Stopping ice-grain damage during thaw without triggering freezing
Most cryopreservation loss happens on the way back up: tiny ice crystals merge into larger, sharper grains that shear cell membranes and aggregate biologics. DB-IRI1 is a panel of narrow, disulfide-free repeat rods (184–608 AA) and short chemically synthesizable helices (33–44 AA) built to sit on ice grain boundaries and slow recrystallization during thawing while staying far too narrow to nucleate new ice.
Where it stands: DB-IRI1 constructs have been folded at full length and evaluated in explicit ice–water coexistence simulations at −10 °C and −13 °C. No material has been ordered and no supplier has been engaged; wet-lab ice-recrystallization (splat) assays and cell-thaw recovery tests have not yet been run and are funded to order under a bilateral evaluation option.
DB-IRI1: narrow rod built to pin ice without nucleating it
A 184-residue cytosolic alpha-helical repeat rod (~13–15 Å wide) carrying eleven aligned valine methyl rungs along one flat face at 4.53 Å per-helix spacing.
1. Narrow enough to avoid intracellular freezing
Triggering new ice at warm subzero temperatures requires a wide two-dimensional template (roughly 225–560 Å across). At ~13.4 Å wide, a monomeric DB-IRI1 rod sits more than fifteen-fold below that threshold, so it is modeled to bind existing grain boundaries during thawing without initiating lethal intracellular ice.
2. Hydrophobic hydration cages without disulfides or TxT motifs
Classical fish and insect antifreeze proteins rely on threonine-rich TxT ladders and multiple disulfide bonds, and often trigger sharp needle-like ice burst below their hysteresis point. Replacing hydroxyl rungs with aligned valine and alanine methyl groups wraps surrounding water into longer-lived hydration cages while keeping zero cysteines and zero TxT motifs.
3. Compare against controlled freeze-drying (DB-INP1)
Use the toolbar toggle to compare DB-IRI1 against DB-INP1, a three-chain assembly built for the opposite bioprocess job: triggering predictable, uniform vial freezing near −2 to −4 °C during pharmaceutical lyophilization.
Lead program · Non-nucleating thawing protection (DB-IRI1)
Cell therapies (CAR-T, NK, iPSCs), organoids, and biologics rely heavily on 5–10% DMSO to survive freezing and thawing, adding wash steps, infusion side effects, and formulation constraints. Natural hyperactive antifreeze proteins rarely solve this cleanly because disulfide-rich folds mispair in reducing environments and basal-plane clamping can drive destructive needle-shaped ice growth.
Across paired explicit ice–water coexistence simulations at −10 °C and −13 °C, replacing classical polar threonine/serine ladders with aligned valine and alanine methyl faces held first-shell water molecules in place up to 27.4% longer and slowed advancing prism-plane ice growth by up to 29.7%, while keeping protein backbone drift under 1.5 Å RMSD. Alongside full-length recombinant rods (184–608 AA), the panel includes 33–44 AA zero-threonine, zero-cysteine single helices sized for standard solid-phase peptide synthesis as chemically defined cryoprotectant additives.
| Parameter | Measured on model / simulation | Notes |
|---|---|---|
| Scaffold formats | 184–608 AA repeat rods & 33–44 AA peptides | Recombinant single-chain alpha-helical rods (viewer shows the 184-AA lead) plus short single-helix peptides sized for solid-phase chemical synthesis. |
| Prism-plane lattice match | 4.53 Å per-helix spacing (8.95–9.98 Å full coil across panel) | Matches the 4.52 Å primary prism spacing of hexagonal ice while keeping transverse rod width (~13.4 Å) well below critical nucleation dimensions. |
| Model confidence & sequence liabilities | Cα mean pLDDT 94.2 (all-atom 93.8); up to 98.0 across panel | Zero cysteines (no disulfide mispairing), no secretion leader, and zero canonical TxT antifreeze motifs on the methyl-ladder leads. |
| Ice–water coexistence simulation | Up to +27.4% water residence; −29.7% prism growth rate | Measured across paired −10 °C (263 K) and −13 °C (260 K) explicit ice–water simulations with 1.07–1.41 Å backbone Cα RMSD. |
| Target applications | DMSO-reduced cell therapy, organoid, tissue & biologics thaw recovery | Designed as a non-nucleating extracellular or intracellular additive to curb Ostwald grain coarsening during warming and transient cold-chain excursions. |
| Stage, IP & material status | Scored and simulated · Provisionals on file | U.S. provisional patent applications on file. No physical stock is held; recombinant expression or solid-phase peptide synthesis is performed to order under option, and wet-lab IRI splat and cell-viability assays are pending. |
Complementary bioprocess context · Controlled freeze-drying (DB-INP1 & DB-INP2)
In pharmaceutical lyophilization, the engineering problem flips: sterile vials supercool unpredictably down to −15 to −20 °C before freezing all at once, producing fine ice pores that slow primary drying and cause vial-to-vial cake variation. Where DB-IRI1 stays narrow to avoid nucleation during thawing, DarkBiome’s wider multi-chain assemblies (DB-INP1, a soluble 3 × 514 AA bacterial homotrimer at CRO Synthesis Queued, and DB-INP2, a 67-AA fungal beta-hairpin sandwich with a 39-AA peptide derivative) present an extended water-ordering footprint designed to trigger uniform freezing near −2 to −4 °C without bacterial cell-wall debris.
For biologics freeze-drying workflows that require both predictable freezing onset and protection against ice-grain coarsening during annealing, trace levels of a warm-onset nucleator (DB-INP1/DB-INP2) can be evaluated individually or paired alongside bulk DB-IRI1 under the same confidential evaluation framework.