Prometheus is the new gold standard
for protein stability characterizations

PDI

Polydispersity index represents distribution of size populations

Low PDI values indicate prep is free of large aggregates or multiple protein populations

T_m (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

T_onset (for ratio)

Temperature at which thermal unfolding begins, measured from fluorescence ratio

More thermostable proteins have higher onset of unfolding; furthermore, proteins with T_onset values close to their T_ms are more uniformly folded and therefore more stable

E_a(*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

 r_H

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

T_size

Temperature at which average particle size begins to increase – measured from from growth of r_H in cumulants fit Indicates temperature at which protein begins to unfold, thereby indicating how stable it is; higher T_size 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

T_turbidity

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 T_turbidity values or no change in turbidity signal

T_scattering

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

k_D

Diffusion interaction identifies onset of unfolding and impact on colloidal stability

Negative k_D values indicate propensity towards self-interaction, and therefore greater likelihood of aggregating at the high concentrations required for clinical use

C_m or C₅₀

The concentration of a denaturant that causes 50% of proteins to unfold

Higher C_ms 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.