Category: Formulation and Quality
Purpose: To assess correlations between intrinsic fluorescence of bovine serum albumin (BSA) and its aggregation upon accelerated and long-term storage, using multivariate experimentation.
Methods: A central composite multivariate experimental design was used to prepare BSA in formulations with varied pH, sodium chloride, sucrose, methionine, and polysorbate 80 levels. Room temperature fluorescence spectra and mid-points of thermal transitions of BSA were measured for each formulation. Fluorescence parameters for room temperature measurements included wavelength of fluorescence emission maximum (lmax) and initial emission intensity ratio 330nm/350nm (IIR330/350). Fluorescence parameters for thermal transitions included mid-points (Tms) obtained from monitoring intensities at 330nm (FI330), 350nm (FI350), and the 330nm/350nm ratio (IR330/350). The formulations were also prepared and stored at 40°C for 1 month and 4°C for approximately 3 years, to determine aggregation measured by size-exclusion high-pressure liquid chromatography (SE-HPLC). Statistical correlations between fluorescence and SE-HPLC parameters (% aggregates and SE-HPLC total area) were analyzed using JMP.
Results: Multivariate analysis showed several significant (p-value < 0.05) correlations between fluorescence and SE-HPLC parameters. All three Tm parameters showed a positive correlation to SE-HPLC total area at 40°C. All except the Tm determined from monitoring Fl330 showed a positive correlation to SE-HPLC total area at 4°C. The Tm determined from IR330/350 correlated inversely to % aggregates at 40°C. However, positive correlations between Tms (all except the Tm determined from Fl330) and % aggregates were observed at 4°C. Room temperature fluorescence (lmax and IIR330/350) correlated to SE-HPLC total area at 40°C% and % aggregates at 4°C. Assessment of the JMP prediction and interaction profiles showed that excipients that had a significant effect on IIR330nm/350nm also had a significant effect on % aggregates at 4°C storage
Conclusion: Altogether, the data suggests that fluorescence measurements at room temperature, rather than Tms, are a better predictor for % aggregates at 4°C (long-term) storage. In addition, in our case, IR330/350 fluorescence data provided more meaningful data, rather than fluorescence intensity at 330nm or 350 nm alone. The results illustrate the value in using multivariate experimentation when assessing the effects of excipient combinations on protein stability.