 1.27%

223 36.0216.84 6.77%

142 144.05 6.57 4.77%

Intellectual Property – Driven InnovationRapiplex: A Novel Multiplex Rapid Assay Platform

• RapiPlex: An integrated Point-of-Care in vitro diagnostic platform;

• Unique intellectual property position

• Multiplex, Quantitative and/or Qualitative, Versatile, Portable

• Three integrating technologies to provide a robust platform

• Fluidics


• Fluidics

• Immunodiagnostic assay systems

• Integrated fluorescent reade

+

Fluidic Chip

• Proprietary fluidic strip & chip not constrained by lateral flow IP

• Disposable oriented in the vertical position when an assay is run

• No overlapping capillary materials – gaps between materials

• Enables simultaneous multiplex analysis from a single sample

• Defined individual channels in which sample flow can be controlled

• Individual channel entry ports for each channel


• Flow control zone ensures accurate and controlled flow in each

channel

• Injection molded – High Impact Polystyrene

• Suitable for all assay formats and multiple biological fluid sample

types – demonstrated on saliva, serum and whole blood to date

400,000

500,000

600,000

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800,000

Avg

. Pea

k H

eigh

t (R

FU)

Biosensia Myoglobin

Myoglobin assayRepresentative DCN Data


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300,000

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Avg

. Pea

k H

eigh

t (R

FU)

Myoglobin Conc. (ng/ml)

Error Bars – 95% Confidence

Comparison of assay results before and after CVs have been reduced and the dynamic range increased

150,000

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300,000

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Biosensia Myoglobin – Low End

Myoglobin assayRepresentative DCN Data


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k H

eigh

t (R

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Error Bars – 95% Confidence

Comparison of results at lower concentrations

Myoglobin Conc. (ng/ml) 95% Confidence Limits

5 16,661 10,504

10 37,698 22,495

25 82,432 41,144

Myoglobin AssayCurrent DCN Sensitivity Data


25 82,432 41,144

50 145,149 106,220

100 295,043 183,477

350 764,314 483,319

Sensitivity after DCN/SPD improvements. Sensitivity has been lowered to 5 ng/ml from 1000 ng/ml through a combination of improved reproducibility and increased dynamic range. Efforts continue to reduce CVs in the 5-10% range

Myoglobin Dilution Series (N=12)

R² = 0.9985R² = 1

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600,000

700,000

800,000

900,000

Avg. Peak Height (RFU)

Biosensia Myoglobin

Before

After


0

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300,000

400,000

10 100 1000 10000

Avg. Peak Height (RFU)

log Myoglobin Conc. (ng/ml)

After

Poly. (Before)

Poly. (After)

Summary

• Solved major mechanical design issues• Assay CV’s controlled• Good Slope of Dose Response• Clinically relevant sensitivity (~5 ng/ml)


Manufacturing Process Design

Membrane Conjugate Pad

Dispense test and control lines

Block membrane if necessary

Pre-treat pad

Dispense / dip conjugate

Sample Pad

Pre-treat pad

Dry

Dry Dry

DryDry


Assemble membrane, conjugate pad, sample pad, wick and backing into cards

Cut cards into strips

Assemble strips into cassettes

Package cassettes

Process Control Batch Vs Continuous Processes

XYZ 3050 dispenser with BioJets and AirJets

Dispensing Dipping Drying


BioJets and AirJets

Clamshell benchtop laminator

Manual Dip Tray

CM 4000 Guillotine Cutter

Lamination Cutting

Forced Air Oven

Equipment and ProcessesBatch Vs Continuous

Dispensing, Dipping, Drying


Reel-to-reel web handling and dispensing platform

Lamination, Cutting

In-line processing provides for greater process control and equivalent processing

In line camera and bad part marking system on a BioDotRTR 4500 inline dispensing system

Equipment and ProcessesBatch Vs Continuous

Complete, creative solutions for developers and manufacturers of rapid assays10

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1 64 127

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505

568

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946

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Pixels

Mean Pixel Width Std Dev %CV17.57 0.89 5.1

Output from a quality control camera system on a RTR 4500. Dispensing run over a 50m dispense length, 50mm/s, human IgG at 1mg/ml, 0.8ul/cm on nitrocellulose

All the elements of an integrated device are available over the counter

• High quality test development• Reader development• Device development• Manufacturing equipment


• A partnering approach is generally required to bring the specialized expertise and components into the assay

DCN’s Network Partners

DCN’s Network of Affiliates is a group of closely knit companies

that work together to provide all of the key elements necessary to

design, develop manufacture and commercialize point of care

diagnostic systems. Member companies include:

• Manufacturing equipment suppliers


• Cassette and device designers

• High volume molding companies

• Materials converters

• Reader developers and manufacturers

• Contract manufacturers

• Platform technology suppliers

A reader based semi quantitative serological assay: Rapid Assay for anti-PF4:Heparin in human plasma (HIT)

• Fluorescent, reader based, integrated RFID tag and novel cassette• ESEQuant fluorescent reader from Qiagen Lake Constance• Threshold assay, with Positive/Negative reporting• Predicate ELISA in market. Rapid assay must be as sensitive.• No known standards• Intended for the professional clinical laboratory market• Will be labeled as an IVD :developed under full design controls


• Will be labeled as an IVD :developed under full design controls• Assay and device developed for large scale manufacturing

Major Challenges

• Highly charged specific molecule• Total immunoglobulin assay imparts formatting requirement

– sample and conjugate cannot mix prior to interaction with test line

• High sensitivity needed• Lack of standards, so establishment of cutoff requires

comparison to performance of predicate ELISA =


comparison to performance of predicate ELISA = Requirement for high correlation to ELISA result.

• Requirement for tight CVs• Integration of novel device design elements for IP generation• Predicate rapid assays in the field require multiple

additions, timing steps and have poor user interface. The aim of the program is to generate a device without these attributes

Customer wanted to convert ELISA to detect Human Immunoglobulins into a fluorescent based lateral flow. Objective was to produce a lateral flow assay that was equal or better than their ELISA. (CAN WE MENTION GTI)? After several months of development feasibility testing was conducted with 84 characterized serums. Negative samples may actually contain immunoglobulins but are below the detectable limit of the ELISA.

Comparative results of lateral flow and ELISA data on all clinical samples

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Test

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ak H

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OD vs Test Line Peak Height(All Data)

OD vs TL


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Test

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ELISA OD

OD vs TL

• A panel of 83 samples, characterized by predicate ELISA• All samples came from patients who had received heparin• Testing in triplicate / quadruplicate if adequate volume• Average values shown.

Scatter plot of Test Line Peak Height against ELISA OD of all data

Cutoff: Comparative

0.400

0.500

0.600

0.700

TL/CL

OD vs TL/CL (OD <1.0)

OD vs TL/CL


0.000

0.100

0.200

0.300

0 0.2 0.4 0.6 0.8 1

TL/CL

ELISA OD

OD vs TL/CL

0.3 od Cutoff

OD = 0.3ELISA TL/CL TL

True positives 41 39 37

False Negative 0 2 4

True Negatives 42 42 42

False Positive 0 0 0

n 83 83 83

Collated Testing Results: ELISA vs LFIA using various cutoffs


True Positive = Samples with OD > cutoff and detected as positive

False negative= Samples with OD > cutoff and detected as negative

True Negative= Samples with OD < cutoff and detected as negative

False Positive= Samples with OD < cutoff and detected as positive

n 83 83 83

PPV 100.00 100.00 100.00

NPV 100.00 95.45 91.30

Sensitivity100.00 95.12 90.24

Specificity100.00 100.00 100.00

Dilution Series: High positive Diluted in Negative

R² = 0.9975

0.0

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1.0

1.5

2.0

2.5

3.0

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TL/CL Ratio

TL/CL Ratio Relative to ELISA OD, n=5

TL/CL ratio

Poly. (TL/CL ratio)


R² = 0.9989

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0.00 0.50 1.00 1.50 2.00 2.50 3.00

Peak Height (rfu)

ELISA OD

Peak Test Line Height Relative to ELISA OD, n=5

5-14 serum TL Ht

Poly. (5-14 serum TL Ht)

0.0

0.00 0.50 1.00 1.50 2.00 2.50 3.00

ELISA OD

Integrated System DevelopmentUsing the DCN Network to develop complete assay systems

• Project Management• System requirement definition• Product specification

• Cassette design• Rapid prototyping•Mold design and development

• Reader development• GUI design

• Reagent Development• Assay design and development

• Regulatory consulting• Quality system management

Program Setup and Management

System Development

The client provides the innovation in reagents and application areaDCN provides the rest


•Mold design and development•Mechanical engineering design

• GUI design • Assay design and development• Calibration system development• Documentation•Manufacturing process design

• Quality system management• FMEA / Risk analysis

• High volume plastic molding •Manufacturing equipment supply

•Manufacturing facility setup• Scale-up and manufacturing transfer• Reagent manufacture• Gap manufacturing

Commercialization•Market research• Board level advice• Networking to finance

• Branding• Troubleshooting• Patent Landscaping

Manufacturing Transfer

Key Points

1. Opportunities are available in numerous market segments for rapid assays with the right level of performance

2. Assays can be developed to handle almost any reasonable market condition, based on existing technology– Simple, low cost lateral flow assays for simple, qualitative

low resolution applications– More complex, more expensive lateral flow systems for


– More complex, more expensive lateral flow systems for high sensitivity, more quantitative, difficult applications

– These two types of assay are very different in how they are developed and manufactured

– Other approaches to high performance rapid testing are possible, but with a much longer outlook.

3. All of the elements needed to develop and manufacture high performance tests are available today

Acknowledgements

• DCN– Roy Chung– Jerrold Loo– Wendy Mendiola– Michael Fiechtner

• Symbient Product Development– John Zeis


– John Zeis– Scott Castenon

• Shara Rosen for marketing information• Thanks to GTI and Biosensia for use of data• Thanks to Cynthia Rocha, Camila Resende Duarte,

Gislaine Silva, Regina Fernandes from SECTES and Christiane Contigli from CETEC and all who helped to coordinate my attendance at INOVATEC

www.dcndx.com

Contact :Brendan O’Farrell at

[email protected]

Diagnostic Consulting Network Inc6354 Corte del Abeto, Suite B-C

Contact InformationPlease email to receive copies of the slides


6354 Corte del Abeto, Suite B-CCarlsbad, CA 92011Tel: 760-804-3886Fax: 760-804-3866

• Quickly develop and commercialize rapid assays for any application

• Experienced with fluorescent, visual (colloidal gold / latex) and

paramagnetic labels

– From concept to commercialized assay in under 12 months

We can develop assays for you:Assay Development Services


– From concept to commercialized assay in under 12 months

– ELISA conversion to rapid assay formats in short timeframes

– Reagent sourcing and development

– Rapid prototyping

– Feasibility studies

– Complete development

– Manufacturing transfer

• The plastic devices used in these assays can determine their ability to

perform in demanding applications and need to be custom developed

in virtually any high performance application

• DCN partners with product designers, such as Symbient Product

Development and molding companies such as HK Plastics to integrate

We can design and develop your plastic devices:Cartridge and Device Design Service


new cassettes and novel device designs into programs for clients

• Partners can be contracted through DCN, and DCN will manage the

entire development program on behalf of the client

• DCN partners with the major suppliers of readers to the point of care

marketplace to provide reader solutions for our customers.

• We can provide readers for:

– Visual labels

– Fluorescent labels

– Paramagnetic labels

We can help you develop reader systems for your assaysReader Development and Integration


– Paramagnetic labels

• Our primary partners for off the shelf and custom reader supply are QLC

(formerly ESE) and LRE (Esterline), and we distribute for both companies in the

US.

• We also work with custom reader design and development houses including

Source Scientific and Polygenesis.

• DCN can develop and

commercialize all aspects of

a point of care diagnostics

platform, through our in-

house capabilities and our

Network of Affiliates

We can design and develop your complete assay systemIntegrated System Design and Development

Cartridge top housing

RF id tag

Cartridge bottom housingStrip and Assay


• Custom design and

integration for high

performance assays our

specialty

Desiccant

We can connect you to all of the best suppliers:The Network of Affiliates

DCN’s Network of Affiliates is a group of closely knit companies

that work together to provide all of the key elements necessary to

design, develop manufacture and commercialize point of care

diagnostic systems. Member companies include:

• Manufacturing equipment suppliers


• Cassette and device designers

• High volume molding companies

• Materials converters

• Reader developers and manufacturers

• Contract manufacturers

• Platform technology suppliers

• Learn how to develop and manufacture lateral flow assays

• Customized one-on-one courses held at DCN’s core facility in

Carlsbad, California

• Scheduled on demand

• Duration 3 – 5 days

We can teach you to develop and make your own assaysEducation and Training Services


• Theoretical and practical elements dealt with

• Bring your reagents and optimize an assay during the course

We can help with any aspect of commercialization Consulting Services

DCN has over 20 consultants on staff that can provide a wide range of

services related to the point of need diagnostics or detection business:

• Regulatory Affairs

• Project management

• Risk assessment

• Intellectual property landscaping


• Intellectual property landscaping

• Manufacturing process development

• Assay troubleshooting

• Quality system establishment for diagnostic companies

• Device design

• Facility setup

• Equipment specification and procurement

• Market evaluation

• Technology evaluation

• DCN’s facility is completely equipped for manufacture of lateral flow, flow through, and

other format point of need assays

• The facility:

– 8000 square feet, containing 3000 square feet of wet lab, housed in a class 10,000

capable cleanroom, and an associated controlled humidity room

– Currently ISO 9001:2008 compliant and will complete EN 13485 by end Q4 2010

• Manufacture non-FDA regulated product for several clients in the US and Europe, and

We can manufacture your tests for youContract Manufacturing


components and reagents for clients in the US and outside

• DCN partners with several dedicated contract manufacturers to whom

it can transfer product for manufacturing.

Reagent Prep Area

Main Lab (Class 10,000 capable cleanroom)

Component Processing Area (Temperature / Humidity Controlled)

• DCN provides everything needed to develop or manufacture lateral flow or

flow through assays

– Colloidal gold, colored and fluorescent latex beads and conjugates

– Assay materials: membranes, pads, backing, cassettes wicks, etc

– Antibodies and antigens

– Small molecule conjugates

We can supply you with key componentsMaterials, Reagents and Kits


– Small molecule conjugates

– Test strip readers: Handheld and benchtop

• Kits of materials and reagents

– Colloidal gold

– Material Starter Kit

– Colloidal gold conjugation kit

– Latex conjugation kit

– Gold conjugation kit

We can make the critical reagents for youCustom Conjugate Development and Manufacture

• Expertise in covalent conjugations

– Protein / peptide /small molecule –Particle

• Colored, fluorescent or paramagnetic latex, gold, quantum dots,

etc.

– Protein – ProteinStreptavidin Bead #2

2.5Strep #2 (1X)


• Full optimization process

• Full QC including binding

assays where reagents are

available

• For diagnostics or separations (magnetic latex)

• Small to full prep scale

-0.5

0

0.5

1

1.5

2

0 5 10 15 20 25

ng Biotin

OD

Strep #2 (1X)

Strep #2 (10X)

Strep #2 (20X)

Merck Strep REC/1.0umCandidat 2

Merck Strep C-REC/ 1.0umCandidat 4

Merck Strep T REC/1.0umCandidat 4

Merck Strep 0.2um Candidat 1

• Dr Roy Chung: Over 25 years of assay development and manufacturing

experience. Previously Lead Scientist at Nanogen

• Dr Hans Boehringer: Over 20 years of assay development and manufacturing

experience. Most recently Scientific Director at Genzyme Diagnostics

• Dr Brendan O’Farrell: 20 years of assay development, manufacturing,

equipment and manufacturing process development. Previously VP of

Technology Development at BioDot Inc and Director of R&D and Manufacturing

at SDS Inc.

Our People


at SDS Inc.

• Michael Surmanian: 20 years of biotech technology development experience.

Formerly President of BioDot Inc.

• Assay Development Team of 10 scientists, all of whom worked for major

human and veterinary diagnostic companies. Minimum of 5 years of experience

Consulting Group.: Over 25 industry experts available. Examples:

• Dr Judith Howard: Ex VP of Quality, Regulatory at Inverness Medical

Innovations

• Dr David Boudreau: Ex Director of Operations, Nanogen Inc

• Michael Weinholt: President of Regulatory Consulting Inc

Our Strengths

• Expertise in reagent development, assay development, device design

and reader integration

• Experience in all aspects of system development, regulation,

commercialization and manufacturing

• Modular menu of services, allowing for flexible project execution –


• Modular menu of services, allowing for flexible project execution –

select only the services you need

• Parallel development strategies for device and assay development –

science and engineering combined

• Networked to all of the partners you will need to get to market

• Full design controls and documentation.

• Full support for registration activities

• Speed to commercialization

1) Assay

• Materials for fluid movement and reaction matrices

• Biological recognition reagents (antibodies/antigens)

• Signal generation reagents (labels)

2) Reader system

Challenges in Designing and Manufacturing High Performance POC Assays


• Technology

• Supplier

3) Device

• Sample collection and processing

• Conjugate handling and mixing

• Cassettes

4) Manufacturing process design

Market Penetration by Application

25

30

35

40

45

% of Available Market, 2007(compared to lab based immunoassays that have a rapid segment)


0

5

10

15

20

Medical Diagnostics

Veterinary Food and Beverage

Pharmceuticals Envionmental Water

60

70

80

90

Percentage of Available Market by Geographical Region

Market Penetration by Geographical Region


0

10

20

30

40

50

US

EU

ROW

• Sensitivity:

• Fluorescence based tests can be more sensitive than colorimetric

• Higher sensitivity allows new applications and markets

• Control

• Fluorescent assays are based on latex particles which can be

Why we like fluorescent labels for reader based applications


coupled covalently. Colloidal gold conjugation is passive.

• This allows for better control of the conjugates and improved CV‘s

• Availability

• High quality, high performance beads available

• Multiple sizes, chemistries

• Availability of off-the shelf readers

• Makes development easy and low cost

• Makes fluorescence a comparatively open system

Device Design Trends and Implications

• IP considerations have driven a trend towards novel device design:

– clear pathways to designing non-infringing devices exist

• For many applications, sample handling, sample preparation,

extraction and delivery to devices need to be performed


– E.g. Tests for agriculture, biowarfare, food pathogen testing,

environmental testing involve handling difficult samples, treating

them and delivering them to devices in difficult field conditions.

– Devices can be designed and integrated to cassettes to perform

these functions


nAvg Peak

HeightCV

0 15 0 na

5 15 13,583 11.3%

10 15 30,097 12.6%

Myoglobin assay dataRepresentative DCN Data


10 15 30,097 12.6%

25 15 61,788 16.7%

50 15 125,685 7.7%

100 15 239,260 11.7%

350 15 623,817 11.3%

DCN in conjunction with Symbient implemented multiple changes to the device and reagents. CVs in the clinically relevant range have been reduced by 50%

Prototyping Capabilities

CNC Machining Steriolithography (SLA)


CNC Machining

Prototype Mold Fabrication Injection Molding

Steriolithography (SLA)

Assembly and Testing


Myo Conc. (ng/ml) N Avg. TL pk ht TL %CV

25 12 64,948 11.0%

50 12 118,659 5.9%

Myoglobin Dilution Series (N=12)


100 12 229,002 10.4%

250 12 457,563 4.1%

500 12 749,168 6.9%

Overall Conclusion

• Doing so requires careful design of all aspects of the product and the integration of high quality fluorescent label, cassette and reader technologies

• The design and development of products of this nature is a highly specialized task

• That is where a company like DCN, with specialist knowledge and experience, comes into the picture

• How can we help you?........


• How can we help you?........

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Documents

closely knit companies

large animal testing

care diagnostic systems

myoglobin dilution series

member companies include

sample handling

technology development

assay development