3636 Boulevard of the Allies Pittsburgh, PA 15213

Applying Protein Plastics in Chemical Defense

October 2003

Keith LeJeune, PhD CEO and Co-founder 412-209-7298 [email protected]

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Background

• Founded Dec 1998 – Keith LeJeune, CEO, Agentase LLC – Alan Russell, Director of McGowan Institute for Regenerative Medicine, University of Pittsburgh and Pittsburgh Tissue Engineering Initiative • Proprietary technology permitting the direct chemical incorporation of enzymes within polymers during polymer synthesis • Advantages over conventional techniques for enzyme immobilization – No leaching of activity from support material – Desensitization of enzyme to environment – Durability and reusability – Substantial loading capacity for protein – High degree of activity retention – Opportunity to incorporate indicating compounds within polymers (sensor) 2

1 Technology Applications

• Chemically active surfaces • Decontamination of hazardous chemicals – Pesticides – Chemical weapons (DFPase, OPAA, OpdA, OPH) • Sensors – Co-polymerize enzymes and indicating compounds – Color change illustrates enzyme activity – Used to detect • Substrates (Reactants) • Inhibitors

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Decontamination

• Optimization of enzyme-polyurethane for agent detoxification – Improve physical properties for removing agent from surfaces • Moisten polymers in tap water • Achieve > 99.5% removal of 40mg GD on a 80 cm2 surface in 3rd party experiments – Steel plates – Plastic surfaces – Ceramic tile – Improve catalytic activity of enzyme polymers

60000

g) 50000 m 40000 30000 GD 20000 Pinacolyl methylphosphonate 10000 in solution ( Mass of analyte 0 0 50 100 150 200 250 300 Time (min) 55 mg GD in 100 ml buffer with ~1.0g polymer containing less than 3mg of enzyme

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2 Issues limiting practical utility of employing polymers for CW decontamination

• Enzyme specificity • Need for buffer capacity – Hydrolysis of moderate concentrations of agent reduces pH – Initial pH outside acceptable range for enzyme catalysis – Not practical to be required to “buffer” a surface or solution prior to decontamination • Incorporation of buffer capacity within polymers during synthesis 100% 75% 50% Enzyme polymer 25% Buffered enzyme polymer Detoxification 0% 0 1 2 3 4 5 6 Initial methyl parathion Concentration (mM)

– Buffered polymers ~1g moistened in tap water decontaminate (>99.5%) 40mg GD in < 2 hrs. 5

First Product: Nerve agent Sensor

Clean Contamination Agentase’s sensor provides a clear, intuitive, and easy to read response for nerve agent contamination 6

3 How does the TL Sensor work?

• Two polyurethane-based fabric layers – Enzyme Polymer Layer – Substrate Polymer Layer

2.5 • Dynamic pH equilibrium pH equilibrium – Cholinesterase 2

• Produces butyric acid 1.5 • pH optimum at ~8.0 1 – Urease Urease + - ChE • Produces NH4 and OH 0.5

• pH optimum below 7 Enzyme activity [relative] 0 5 6 7 8 9 10

pH

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Benefits of the described technology

• Versatility in sampling – Surfaces – Water – Air • Response – Positive response in seconds – Response intensity proportional to contamination level • Surface compatibility – Steel Plates, flat and grooved – Plastic – Aluminum – Finished Wood – Ceramic floor tile – Glass – Leaves – Skin Tissue – Soil • Great resistance to interference because of dynamic equilibrium approach

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4 Performance of Nerve Agent sensor

• Storage stability and shelf-life – > 31 days at 140oF Correlates to more than 18 months at – > 6 months at 105oF room temperature • Temperature effects – Sensor operational at subzero and high temperatures (tested at 158oF) • High resistance to chemical interference – Environment tested include: • Diesel, auto & aviation fuel, antifreeze, ethanol, vinegar, toluene, fire-fighting foam, sea water and several pesticides & household cleaners • Tests conducted in 1% or greater concentrations in solution and and 1% interferent saturated air • Training version available – Classroom and operational exercises 9

Live agent validation Color Color Agent Total Mass Mass / cm2 Green (Y/N) Result (2 min) (25 min) GD 100 ug 1.25 mg/cm2 Red Red N 20 ug 0.25 mg/cm2 Red Red N 8.0 ug 0.10 mg/cm2 Red Red N 4.0 ug 0.05 mg/cm2 Some Red Red N 1.0 ug 0.0125 mg/cm2 Some Red Red N Limit 0.2 ug 0.0025 mg/cm2 Yellow Yellow N

VX 100 ug 1.25 mg/cm2 Red Red N 20 ug 0.25 mg/cm2 Red Red N 8.0 ug 0.10 mg/cm2 Some Red Red N 4.0 ug 0.05 mg/cm2 Slight Red Red N Limit 1.0 ug 0.0125 mg/cm2 Yellow Yellow N 0.2 ug 0.0025 mg/cm2 Yellow Yellow N

Water Blank Plate a 0.00 mg/cm2 Yellow Lime green Y Blank Plate b 0.00 mg/cm2 Yellow Lime green Y Blank Activated Only 0.00 mg/cm2 Yellow Lime green Y Summary of TL Sensor data from ECBC Report 0033-080702: 7 August 2002

• Surface detection limits verified in UK • GA - between 99 and 22 ng/cm2 • GB - between 99 and 22 ng/cm2 • GD - 22 ng/cm2 • VX - between 180 and 99 ng/cm2 10

5 Practical limitations

• Temperature sensitivity – High temperature stability issue • Shelf life is function of temperature • Co-package with Temp indicator – Low temperature operation • Water in reservoir cap must be liquid • Thawed sensor can be used on frozen surface

• Incompatible environments – Excessively dirty surfaces • Block ability to see polymer color – Extremely acidic or basic environments • Immediate color change (purple/pink)

• Nerve agent point detection Only

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Additional applications of sensor technology • Expansion of detection capabilities to additional CW and TIC – Polymerization chemistry compatible with most enzymes – Many enzymes identified with sensitivity to target chemical hazards Product Concept • Pencil box for detection of CW agents and high-risk TICs • Real-time continuous monitoring – Equilibrium reaction scheme used to trigger response – Covalent attachment of sensing components within polymer Product Concepts • Water Monitor • Air monitor • Wearable badge • Combination of parallel efforts • Array of wearable badges for operationally identifying chemical hazards • Public venue air monitor for chemical hazards 12

6 Additional applications of sensor technology

Water monitor for nerve agents Real-time continuous water monitor for all CW agents

Blister Indicator lights Blood Nerve Clean Inlet Effluent collector

Renewable substrate cartridge

Single-use sensors for all CW agents

TL Sensor for nerve agent CW

12 h

6 h

Wearable badge for 0 h Public venue gas nerve gases monitor for CW agents 13

Point detection capabilities - Blood Agent sensor

• Prototype cyanide sensor – Identical in form and use to nerve agent sensor – Slightly improved shelf-life – Detection limits well below IDLH (mg) Clean surface Cyanide surface • Excellent operational utility for example ... – Car – Brick – Wood – Steel – Concrete – Fire fighting foam

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7 Performance

• Sensitivity – OSHA and IDLH levels (cyanogen bromide)

5mg/m3 (OSHA)

Control 25mg/m3 (IDLH)

• Live agent tests scheduled at 3rd party facility – Hydrogen cyanide – Cyanogen chloride

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Demonstration of Blood Agent sensor

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8 Continuing expansion of agent detection capabilities

• Identified additional CW agents / TIC compatible with enzyme-based detection • Blister • Acetaldehyde • Hydrazine • Acrolein • Ammonia • Formaldehyde

• Development of Prototype kit for detection of CW / TIC

A B C D E F

– Sensors for Nerve agents (B) and Blood agents (E) are fully functional – Present activity • Devising kit and sensor construct to best fit end-user needs • Adding additional hazardous agent detection capability 17

Continuous monitoring capabilities - Water monitor

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9 Response to ChE inhibitor

Signal Response - Effluent pH 7

6 pH 5 Addition of DFP to aqueous inlet 4 0 1 2 3 Time (hours)

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Envisioned prototype - (Under construction)

Alarm and communication device

Substrate Power supply Cartridge and controller

Color Effluent reader Reservoir View cell with polymer

Water Micro-pumps pH monitors Inlet

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10 Proof-of-concept demonstration: Wearable badge

• Model badge capable of detecting DFP vapor at low ppb levels • Product concept – Single lightweight sensor (<50g) housing with multi-agent compatibility – Maintenance free operation (>12 hrs) – No external power requirement – Resistant to chemical interference – Integrated communications capability

Gas-tight syringe with valve to insert hazard Lid with Substrate Feed injection port

Polymer Sensor 1 Liter

container Waste Reservoir 0 0.5 2.5 5.0 10.0 min Polymer response - post DFP vapor injection

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Contributors

• Supporting Agencies – Army Research Office (ARO) - Lee – Defense Threat Reduction Agency (DTRA) - Pollack, Hoefler – USMC - Becker – Defense Advanced Research Projects Agency (DARPA) - Rudolph

• Live agent testing facilities – UK Defense Evaluation and Research Agency (now dstl) – U.S. Army Soldier and Biological Chemical Command; Edgewood Chemical Biological Forensic Analytical Center – Centre d'Etudes du Bouchet - Cazaux France

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11 Summary

• Enzyme polymerization has utility in decontamination and detection • Agentase nerve agent sensor is a proven product – Product validated by third parties • Live agent (V and G-series) • Operational exercises – Fielded product in both military and civilian environments • Encouraging proof-of-concept data on 2nd generation products – Continuous monitoring devices • Water • Air – Detection kit with expanded CW/TIC capabilities • Blood • Blister • TICs 23

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