Fig 1.
Glass transition temperatures of freeze-dried plasma samples.
DSC thermograms of plasma samples that were freeze-dried without protectants (green), or freeze-dried with 10% glucose (blue), sucrose (orange), or trehalose (red). Representative thermograms are shown (A). Tg-values were determined as the midpoint of the temperature ranges were glass transition occurred. Measurements were done in triplicate, and mean values ± standard deviations are presented (B).
Fig 2.
Turbidity of plasma after freeze-drying and rehydration.
Human plasma (A−C) and pure IgG (D,E) were freeze-dried without (green) protectants as well as 2.5−10% glucose (blue), sucrose (orange) or trehalose (red). Samples were analyzed before (bars with diagonals) and directly after (filled bars) freeze-drying (A,B,D,E) or after 7 d dried storage at temperatures ranging from 4−60°C (C). Protein aggregation was evaluated by means of turbidity as the absorbance at 550 nm (human plasma) or 350 nm (IgG). Mean values ± standard deviations are presented. For plasma, three technical replicates were performed for plasma obtained from six different donors. For IgG, measurements were done in triplicate.
Table 1.
Turbidity of plasma after freeze-drying and rehydration.
Spectroscopic analysis of plasma turbidity (i.e. absorbance at 550 nm), both before and after freeze-drying of plasma without supplements (-) as well as supplemented with 2.5−10% glucose (GLU), sucrose (SUC) or trehalose (TRE). Plasma from six different donors was analyzed (L1−6), with performing three technical replicates each. Mean ± standard deviations are presented, while statistically significant (p<0.05) differences amongst the time point of analysis (i.e. pre versus post freeze-drying) are indicated with an asterisk, and those between different sugar concentrations used (post freeze-drying data only) are indicated with different superscript letters. Values between different donors were statistically significant for all cases.
Fig 3.
Infrared spectroscopic analysis of freeze-dried plasma.
Infrared spectroscopic analysis of plasma freeze-dried without protectants (green), or freeze-dried with glucose (blue), sucrose (orange), or trehalose (red). Full original spectra are presented (A) as well as normalized second derivative spectra of the amide-I protein region (B). To reveal relative contributions of α-helical and β-sheet structures at ~1650 and ~1630 cm‒1, respectively, band intensity ratios were calculated; for plasma freeze dried with increasing trehalose concentrations, as well as 10% glucose and sucrose (C). Furthermore, the amide-I protein region (1700−1600 cm‒1) was subjected to PCA and scores plots of the first two principal components were prepared (D). Measurements were repeated 3−6 times, and mean values ± standard deviations are presented.
Fig 4.
Storage stability of freeze-dried plasma samples.
Turbidity/protein aggregation characteristics of freeze-dried plasma samples stored for up to 1 month at temperatures ranging from 4−60°C (A), as well as plasma IgG contents after storage under different conditions (B). Freeze-drying was done without supplements (green) as well as with supplementation of 10% trehalose (red). Turbidity was assayed directly after rehydration, as the absorbance at 550 nm (A). IgG contents were determined for plasma samples after 30 d storage; for hydrated sample and freeze-dried samples stored at 22°C, as well as frozen samples stored at −80°C. Mean values ± standard deviations are presented from replicate measurements performed using plasma from three different donors.
Fig 5.
Infrared spectroscopic analysis of freeze-dried plasma during storage.
Storage-related changes in infrared spectra of plasma freeze-dried without protectants (green) or with 10% trehalose (red). Spectra were acquired immediately after freeze-drying (solid lines) as well as 6 weeks storage (dashed lines) at 22°C; and normalized second derivative spectra were calculated for the 1700−1600 cm−1 region (A). The ratio between the band intensities at ~1630 and ~1650 cm‒1, representing respectively α-helical and β-sheet structures, was calculated to reveal changes in the protein secondary structure during storage at 22 or 37°C (B). Mean values ± standard deviations were calculated from three replicates/measurements performed using plasma from three different donors.
Fig 6.
Oxidative damage of freeze-dried plasma during storage.
Accumulation of oxidative stress/damage as ROS (A) and protein carbonyl contents (B), in plasma samples during hydrated storage (circles) as well as storage in the freeze-dried state (squares). Plasma without supplements (green) as well as supplemented with 10% trehalose (red), was stored at temperatures ranging from 4−60°C for 7 d (A) or 30 d at 37°C (B). In case of determining plasma carbonyl contents also fresh specimens were analyzed (bars with diagonals) Mean values ± standard deviations are presented for replicate measurements using plasma obtained from three different donors.