Analytical Ultracentrifugation (AUC)

Analytical ultracentrifugation (AUC) is a first-principles, solution-phase technique for characterizing macromolecules and nanoparticles without a stationary phase (“matrix-free”). In a strong centrifugal field, AUC measures either sedimentation velocity (SV-AUC)—the rate at which species move—or sedimentation equilibrium (SE-AUC)—the concentration distribution at equilibrium. From these data, AUC resolves heterogeneity (e.g., monomer vs. aggregates), determines hydrodynamic properties, and, with appropriate models, reports absolute or apparent molar masses and association parameters.

• Legacy: Preparative/density-gradient ultracentrifugation—useful, but limited quantitatively.

• Established standard practice: SV-AUC for high-resolution size/aggregation distributions (e.g., c(s) analysis via SEDFIT); SE-AUC for absolute/weight-average molar mass and reversible association analyses.

• Emerging/orthogonal: SEC-MALS (higher throughput, mass from scattering) and Mass Photometry (MP) (single-particle mass distributions; increasingly used orthogonally for AAV empty/partial/full).

• SV-AUC: At high rotor speeds, species sediment and broaden by diffusion. Fitting the Lamm equation yields a sedimentation-coefficient distribution (c(s)), cleanly separating monomer from oligomers/aggregates; multi-wavelength SV-AUC can deconvolute signals from components (e.g., AAV protein vs. genome at 280/260 nm).

• SE-AUC: At lower speeds and long times, sedimentation is balanced by diffusion; fitting the exponential radial concentration profile provides molar mass and association constants (Kd) without shape assumptions.

• SV-AUC: Primary output is c(s) vs. s (Svedbergs). The main peak corresponds to the principal species; higher-s peaks are larger particles/aggregates. Relative areas (after appropriate baselines/optics) report species % composition.

• SE-AUC: Global fits of radial concentration gradients to thermodynamic models yield molar mass (and, for reversible self-association, Kd); random residuals indicate good model agreement.

• First-principles, matrix-free solution analysis in native buffers (no column interactions).

• High resolution for polydisperse mixtures; detects species missed by SEC when column interactions bias results.

• Widely relied upon in AAV analytics for empty/partial/full assessment; multi-method corroboration is encouraged.

• Throughput: Runs are hours to (for SE) days; not ideal for routine high-volume QC.

• Method expertise: Careful experimental setup and analysis are required (e.g., solvent density/viscosity, temperature, optics, cell alignment).

• Dynamic range/LOQ: Practical LOQ for minor aggregates is usually ~1–3% (system-dependent; best practice needed)—so report sub-percent levels cautiously.

• Specificity: Demonstrate resolution of monomer vs. relevant aggregates (e.g., stress studies); for AAV, show discrimination of empty/partial/full and any co-sedimenting impurities.

• Precision & Accuracy: Establish repeatability/intermediate precision; document analysis parameters (e.g., SEDFIT settings), and verify against orthogonal methods (SEC-MALS, MP, AEX).

• LOD/LOQ & Linearity: Determine empirically; LOD/LOQ studies rather than assuming fixed thresholds.

• System suitability: Periodic checks with standards (e.g., BSA or NIST mAb), instrument calibration, and predefined fit/RMSD criteria.

Standardize SV-AUC conditions and reporting: verify rotor speed and temperature, document optical/radial calibration/meniscus determination, and correct sedimentation coefficients to s₍20,w₎ using measured buffer density/viscosity (SEDNTERP-equivalent). Include a run-level system suitability control (e.g., BSA or NISTmAb, RM 8671) to confirm expected peak position(s), resolution, and fit quality (RMSD/residuals), and lock analysis parameters (SEDFIT model/regularization, bounds, and c(s) integration ranges). For AAV, qualify an in-house reference panel of empty/partial/full capsids by orthogonal methods (e.g., AEX, ddPCR/qPCR, MP/TEM) and use it to set integration masks and verify 260/280 multi-wavelength agreement. Define traceable acceptance criteria (e.g., allowable s-value windows, residual thresholds) and trend them over time. For SE-AUC, check molar-mass accuracy with well-characterized proteins under the same buffer conditions. Where no public RMs exist (e.g., universally accepted AAV empty/full standards), rely on qualified internal standards and inter-lab cross-checks; use public RMs (e.g., NISTmAb) where available to harmonize performance across sites.

• mAbs & proteins: Quantitative %HMW, oligomer states, and self-association; powerful orthogonal method to SEC.
• Gene therapy (AAV): SV-AUC is widely used to quantify empty/partial/full capsids and detect capsid aggregates; often combined with multi-wavelength detection (260/280 nm) for enhanced specificity.
• LNPs/mRNA: (Multi-wavelength / density-matching) SV-AUC can probe drug (RNA) loading and heterogeneity, complementing DLS/AF4-MALS/EM.