Prometheus for Protein Stability Characterizations

· Purity
PDI
Polydispersity index represents distribution of size populations
Low PDI values indicate prep is free of large aggregates or multiple protein populations
· Thermal Unfolding
Tm (for 330 nm, 350 nm, and ratio)
Melting temperature, or point at which 50% is unfolded
Ranking candidates by melting temperature is a well-established method for finding most thermostable constructs, considered better selections for further experimentation

Tonset (for ratio)
Temperature at which thermal unfolding begins, measured from fluorescence ratio
More thermostable proteins have higher onset of unfolding; furthermore, proteins with Tonset values close to their Tms are more uniformly folded and therefore more stable

Ea(parameter derived from data)
Activation energy of unfolding (*parameter derived from data)
The more energy required to drive unfolding of a protein, the more stable it is

Reversibility of unfolding
Point at which irreversibility happens when unfolding, thereby making aggregation more likely
Engineer or formulate proteins to avoid or delay irreversible unfolding and loss of function

· Sizing Information
rH
Hydrodynamic radius shows size of particle in solvated state
Establishes baseline sizing parameter about a protein of interest, used to compare before and after changes to formulation buffer or production processes

Molecular weight
Average molecular weight of all particles in solution Establishes baseline sizing parameter about a protein of interest, used to compare before and after changes to formulation buffer or purification processes

Tsize
Temperature at which average particle size begins to increase – measured from from growth of rH in cumulants fit Indicates temperature at which protein begins to unfold, thereby indicating how stable it is; higher Tsize indicates greater colloidal stability

Average scattering intensity
Identifies if concentration is too high or low for proper size distribution analysis; high-quality DLS data starts with good signal quality

· Aggregation and Self-Association
Tturbidity
Onset temperature of turbidity, or large aggregates >12.5 nm radius
Large, amorphous aggregates contaminate preps and lower safety, activity, and stability of a prep; aim for high Tturbidity values or no change in turbidity signal

Tscattering
Temperature at which scattering intensity begins to change, indicating increased size and aggregation
Additional parameter for evaluating colloidal and conformational destabilization of a protein; higher Tscattering means a more stable protein

B22
Second virial coefficient used to extrapolate self-association behavior at higher concentrations
Negative B22 values indicate propensity towards self-interaction, and therefore greater likelihood of aggregating at the high concentrations required for clinical use

kD
Diffusion interaction identifies onset of unfolding and impact on colloidal stability
Negative kD values indicate propensity towards self-interaction, and therefore greater likelihood of aggregating at the high concentrations required for clinical use

· Chemical Denaturation
Cm or C50
The concentration of a denaturant that causes 50% of proteins to unfold
Higher Cms indicate a more stable protein

ΔG
The Gibbs free energy of protein unfolding, a thermodynamic measure of the likelihood a folding event may occur
Proteins with more negative ΔG are more likely to fold. Changes in the Gibbs free energy are measured with ΔΔG, which shows the relative stability of a protein at various concentrations or conditions.

General Specifications
Prometheus Panta
Sample volume: 10 µL
Sample handling format: Individual capillaries or capillary chip
Throughput in one run: Up to 48 capillaries or 24 in capillary chip
Temperature range: 15 – 110°C
Heating rate range: 0.1 – 7°C/min
Precision of 1°C/min thermal ramp: ± 0.1°C
Optional high-throughput: Yes
Dimensions: 35 cm W x 51 cm H x 52 cm D
Weight: 35 kg

Prometheus Panta + Robotic Autosampler
Prometheus Panta
Sample volume: 10 µL
Sample handling format: Capillary chip
Throughput in one run: U64 capillary chips (1536 samples, four 384-well plates) before need to reload
Temperature range: 15 – 110°C
Heating rate range: 0.1 – 7°C/min
Precision of 1°C/min thermal ramp: ± 0.1°C
Dimensions: 110 cm W x 188 cm H x 90 cm D (stand-alone enclosure)
Weight: 200 kg

Technical Specifications--Prometheus Panta
nanoDSF: Included
Measurement parameters: ratio: Tonset, Tm, Ea, reversibility of unfolding 330 nm, 350 nm: Tm; Excitation: 280 nm
Concentration range: 0.005 mg/mL – 250 mg/mL
Inflection point precision @ 75°C: ± 0.1°C
Initial ratio repeatability: 0.008
DLS: Included
Measurement parameters: Tscattering, Tsize, rH, PDI, kD
Laser wavelength: 405 nm ± 5 nm
Concentration range (DLS/SLS): 0.5 mg/mL for a 15 kDa protein, up to 40% w/v
Size resolution: Down to 0.5 nm – 2 μm
SLS: Included
Measurement parameters: Tscattering, average scattering intensity, molecular weight, B22
Measurement accuracy: ≤ 10% molecular weight
Backreflection: Included
Measurement parameters: Tturbidity
Measurement accuracy: Larger than 12.5 nm radius

Protein Stability Characterization

Create biologics developability profiles during early-stage screening.
Perform comparability studies to ensure that changes to manufacturing processes or sites don't affect the drug product.
Use combinatorial stability parameters to find the optimal storage buffers for your drug candidates.

Scientific areas
· Therapeutic drug discovery & development
Create biologics developability profiles during early-stage screening.
· Gene Therapy
Differentiate AAV serotypes and determine the purity of your vector preps
· Structural biology
Maximize protein solubility and stability before crystallization or Cryo-EM to improve the quality of your structural data