(T1430-03-21) Phase Transitions and Apparent Acidity in HSA/Saline Solution during Freezing and Post Freeze-Drying

Purpose: The purpose of this research was to study phase transitions and pH shifts in HSA/saline solution during freezing and post freeze-drying.

Methods: Direct measurement of pH during sample freezing was performed by low temperature pH electrode. Phase transitions during freezing, post freeze drying, and upon reconstitution of the freeze-dried sample were studied using Small-angle X-ray Scattering (SAXS) and Wide-angle X-ray Scattering (WAXS) in which a saline sample was also included as a control. FTIR analysis of the freeze-dried solid and reconstituted sample was performed to assess changes in secondary structure of HSA. For UV-Visible spectroscopic measurements, Bromocresol purple was used as a molecular probe/chemical indicator in the samples. A calibration curve was constructed using UV-Visible spectra of samples containing Bromocresol purple, adjusted to pH values ranging from about 4.6 to 6.8. Of these samples, the one with native pH was freeze dried using Lyostar 3 freeze dryer, and solid-state UV-visible diffuse reflectance spectra of the freeze-dried samples were recorded using UV-Vis spectrophotometer equipped with a 60 mm integrating sphere accessory. The apparent pH of the freeze-dried sample was then calculated using the calibration curve.

Results: From the initial pH of about 6.7, a minor gradual increase in the pH up to about 7, was observed during freezing of the samples from 20ºC to -10ºC. A spike in temperature caused due to ice nucleation after reaching -10 ºC, didn’t cause noticeable pH change in the sample. Cooling further down to -30 ºC showed the pH to remain between 6.9 to 7.1. Further cooling to -50ºC showed pH readings reaching a high of 8.1. During sample freezing, the WAXS data showed formation of hexagonal ice at -20°C (HSA/Saline) and -15°C (Saline). Upon further cooling, characteristic peaks of NaCl dihydrate were detected at -35°C (HSA/Saline) and at -25°C (Saline). SAXS patterns complemented the WAXS data showing an increase in intensity at around -20°C and additional increase at around -35°C in the low-angle scattering. For the freeze-dried solid, the WAXS pattern indicated formation of crystalline sodium chloride (NaCl) implying loss of water by NaCl hydrate during freeze drying and conversion to anhydrous crystal form. The SAXS plots indicated 2 structures in the freeze-dried solid, with radius of gyration ~ 12 nm (with sharp interfaces, porod invariant =4) and 900nm (surface fractal, porod invariant=3.3). Higher scattering was evident in the SAXS pattern for the reconstituted freeze-dried sample possibly due to presence of HSA aggregates. The Kratky plot for the reconstituted sample indicated presence of folded HSA structure. Comparison of FTIR spectra for freeze-dried solid and reconstituted sample were indicative of difference in the secondary structure of HSA in the two samples. Corelating these results with SAXS data which indicated folded HSA structure, imply that the secondary structure of HSA was disrupted during freeze drying which was restored upon reconstitution. A linear UV-Vis absorbance profile was obtained for Bromocresol purple containing HSA solution samples in the studied pH range of ~4.6 to 6.8. The apparent pH value for the freeze-dried HSA sample was found to be ~6.

Conclusion: During sample freezing, the WAXS pattern indicate possible hinderance but no prevention in crystallization of NaCl dihydrate by HSA; while the SAXS data were indicative of formation of additional interfaces, i.e., ice/solution and probably ice/air/solution in the samples. A minor increase in pH was observed during freezing till -30°C measured by the low temperature electrode, which spiked sharply upon continuing the freezing to -50°C. Although, this was beyond the lower end temperature specification of the pH electrode, crystallization of NaCl may impact the ionic strength and hence the pH of the sample in this region as complemented by the WAXS and SAXS data. This could contribute to potential destabilization of proteins in the sample and could have practical implications on the sample stability. Characterization and comparison of the free-dried solid and reconstituted solution samples by WAXS, SAXS and FTIR collectively suggested possible aggregation and disruption of secondary structure of HSA after freeze drying. The apparent pH value of the freeze-dried HSA sample calculated using molecular probe was found to be about 0.8 unit lower compared to the native pH of the sample in solution before freeze drying. This highlights the difference between freeze concentrated solution showing higher pH shift versus the freeze-dried sample showing apparent acidic pH shift due to removal of water which is consistent with trehalose citrate lyophile results reported in the literature.

Figure 1. pH during freezing. The blue arrow in the top graph marks the temperature of NaCl*2H2O+water crystallization as detected in a separate WAXS experiment.

Figure 2. Top: WAXS patterns during cooling of HSA/Saline and Saline solutions. Bottom: SAXS patterns during cooling of HSA/Saline solution

Figure 6. UV-Vis spectra for Freeze-dried HSA/Saline sample containing Bromocresol purple (triplicate)