HR001122S0047-Amendment-01.pdf

The purpose of Amendment 1 to HR001122S0047, the Ice control for Cold Environments (ICE) Broad Agency Announcement, is to clarify the proposal abstract deadline, molecule classes of interest, and other eligibility criteria. Changes bolded/highlighted/underlined below.

Part I Overview Information is updated as follows:

Proposal Abstract Due Date and Time: September 22, 2022, 4:00 PM ET

Section 1 Funding Opportunity Description is updated as follows:

Program Overview The Defense Advanced Research Projects Agency (DARPA) is soliciting innovative research proposals to investigate the physical properties of ice crystals for the development of new solutions to protect warfighters and infrastructure and enhance operational capabilities in extreme cold weather (ECW) environments. The Ice Control for Cold Environments (ICE) program aims to leverage biological adaptations to cold environments for novel biologically sourced and inspired materials to facilitate Department of Defense (DoD) operations in ECW. The ICE program structure is organized into two phases with a potential third phase. Phases I and II are the focus of this BAA, and Phase III may be solicited under a separate BAA.

The goal of the ICE program is to deliver novel biologically sourced or inspired molecules that can control or exploit ice crystallization. In Phases I and II, the program seeks to discover and/or improve known molecules with enhanced activity corresponding to three broad activity classes of ice control: inhibition of crystallization/re-crystallization, ice nucleation, and molecular adhesion to ice. In support of these objectives, ICE performers will design robust assays to identify and test candidates for ice control activity that should be specific to each of the three activity classes;

execute iterative design, build, and test cycles to improve activity; optimize candidate molecule performance; and explore methods and approaches to functionalize these molecules to enable incorporation into different materials and form factors for future prototyping efforts in a Phase III effort, which may be solicited under a separate BAA.

BACKGROUND INFORMATION

The warming of the arctic has opened access to new trade routes and necessitated an expanded operational area where the U.S. military must counter peer adversaries seeking to exploit emerging theaters in ECW areas. Significant physiological and material barriers exist to establishing and maintaining a force capable of sustained operations in ice-prone environments. Many of these challenges are a consequence of the physical properties of ice such as ice crystal formation, recrystallization, and propagation, as well as the impact these phenomena have on the surrounding operational environment and force readiness. Ice control capabilities could include, but are not limited to, the prevention of frostbite injuries, reduction of ice accretion on vehicles, vessels, and aircraft, decreased damage to infrastructure, maintaining aqueous solutions (potable water, medicines), solving transportation and logistics challenges (ice bridges, roads, runways), and enabling field operations.

The dynamic formation and dissolution of ice is a ubiquitous process with wide-ranging effects on both natural and built environments, including creating structural challenges to both living and non-living systems and materials due to the growth and expansion of ice crystals. To cope with these challenges, organisms that inhabit environments prone to ice formation have evolved unique biological adaptations to mitigate, and in some instances exploit, the physical properties of ice in order to survive and flourish in harsh conditions. The ICE program seeks to utilize diverse and ingenious biological solutions to operating in extreme environmental conditions.

Proposers may seek to leverage a series of technological advances across disparate fields that have produced a confluence of biotechnology capabilities enabling the identification, engineering, optimization, and scaling of new biologically sourced or inspired molecules displaying ice control properties, including:

ice-binding proteins (fish, insects, fungi, bacteria, and plants), capable of modulating the physical and kinetic properties of ice formation in a dynamic fashion at the molecular level;

pigments capable of absorption of defined wavelengths of light and radiative heat transfer to selectively melt snow versus ice (algae and bacteria);

cryoprotective polysaccharides (bacteria, algae, insects, and plants); and small molecule cryoprotectants and eutectic mixtures (animals, insects, and plants).

Although only a limited number of these compounds have been identified and experimentally validated to date, these molecules hail from a diverse set of organisms and have demonstrated unique functionalities by divergent mechanisms such as instigating ice formation at elevated temperatures, decreasing ice formation at lower temperatures (with no effect on the melting point), selectively adhering to ice at ice/water interfaces, enabling cryoprotection and anti-desiccation activities.

ICE performers will characterize candidates based on their method of ice control, broadly grouped under inhibition, induction, and adhesion, regardless of the type of molecule (e.g., proteins, small molecules, polysaccharides). Some of the challenges to be addressed for each activity class of ice control molecule include, but are not limited to:

1) Discovering and characterizing new ice control molecules.

2) Measuring key physical properties of ice crystal formation and maturation and corresponding modulation by exogenous agents in standardized, quantitative, high throughput, and reproducible assays.

3) Optimizing function of ice control molecules at varied temperature ranges, improving stability, and enabling identification, isolation, validation, and optimization of novel molecules.

4) Improving the dynamic functional range of molecules to expand suitability for diverse DoD applications.

The field lacks standardized, quantitative, and reproducible assays to measure key physical properties of ice crystal formation and maturation, as well as their corresponding modulation by exogenous agents. Current approaches are time consuming, require expert execution for reliable results, can be dependent on qualitative observation/scoring, are low throughput, and can be prone to either false positive or negative results depending on method and protocol.1,2

Standardized testing methodologies capable of robust, reproducible quantification of molecule performance related to ice induction, adhesion and inhibition activity classes respectively, would be advantageous to identifying and developing novel materials capable of inhibiting or accelerating ice crystal formation/propagation or binding to ice for DoD ECW applications.

TECHNICAL APPROACH AND STRUCTURE

The ability to modulate specific properties of ice such as the type, size, shape, texture, freezing point, melting point, kinetics, strength, and thickness would be advantageous for a wide variety of DoD applications. Studies by a diverse cadre of investigators focused on elucidating the biochemical, and physiological adaptations that microbes, plants, and animals display to survive extreme cold have identified a number of biological molecules (proteins, polysaccharides, and small molecules) that exhibit the ability to modulate or exploit the properties of ice. These activities include antifreeze, ice nucleation, ice recrystallization inhibition, ice structuring, and ice adhesion.

While some ice-modulating molecules and their associated properties have been previously characterized and reported in literature, significant foundational research and development efforts are required to screen for activity in a robust, standardized, reproducible manner and to optimize molecules for performance. To systematically address these capability gaps, ICE program performers must sequentially develop solutions to expand discovery and standardize performance screening of molecules capable of inhibiting ice crystallization/re-crystallization, nucleation, and molecular adhesion to ice.

ICE is anticipated to be a three-phase program. Phase I will be 18 months, and Phase II will be a 12-month option, for a total of 30 months of discovery, screening, and optimization. Phase III (not solicited under this BAA) may include application-specific testing to enable transition to DoD stakeholders.

In Phase I, performers design or discover new biologically sourced or inspired methods of controlling ice and develop new assays and test beds to monitor crystallization and performance to determine ice control properties. By the end of Phase I, successful performer teams should establish a high-throughput and quantitative testbed for screening performance of ice control candidates; identify at least 25 molecules in each activity class of ice crystallization: 1) inhibition

2) induction, and 3) adhesion; and optimize a subset of at least 10 molecules in each activity class to broaden the operational range of ice modulation.

Phase II will focus on screening the candidates based on performance for potential DoD-relevant applications, including the generation of safety data. Intermediate and end-of-phase milestones, outlined in Section 1.3, will be required in each phase to evaluate progress throughout the program.

Proposals should contain, subject to DARPA agreement, quantitative- and application-relevant metrics to assess technical performance toward milestones (see Section 1.3 for details). By the end of Phase II, performers will be asked to demonstrate ≥75% improvement of ice modulation based on application (ice inhibition, induction, or adhesion), and demonstrate approaches and methods to incorporate and/or formulate Phase II molecules into an appropriate material and form factor for testing in a Phase III. To assist with the development of a framework compatible for a potential Phase III (not solicited under this BAA), DARPA will highly recommend that proposers integrate at least one of the DoD ECW application areas outlined in Section 1.2.3 into their proposed research and development efforts. Proposals that could enable multiple future applications and materials formulations are highly encouraged.

Phase III may include application-specific testing to enable transition to DoD stakeholders.

DARPA anticipates potentially releasing the Phase III solicitation during the Phase II period of performance. The timeline for Phase III work is tentatively scheduled to be 12-18 months in duration, dependent upon application and associated testing requirements. Candidates for testing in Phase III are not limited to those discovered and screened in Phases I and II. However, data from these phases may inform the application(s), scope, testing parameters, and metrics for a Phase

III BAA.

Beginning in Phase I and throughout all screening and optimization, DARPA anticipates consistency in the evaluation of all candidate molecules through collaboration and concurrent testing with a U.S. Government Independent Verification and Validation (IV&V) partner.

DARPA’s IV&V partner will complement performer testing and independently evaluate candidate molecules to inform DARPA program decisions. All performers are expected to work with and provide samples to the IV&V partner on schedule in the format required.

1.2.1 Phase I: Quantitative Test Bed Infrastructure, Candidate Discovery, and Characterization (18 months)

Phase I comprises two parallel research and development tracks in the first 12 months: 1) quantitative testbed infrastructure and 2) candidate discovery and library generation. Proposers must address both tracks. These tracks will converge at month 12 wherein libraries of candidate molecules will be screened for ice crystal modulation capabilities. By the end of Phase I, the best performing candidates will be down selected in consultation with DARPA and subjected to iterative rounds of focused discovery, design, and performance improvement. At least 10 molecules per activity class (inhibition, induction, and adhesion) will be down selected by each performer team in consultation with DARPA for transition to Phase II.

Track 1 Quantitative Testbed Infrastructure (0-12 months) A key challenge to discovering, developing, and optimizing new molecules and materials with the ability to modulate ice crystal size, shape, and growth kinetics are the bespoke, variable, semi-quantitative assays currently employed to measure key physical properties of ice crystal formation and maturation, as well as their corresponding modulation by exogenous agents. ICE encourages the development of robust standardized, quantitative, and reproducible assays that can be multiplexed or used in high throughput approaches to enable the rapid identification and prototyping of molecules and materials capable of inhibiting or accelerating ice crystal formation/propagation or binding. Proposers are encouraged to design assays that screen for multiple activity classes of ice modulation, increasing the generalizability of the assay. Assay performance across performer teams will be validated at month 9 using blinded samples provided by IV&V partners.

Proposed assays must be capable of meeting the following specifications:

Molecular diversity - performers are required to demonstrate that their assay is functional, reproducible, and equivalent across at least 1) proteins/peptides, 2) polysaccharides, and

3) small molecules.

Temperature range - assays need to be operational and produce equivalent results over a temperature range of +5°C to -30°C.

Dynamic range - assays must be capable of screening candidate molecules over at least two orders of magnitude in concentration.

Robustness - assays must be capable of performing at least 3 replicates per sample in parallel, achieving results that vary by no more than 5% between replicates, and must include appropriate positive and negative controls.

Reproducibility - assay platforms must utilize quantitative data collection NOT dependent on subjective interpretations by a human (i.e., microscopy-based approaches dependent on human scoring of activity and performance).

Throughput - final assays must be capable of screening a large number of molecules in order to achieve the molecular diversity required to identify candidates with ice modulating properties. It is expected that assays demonstrate the capability to screen up to 500 samples (in triplicate) over a continuous 24-hour period (pooling of samples and multiplexed approaches are acceptable).

Quality Control – variation between assay runs regarding the accuracy and precision of the testbed must be quantified, mitigated, and minimized with a standard deviation of no less than 5%.

DARPA is agnostic to testing regimes or assay platforms. Potential approaches include but are not limited to:

Microscopy approaches that utilize automated image analysis or AI

Differential Scanning Calorimetry

Fourier Transformed Infrared spectroscopy

Raman Spectroscopy

Osmometry

Combinatorial approaches

In Phase I, IV&V partners will work with DARPA ICE program teams to develop a common framework for measurements and results within each laboratory and support the goals and objectives of the ICE program by measuring ice properties and adhesion across length scales on (multi-functional) technologies provided to IV&V partners by DARPA and ICE performers.

IV&V partners will facilitate selection and distribution of a panel of unknown materials for all performers to test under identical conditions for each team’s proposed assay. Each team will receive materials for testing within 6 months (up to 5 molecules x 5 replicates). All performers must complete studies to return data for comparison and compilation by the end of month nine.

The IV&V partner will aggregate and compare results between performers as well as from equivalent studies, returning reports to DARPA and performers. Possible metrics for testing include:

By the end of Phase I, IV&V partners will work with DARPA and ICE performers to develop the test matrix, including substrate materials, temperature(s), testing mode, and aspect ratio of substrates for small-scale ice adhesion tests. Target sizes include 70 x 150 mm (~3x6 inches).

As Phases I and II are focused on candidate discovery and performance screening, DARPA plans to implement ice adhesion studies on a small scale (cm scale) utilizing freshwater columnar ice.

Track 2 Candidate Discovery and Library Generation (0-12 months) While a wide variety of different biologically sourced molecules with ice modulating properties have been reported in the literature over the last several decades (i.e., proteins, polysaccharides, and small molecules), the relative number of examples are quite small. However, published data (genomic sequences, amino acid motifs, macromolecule crystal/NMR structures, polysaccharide composition, small molecule substituents/ratios, and physiological data), coupled with state-of-the-art high throughput biotechnology capabilities have created an opportunity space to discover, engineer, and optimize biologically sourced/inspired molecules with enhanced ice modulation properties. Track 2 aims to leverage existing data and next-generation technologies to develop at least two different libraries of diverse molecules to screen and identify candidates with ice modulating activity.

Potential approaches to library generation include but are not limited to:

Metagenomics approaches to identify new sequences with potential ice modulating activity

High throughput mass spectrometry

In vitro selection, directed evolution, phage display

Synthetic chemistry and/or biochemical approaches

Bioinformatics and/or computationally assisted protein discovery and design

Combinatorial approaches

Proposals must address the following information related to Track 2:

Rationale for approach and selection of molecular targets, including estimated formulations for potential DoD applications

Estimation and justification for library size

Justification and risk mitigation for library generation approach:

o For metagenomics-based approaches and sampling sites, estimated number of sequences, and projected throughput/capacity of sequencing required to generate sufficient data to mine o High throughput mass spectrometry should detail the estimated number of biological samples or mixtures to be screened and how sufficient chemical diversity can be achieved to enable candidate identification o In vitro selection, directed evolution, and phage display based techniques must justify selection approaches and describe how significant molecular diversity can be achieved to mitigate risk o Synthetic chemistry and biochemical approaches must describe the breadth and depth of chemical diversity achievable using the proposed approach(s) o Bioinformatics/computational methodologies must articulate/justify how the proposed approaches identify significant candidate diversity to screen for functionality and mitigate risk o Combinatorial approaches should detail the advantages over stand-alone methods

Biological or synthetic production approaches to produce molecules in quantities sufficient to screen and begin optimization within the 18-month Phase I timeframe

Plans for assay protocol coordination with other ICE teams (secure infrastructure and servers)

Plans for data sharing with IV&V partner and DARPA

Integration of Tracks (12-18 months) By the end of month 12, performers will have completed screening for up to 1000 candidate molecules and have received a report from the IV&V partner with feedback on testbed performance and specifications. DARPA anticipates integration of the two tracks to demonstrate the robustness of the quantitative test bed infrastructure and use this infrastructure to test and characterize the ice modulating candidates. The best performing candidates will be down selected in consultation with DARPA to obtain ≥10 molecules for each of the three ice modulation activity classes for transition to Phase II.

Proposers must provide the following details for each activity class of molecules:

Performance for each activity class

Initial demonstration of the performance of the test bed and plans to expand its capabilities

Approach to optimization of ice modulation capabilities of the top candidates

A coordination and data sharing plan with IV&V partner

Delivery of sufficient quantities of the material to enable multiple tests that can produce statistically significant data

1.2.2 Phase II: Accelerated Molecule Engineering for Performance and Formulation (12 months)

Molecules selected for transition into the Phase II pipeline will be subjected to additional rounds of library design, build, and test to further optimize function and performance to meet Phase II benchmark metrics. In parallel, performers must develop methods and approaches to functionalize molecules for potential incorporation into prototype materials for a potential Phase III solicitation.

DARPA highly recommends that proposals integrate/address at least one of the DoD ECW application areas outlined in section 1.2.3 in order to fully assess the impact and applicability of Phases I and II efforts into a potential prototyping work in the future. In addition, proposals must provide details and articulate methods/approaches to increase the scale of molecule production to sufficient quantities to support Phase II research and development efforts. Phase II will conclude with evaluation of materials performance and benchmarking by the IV&V partners, the results of which will inform potential transition to the Phase III testing focused on advanced development (not solicited under this BAA).

Proposals must address the following information related to Phase II:

Detailed rationale on approach and timelines for iterative molecule design, build, and test to improve activity and enhance performance on molecules transitioned to Phase II.

A comprehensive justification and rationale for how the selected activity class of molecules can be functionalized, modified, or incorporated into different formulations/materials such as gels, aqueous solutions, powders, textiles, resins, composites, and coatings, among others that are suitable for use in selected DoD ECW application areas.

Methods and approaches to determining stability, half-life, activity, toxicity, durability, and efficiency of performance and formulation/incorporation into different material form factors and alternative approaches to mitigate risk.

Details on approaches to scale-up and a preliminary technoeconomic analysis of costs associated with production of active molecules and incorporation into proposed materials.

A comprehensive testing plan to assess materials performance relative to selected formulation/ECW application.

Details on alternative approaches and risk mitigation strategies for molecule optimization, materials development, and scale up.

Phase II engages Design, Build, Test, and Optimize (DBTO) cycles to demonstrate continuous, improved performance of ice control candidates in three activity classes of molecules or materials with ice modulation ability: inhibition, induction, and adhesion.

Proposals must address the following:

Anticipated timeline for DBTO cycles and process

Capacity to produce molecules or materials for testing

Approach to scale up quantities of molecules and/or materials

Plans for data sharing with IV&V partner and DARPA (secure infrastructure and servers)

In Phase II, the IV&V partner will focus on small-scale testing of materials using freshwater columnar ice in application relevant testing regimes (i.e., coated substrates, liquid mixtures, skin and tissue surrogates, etc.). In addition, IV&V will perform small-scale ice testing with reference materials/substrates (to be determined) and will collaborate with performers to establish methods to fabricate appropriate substrates and/or mixtures for testing, evaluation, and independent verification. Performers are expected to supply at least 5 candidates per molecule activity class at quantities sufficient for multiple experiments/replicates dependent on testing regime. Approaches to assess the performance of candidate molecules may include scratch and adhesion tests, microscopy/spectroscopy-based analysis, shear force, freezing/melting points, glass transition temperatures, stability and durability analysis, and wettability measurements, among others, by month 28.

To address the different ice types encountered in real field applications, all studies will utilize freshwater columnar ice and, potentially, saline spray ice. Safety, environmental, and toxicity data should be gathered in anticipation of EPA, FDA, or other regulatory requirements. Large scale studies should be carried out on different geometry substrates: such as flat or curved panels or a cable. Animal studies should be included as needed depending on application (i.e., frostbite).

1.2.3 Phase III: Materials formulation, testing, and scale-up (12-18 months) Phase III is NOT solicited in this BAA. Phase III may optimize molecules and materials for testing of applications with transition partners.

Successful demonstration of improved performance and control of ice crystals will inform potential DoD relevant applications to pursue future testing. In preparation for a potential Phase III, proposals should strive to articulate how data for molecule candidates achieving Phase II metrics for each of the three molecule activity classes can inform and enable prototyping efforts for one or more of the ECW application areas listed below. However, Phase III may not be limited to candidate molecules/materials discovered and screened during Phases I and II of ICE.

Innovative approaches including novel functionalization or modifications of selected molecules to incorporate into deployable compounds may be pursued. Scale up predictions of molecules for specific applications must be considered. Applications may be selected for further testing and development based on performance and relevance to DoD requirements. Testing should be designed to enable transition to a DoD stakeholder, but access to candidate molecules/materials by local and vulnerable populations will be considered.

ECW EXAMPLE APPLICATION AREAS

1. Frostbite Prevention: Injuries to service members resulting from exposure to extreme cold weather is a persistent problem. There are currently limited technologies for the prevention of frostbite, which results from formation and expansion of ice crystals on and in skin and peripheral tissues. Biological molecules that prevent the formation or growth of ice crystals could potentially be leveraged as an anti-frostbite countermeasure.

Incorporation of these molecules into topical formulations such as a gel, hydrogel, cream, dermal polymer, or other appropriate medium may provide a solution.

2. Critical Aqueous Solutions: Freezing of critical aqueous solutions such as potable water, IV fluids, and sterile injectable medications is a challenge to sustained operations in ECW.

The addition of biological molecules that could lower the freezing and/or melting points of these critical solutions in a non-toxic manner would be highly desirable.

3. Anti-icing Coatings: Ice accretion on maritime vessels is a persistent operational and safety challenge. Current mitigation procedures for ice accretion employed at sea consist primarily of manual removal with blunt objects. This approach is time consuming, physically intensive, and not suitable for sensitive equipment and devices. The incorporation of anti-icing molecules into coatings and adhesive formulations that could be easily applied over large surface areas represents a potential solution to this ubiquitous problem.

4. Green De-icing Solutions: Current de-icing fluids are comprised of different mixtures of salts or polyethylene glycol. These fluids are corrosive and toxic to the environment.

Biologically sourced and/or inspired molecules could provide a non-toxic, environmentally friendly, and low-cost de-icing alternative for runways and aircraft.

5. Ice Inducers: Ice nucleators can promote freezing and ice strengthening at elevated temperatures. Overland vehicles in extreme cold often utilize frozen rivers and “ice bridges” to traverse the rugged terrain of the Arctic. In addition, the Army Corp of Engineers utilizes cleared snow to construct protective barriers/berms to defend against kinetic weapons. The materials will be evaluated in operational conditions to determine performance and utility.

PROGRAM METRICS

In order for the Government to evaluate the effectiveness of a proposed solution in achieving the stated program objectives, proposers should note that the Government hereby promulgates the following program metrics that may serve as the minimum basis for determining whether satisfactory progress is being made to warrant continued funding of the program. Although the following program metrics are specified, proposers should note that the Government has identified these goals with the intention of affording the maximum flexibility, creativity, and innovation in proposing solutions to the stated problem.

Quantitative performance metrics are designed to improve understanding of the candidate molecules by activity class (e.g., inhibition, induction, adhesion) and optimize performance. Phase II will down select the candidates screened based on performance. Proposers to the ICE program are required to define ambitious, specific, and quantitative metrics in support of program goals, including intermediate metrics (e.g., every 3 months, or sooner) to help further evaluate progress.

Some exemplary milestones are included below for proposers to consider but are not meant to be prescriptive. Final metrics are to be determined at time of award negotiation and are subject to DARPA approval. Proposers should note that program metrics may serve as the basis for determining whether satisfactory progress is being made to warrant continuation of the program.

1.3.1 Phase I

Phase I is an 18-month phase that contains two tracks that will be pursued simultaneously by each performer. The two tracks should be integrated by month 12. Track 1 focuses on developing quantitative test bed infrastructure and Track 2 focuses on discovering novel ice-modulating candidate molecules. The goal of Phase I is to demonstrate the robustness of the quantitative test bed infrastructure and use this infrastructure to test and characterize the novel ice modulating candidates. The metrics listed below are the minimum acceptable criteria, proposers are expected to provide additional rigorous metrics specific to their proposed strategies as needed to effectively ensure that sufficient progress is being made toward the minimum program metrics.

Table 1: Phase I Milestones and Metrics.

Track Month Milestone Description Quantitative Metric

4 Basic test bed performance:

Demonstrate performance of the test bed using commercially available molecules and materials.

The test bed must be capable of assaying:

1) Proteins/peptides.

2) Polysaccharides.

3) Small molecules.

Within a temperature range of +5°C to -10°C.

9 Test bed challenge:

Using blinded samples provided by the ICE IV&V partner, evaluate the accuracy of the test bed.

Using the test bed, successfully identify the performance characteristics of all of the supplied substrates within 10% accuracy of the known parameter (provided by the IV&V partner).

Track 1:

Quantitative Test Bed Development

12 Advanced test bed performance:

Optimize the performance of the test bed and expand its capabilities.

The test bed must be capable of:

1) Assaying a temperature range of +5°C to -30°C.

2) Assaying over two orders of magnitude of concentration.

3) Assaying 3 technical replicates per molecule with less than 5% variation in relevant assay metric.

4) Producing automated and quantitative data collection that is NOT dependent on subjective interpretations by a human.

5) Screening of ice modulation activity for at least 500 samples, in triplicate, over a continuous 24-hour period.

6) Achieving a run to run variability of less than 5% for the same sample assessed on different days.

Track 2:

Candidate Discovery and

6 Candidate Discovery Method 1

Prepare a minimum of 250 candidates for testing by a single method of synthesis, production, collection, etc.

Track Month Milestone Description Quantitative Metric Characterization 12 Candidate Discovery

Method 2 Prepare a minimum of 750 additional candidates by either:

1) A second method of synthesis, production, collection, etc. (unique from the month 6 milestone).

OR

2) A combination of the first and second methods (250 new candidates from the first method and 500 from a second method).

15 Candidate Screening Screen at least 1000 candidate molecules to obtain ≥ 25 molecules per activity class with ice modulation abilities.

18 Candidate Characterization

Demonstrate advanced ice modulation capabilities from the top 10 candidates from each activity class by methods such as:

1) Inhibition of ice crystallization measured by size and shear force for surface removal <100kPA

2) Induction of ice crystallization measured by time to crystal formation, size, and shear force for surface removal

3) Adhesion to ice measured temporally and shear force strength (shear force and peel test)

Integration of Tracks

18 Validation of high performing candidates by IV&V partner.

Verification of 5 high performing candidates Delivery of sufficient quantities of the material to enable testing

1.3.2 Phase II

The overall phase goals are listed in Table 2. The metrics listed below are the minimum acceptable criteria. Proposers are expected to provide additional rigorous metrics specific to their proposed strategies as needed to effectively ensure that sufficient progress is being made toward the minimum program metrics.

Table 2: Phase II Milestones and Metrics Month Milestones Metrics 28 Performance Optimization Screen ≥5 candidates per activity class for form and function with application relevant metrics. For example:

1) Ice crystal modulation: -20C/- 60C

2) Ice adhesion: <100kPA

3) Demonstrate ≥75% reduction in tissue necrosis

4) Demonstrate ≥75% reduction in ice accumulation on a surface

5) Safety/environmental data based on surfaces selected for testing

6) Minimal tissue reactivity to formulation

7) Toxicity assays to produce data for regulatory engagement

30 Validation of candidates by IV&V partner Metrics for the three activity classes of ice modulation molecules will be determined based on the analytical testing regime employed. These will be determined in collaboration between the DARPA PM, IV&V partner, and any relevant government stakeholders.

1.3.3 Phase III

Phase III is NOT solicited in this BAA. Potential phase III goals are listed in Table 3 to inform the schedule and milestones proposed to this solicitation for Phases I and II. The metrics listed below are notional and intended to provide intellectual inspiration to proposers to develop well-rounded, focused proposals. A potential Phase III may be informed by results of Phases I and II regarding the selection of DoD relevant application and testing requirements (See section 1.2.3).

Table 3: Phase III Milestones and Metrics Month Milestone Metrics 42 Operationally relevant testing with IV&V and/or DoD partners.

Test at least 5 candidates scaled up formulations for application specific conditions with IV&V and/or DoD partner.

1) Demonstrate at least 75% modulation based on DoD ECW application.

2) Provide all safety, toxicity, and environmental impact data, based on the surfaces and operation environments for testing.

48-54 Final demonstration with IV&V and/or DoD partners.

Test at least 2 candidate formulations for application specific conditions with IV&V and/or DoD partner.

1) Demonstrate at least 90% modulation.

2) Operationally persistent (time or ruggedness to be determined by what would be necessary for stakeholder adoption).

3) Determine the operationally relevant scale up and production plan.

4) Provide all safety, toxicity, and environmental impact data to stakeholders for necessary FDA engagement.

Section 3.4 OTHER ELIGIBILITY CRITERIA – COLLABORATIVE EFFORTS It is anticipated that successful proposals will be comprised of multi-disciplinary teams, and that successful implementation may require academic and industrial collaborations. Teams may be led by industrial, academic, or non-profit entities, among others. It is expected that the proposed leadership team will include individuals with significant experience and expertise in directing technology development, leading large and diverse teams with both academic and industrial partners, and have significant experience in the discovery and characterization of biologically sourced or inspired molecules capable of modulating or controlling ice and assays to measure ice control properties of diverse molecules.