Analytical ultracentrifugation (AUC)

Analytical ultracentrifugation was used to characterize aggregates and fragments in terms of heterogeneity and particle size. MYL-1501D and reference products were loaded into cells with 2-channel charcoal-epon centerpieces and a 12-mm optical path length. Dilution buffer was loaded into the reference cell to nullify any effects due to excipients in the spectrophotometer. Loaded cells were placed into an AN-60Ti analytical rotor (Beckman Coulter, Brea, CA), loaded into a Proteome Lam XL-A analytical ultracentrifuge (Beckman Coulter), and brought to 20°C. When the rotor reached 3000 rpm, the samples were scanned at 230 nm to confirm proper cell loading before the rotor reached a final run speed of 60,000 rpm. Scans were recorded every 4 minutes for approximately 4 hours (approximately 50 scans per sample); the scan rate was then dropped to every 16 minutes for an additional 8 hours.

Data were analyzed using SEDFIT software version 11.3 [23]. Sedimentation coefficients were derived by fitting the raw data while modeling the influence of diffusion. Briefly, a diffusion coefficient was assigned to each value of sedimentation coefficient based on the assumption that all species have the same overall hydrodynamic shape, where the shape was defined by the frictional coefficient ratio relative to sphere, f/f₀. The f/f₀ values were varied to find the best overall fit of the data for each sample. A maximum entropy regularization probability of 0.683 (1 σ) was used, and time-invariant noise was removed. The resultant size distributions were graphed, and the peaks were integrated using Origin software (OriginLab Version 2018b).