Prometheus Panta for AAV stability profiling with particle sizing and molecular weight determination by NanoTemper Technologies by NanoTemper Technologies

Features:

• Measure thermal unfolding, particle sizing, molecular weight, and aggregation simultaneously, throughout an entire thermal ramp
• High-resolution data about your AAV’s stability profile
• Flexible throughput, with multiple options for sample handling

Parameters that Prometheus Panta measures:

Dynamic Light Scattering (DLS)

SIZE ANALYSIS

T_size (from growth of r_H in cumulants fit): Temperature at which average particle size begins to increase, indicating unfolding

Average scattering intensity: Identifies if concentration is too high or low for proper size distribution analysis

CUMULANT OR SIZE DISTRIBUTION/REGULARIZATION ANALYSIS

r_H: Hydrodynamic radius, shows size of particle in solvated state

PDI: Polydispersity index represents distribution of size populations, a low value is indicative of properly assembled capsids without contaminants

SELF-INTERACTION ANALYSES

k_D: Self-association constant, a parameter used to determine the propensity of a particle to aggregate at higher concentrations

D₀ (*derived from k_D analysis): Theoretical diffusion constant at concentration = 0

nanoDSF

THERMAL UNFOLDING

T_m (for 330 nm, 350 nm, and ratio): Melting temperature, the point at which 50% of the viral particle proteins that comprise your capsid are unfolded; T_m values are a hallmark of certain serotypes, you can also see how small changes to capsid proteins affect their overall stability, as higher T_m values are considered more stable

T_onset/T_on (for ratio): Temperature at which unfolding begins; higher T_on values indicate increased thermal stability, and it is considered favorable to have a sharper slope at the unfolding inflection point, which occurs when the T_on is close in value to the T_m

E_a (*parameter derived from thermal unfolding data): Activation energy of unfolding, a parameter that informs on the stability of a protein in a given buffer environment

Reversibility of unfolding: Point at which aggregated capsid proteins cannot be recovered and refolded without other chemical means

Backreflection

AGGREGATION

T_turbidity: Onset temperature of large (>12.5 nm radius) aggregate formation

SLS

SIZING INFORMATION

Molecular weight: Average molecular weight of all particles in solution; requires a homogenous preparation of a given sample to maximize accuracy

SELF-ASSOCIATION AND AGGREGATION PROPENSITY

T_scattering: Temperature at which scattering intensity begins to change, indicating the increased size and smaller aggregate formation

B₂₂: Second virial coefficient, used to extrapolate self-association behavior at higher concentrations