Lactose

Article pubs.acs.org/jced

Cite This: J. Chem. Eng. Data XXXX, XXX, XXX−XXX

Thermodynamic Properties and Water States in Ternary PVP/ Lactose/Water Frozen Systems Pierre Verlhac, Claudia Cogne,́ * Sev́ erine Vessot, Ghania Degobert, and Julien Andrieu

J. Chem. Eng. Data Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 10/04/18. For personal use only.

Université Lyon, Université Claude Bernard Lyon 1, CNRS, LAGEP UMR 5007, 43 Boulevard du 11 Novembre 1918, F-69100 Villeurbanne, France ABSTRACT: The objective of this article was to present some key thermodynamic and physicochemical data for the rational optimization of freeze-drying cycles. First, a ternary system of poly(vinylpyrrolidone) (PVP) + lactose + water was studied over large concentration and temperature ranges by modulated differential scanning calorimetry (MDSC). In parallel with DSC experiments, the binary system PVP + water was investigated by cryomicroscopy. Both methods showed crystallization during cooling in the PVP concentration range lower than 49% (wt) and ice recrystallization during warming in the PVP range between 49% (wt) and 64% (wt). Second, a large experimental study has been carried out to determine the influence of lactose concentration on freezable water fractions and glass-transition temperatures. Finally, the solid/liquid phase diagram of the ternary PVP/water/lactose system was determined. These new experimental data concern the melting and vitreous transition temperatures of this ternary system and could be very useful in the future for optimizing the operating conditions of freeze-drying cycles of thermosensitive pharmaceutical formulations.

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INTRODUCTION Freeze-drying is a complex soft drying process usually used for fragile and thermosensitive products because it leads to the highest quality and safest results for the final freeze-dried cake.1,2 However, the major drawback of this method is its very high operating costs due to the severe main operating conditions of this batch process which has to be carried out at a very low sublimation temperature and total gas pressure.3,4 Thus, the freeze-drying process is commonly selected for preparing long-term-stabilized drugs (vaccines and serums) and food additives (vitamins, probiotics, etc.) in a growing market.5−7 During this long and complex separation process, three phase changes take place, namely, freezing, sublimation, and desorption. The freezing step is generally the most critical and most damaging step, mainly due to ice crystal growth and osmotic changes in the extracellular medium and, consequently, in the intracellular medium.8−10 That is why the use of cryoprotectants (or lyoprotectants) is usually necessary to achieve adequate quality attributes for the final freeze-dried powder or cake. Many types of cryoprotectants exist and can be classified by their ability to penetrate the membrane of the cells. They are commonly added to the formulation before starting the freezing step in the case of systems with living microorganisms such as vaccines and probiotics. The penetrating agents are small molecules which can diffuse through the membrane cells, reduce the risk of intracellular ice crystal formation, and prevent damage to the cells.11,12 On the other side, the nonpenetrating agents present two main properties: first, because of their large, long chain molecules, they cannot penetrate the cells and they increase © XXXX American Chemical Society

the osmolarity of the extracellular phase, preventing intracellular dehydration and intracellular crystallization. Second, they have the ability to be adsorbed on the bacterial surface, allowing water vitrification around the cells, preventing osmotic shock and slowing down the rehydration kinetics.13−16 Lactose is a well-known and largely used disaccharide, classified as a nonpenetrating agent and isolated from milk. It is already widely used for the preservation of freeze-dried bacteria and also for frozen products.17,18 Poly(vinylpyrrolidone) (PVP) is a long-chain biopolymer that is already used for many medical applications and is classified as a nonpenetrating agent with high glass-transition temperatures and interesting vitrification properties.19,20 Thus, a mixture of these two excipients can provide efficient protection of living cells during the different steps in the freeze-drying process, leading to a final freeze-dried product of high quality with a high cell viability and, on the other side, to a significant reduction in the drying times by increasing the upper limit of the sublimation temperature. That is why an investigation of the state diagram of the three components water + PVP + lactose and its properties was investigated in the framework of a project of optimization of freeze-drying cycles for probiotic aqueous formulations. The state diagram data of the frozen system are usually the key data to set up the appropriate temperatures during the primary drying period (sublimation) and the secondary drying Received: July 16, 2018 Accepted: September 21, 2018

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DOI: 10.1021/acs.jced.8b00613 J. Chem. Eng. Data XXXX, XXX, XXX−XXX

Journal of Chemical & Engineering Data

Article

Table 1. Physicochemical Properties of Used Products chemical name

source

molecular weight

CAS number

solubility

poly(vinylpyrrolidone) monohydrated lactose water

BASF Cooper our laboratory

44 000−54 000 Da 360.312 g·mol−1 18.015 g·mol−1

9003-39-8 10039-26-6 7732-18-5

limited by viscosity 18.90 g·L−1 (25 °C) solvent

Figure 1. MDSC curve of the PVP/water system (22 wt % PVP).

period (desorption) of the freeze-drying cycle.14 As a matter of fact, for amorphous or glassy systems, many experimental data have shown that during the sublimation step the frozen formulation must be maintained below the glass-transition temperature of the maximally concentrated solution, noted as Tg′. These conditions correspond to the temperature at which the liquid solution presents an important increase in the viscosity and solid behavior. Next, during the desorption period, the temperature of the cryoconcentrated phase is usually increased but must be maintained below the vitreous transition temperature, noted as Tg, to avoid the collapse phenomena, as this temperature increases with the dry matter content of the cryoconcentrated phase.4,21 Thus, higher glassy or vitreous transition temperatures result in higher temperatures for sublimation and desorption steps and, consequently, shorter drying times. In this study, an investigation of the ternary system (PVP/ lactose/water) and its properties was carried out. We chose to evaluate the impact of the lactose concentration in different water solutions of PVP in order to better understand its cryoprotectant effect for freeze-drying applications. The first part of this article is devoted to the study of the state diagram of the binary system PVP/water. Modulated differential scanning calorimetry (MDSC) and cryomicroscopy have been implemented to characterize the different phase transitions and physical states. Thus, because these data are not available in the literature, we have investigated the influence of lactose concentration on the melting and glasstransition temperatures as well as the enthalpy of fusion of the water + PVP mixture. Finally, we set up the ternary phase diagram including water, PVP, and lactose, which represent the key data for freezedrying applications.

Cooper (Melun, France). Table 1 summarizes the principal information for these products. Sample Preparation. In order to cover precisely the whole range of the state diagram, the required amount of powder for each component was dried under vacuum for 24 h before use. PVP and lactose were introduced into demineralized water and maintained under constant shaking for 24 h. We prepared samples containing from 4.99% (wt) to 69.47% (wt) polymer in order to plot the state diagram of the binary water + PVP mixture and up to 15% (wt) lactose for the ternary system. The polymer concentrations were calculated by using eq 1 mPVP wPVP = mPVP + mL + m w (1) where wPVP is the polymer weight concentration, mPVP is the mass of PVP, mL is the mass of monohydrated lactose, and mw is the mass of water. Calorimetric Analysis by Modulated Differential Scanning Calorimetry. The DSC apparatus used for the calorimetric measurements was a Q200 type from TA Instruments with a temperature accuracy of ±0.1 °C. Temperature calibration was performed with the melting point of indium (429.78 K) at a heating rate of 10 °C min−1. The energy-scale calibration was performed with the enthalpy of fusion of indium (28.45 J g−1). A nitrogen purge of 50 mL min−1 was employed for all measurements to avoid condensation phenomena in the furnace. Samples (10−20 mg) of mixtures prepared at varying compositions were weighed with a Mettler balance to an accuracy of 0.01 mg and then placed in sealed aluminum pans. To determine the phase-change temperature, the experiments were performed according to the following program: (a) isotherm at 293 K, (b) cooling rate of 20 K·min−1, (c) isotherm at 193 K for 5 min, (d) heating rate of 10 K·min−1, (e) repeated steps a−c, (f) maximum temperature amplitude ±0.20 K every 60 s, and (g) heating rate of 0.5 K·min−1. The results reported in this work corresponded to data taken during

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EXPERIMENTAL METHOD Kollidon30 was generously donated by BASF (Ludwigshafen, Germany). The monohydrated lactose was provided by B



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Figure 2. Phase diagram of the binary PVP + water. The gray line corresponds to the Fox mathematical model of the Tg. Green diamonds represent the Tm and Tg of Furushima et al.,22 blue crosses represent the Tg′ and Tg values, and yellow crosses represent the Tm values experimentally measured in our laboratory. (I) Mixture of ice crystals and a cryo-concentrated solution. (II) Mixture of ice crystals and unfreezable solution. (III) Liquid solution. (IV) Rubbery state. (V) Vitreous state.

the heating phase (steps e−g). These parameter values have been previously proposed by Furushima et al.22 All DSC curves were normalized with respect to the sample mass. The melting temperatures (Tm) were taken at the minimum of the fusion peaks of the heat flow curves, and the glass-transition temperatures (Tg) were taken on the reversed heat flow curves for better accuracy, as is generally the case with reversible thermal events. The enthalpies of fusion (ΔHm) of the mixtures were calculated by integration of the fusion peaks (Figure 1). Cryomicroscopy. The Linkam Scientific FDCS196 freezedrying cryo-stage and optical microscopy were used to observe the freezing behavior of the solutions both during the cooling period up to 173 K and during the warming period. Samples (2−6 μL) of the different concentrations were placed on the microscope slice, spread out on the surface, covered with a cover glass, and then introduced into the microscope chamber. Two different temperature cooling rates were used: a slow rate of 1 K·min−1 and a fast rate of 50 K·min−1. Pictures were taken using a mono CCD camera with 1600 × 1200 pixels from Sony. Three different zones were investigated: the crystallization and fusion zones, namely, the area enclosed by the glass-transition temperature (Tg′), the melting temperature (Tm), and the area above the Tg′ values.

Table 2. Experimental Results Measured in Our Laboratory by DSCa

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RESULTS AND DISCUSSION PVP + Water Binary System Phase Diagram. As already emphasized, the phase diagram is the key data in the freezedrying cycle optimization because it allows us to fix the optimal processing temperatures for a given formulation. In the case of complex mixtures, this setup based on the phase diagram can be quite difficult so that it could be necessary to determine a binary reference system. In our case, we chose to study first the binary PVP/water system because this biocompatible polymer presents some interesting properties for drug and pharmaceutical freeze-drying processes. It has already been currently used in a variety of pharmaceutical formulations as a stabilizing agent, suspending agent and bioprotectant.

a

wPVP (% wt)

Tg (°C)

Tm (°C)

4.99 10.00 15.01 19.98 24.99 30.00 34.81 39.91 44.96 47.88 49.06 49.86 50.80 51.52 51.73 52.85 53.94 54.82 58.01 58.50 59.50 59.47 59.89 65.17 69.47

−22.6 −22.5 −22.6 −21.9 −22.1 −22.1 −23.6 −26.6 −25.1 −61.6 −62.1 −58.1 −59.3 −55.6 −54.3 −48.8 −49.0 −47.3 −40.3 −41.1 −43.1 −36.9 −32.8 −20.5 −5.5

−0.8 −0.8 −1.1 −1.2 −1.5 −2.3 −3.2 −4.3 −6.7 −8.9 −8.6 −10.2 −9.2 −11.4 −11.6 −11.5 −12.5 −12.3 −15.2 −15.9 −14.8

Standard uncertainties (u) are ur(wPVP) = 0.001, u(T) = 0.5 K.

Figure 2 and Table 2 represent the diagram of the physical states of the PVP/water system. The phase diagram of the system consists of the vitreous transition-temperature curve and the melting curve (liquid), which define the domain of the system states encountered during the freeze−drying processes. As described by different authors,4,23,24 a typical freezing cycle of a formulation containing sugar or polymer usually follows the path M → N → O → P → Q. C



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Figure 3. MDSC curve of the system PVP/water (50 wt % PVP) showing the crystallization event during heating.

Figure 4. Cryomicrographs of solutions of 30% (wt) PVP (panels a, c, and e) and 50% (wt) PVP (panels b, d, and f) at different temperatures noted at the top of each photograph. The arrow pointing diagonally down indicates a cooling scan, and the arrow pointing diagonally up indicates a heating scan.

D



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Figure 5. Effect of lactose concentration on the heat of fusion of water in the PVP + water mixture.

The mass concentration of the maximally cryo-concentrated phase was obtained at the intersection between the initial freezing (liquidus) and the vitreous transition-temperature curves. From our experiments, this concentration, noted as wg′, was estimated to 64% (wt), and by extrapolation, the Tg′ value was found to be equal to −24 °C. These data are in good agreement with the literature data of Furushima et al.22 and Levine et al.25 even if the comparison with these literature data could be difficult because the molecular weights of the polymer (PVP) in the two studies are different. In Figure 2, for a concentration lower than 49%, we observe the three states of the material. In region I, a mixture of ice crystals and a cryo-concentrated solution are in an amorphous state. In region II, the system is a mixture of ice crystals and unfreezable solution. It passes to a liquid state after melting in region III. For a concentration above 49% (wt) and below 64% (wt), there was no formation of ice crystals during cooling. This was probably due to the high viscosity of PVP. Indeed, the water molecules could be trapped in small clusters in the polymer network, which did not allow the nucleation and, consequently, the crystallization of water. However, during the heating period, even if the temperature was low, the motion of the polymer chains could probably be restricted to a vibrational mode (cf. Section 3.2). Then, the devitrification of the polymer occurred, and the water molecules were released and their crystallization was then observed (Figure 3). From an economical point of view, it is important to point out the interest in annealing after the freezing step because this heat treatment generally leads to an increase in the glass-transition temperature, which allows the glass transition of cryoconcentrated solution to be reached quickly. Region IV is characteristic of a rubbery state that does not freeze and probably reaches the typical viscosity of the glassy state (1012 Pa s) according to Roos.26 Isothermal annealing treatment is usually used to observe time-dependent ice formation.27,28 Region V corresponds to a solid amorphous (vitreous) state. Cryomicroscopy Studies of the PVP + Water Binary System. The samples with 30% (wt) and 50% (wt) PVP have been chosen to carry out cryomicroscopic observations of the physical states of the PVP + water binary system. Up to −16 °C, we observed for the two PVP weight fractions a liquid state

Table 3. Experimental Heat of Fusion Measured in Our Laboratory by DSCa wPVP (wt)

wW (wt)

wL (wt)

ΔHm (J·g−1)

0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45

0.95 0.90 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.90 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 0.40

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15

346.20 280.30 205.00 185.00 146.70 118.90 96.21 70.03 50.36 253.00 217.00 188.85 144.50 127.65 99.79 82.16 66.48 43.38 211.70 172.10 159.20 145.07 122.00 87.02 75.88 55.68 24.58 194.30 165.55 146.80 130.40 85.98 75.02 56.01 39.27 17.85

a

Standard uncertainties (u) are ur(wPVP) = 0.001 and u(ΔHm) = 0.05 J·g−1.

E



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Figure 6. Effect of the amount and type of solute (PVP + lactose) on the freezable water fraction.

Figure 7. Effect of the lactose concentration on the glass-transition temperature of the PVP + water mixture.

PVP (Figure 4e). This observation confirmed the recrystallization phenomena also observed by DSC in region IV of the state diagram (Figures 2 and 3). Thus, these cryomicroscopic investigations of the PVP + water binary system reasonably confirmed the conclusions obtained on the basis of DSC data analysis concerning the physical states of this system. Ternary System (PVP + Lactose + Water): Influence of Lactose on Water Crystallization. For a ternary solution of PVP + lactose + water, the phase diagram may present slight differences from the binary solution because the vitreous phase could contain the three compounds. To understand the

(Figure 4a,b), represented by region III on the state diagram (Figure 2). The crystallization temperature of the 30% (wt) PVP solution was observed at −18.8 °C (Figure 4c) with the propagation of a freezing front (region II of the state diagram in Figure 2). These observations were in reasonable agreement with our DSC data. The optical density of the 50% (wt) PVP sample did not change (Figure 4b,d) while the sample cooled to −102 °C, which is characteristic of vitrification phenomena according to Zhivotova et al.29 However, the nucleation of ice crystals was observed during warming to −50.3 °C (Figure 4f) for the 50% (wt) PVP sample, and nothing was observed for the 30% (wt) F



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initial amount of water and the freezable amount of water after crystallization. Next, the data of the different water fractions gathered in Figure 6 lead to the following original conclusions: (1) The increase in the quantity of the dry matter (lactose + PVP) led to an important decrease in the amount of freezable water. For the 10% (wt) dry matter samples, 88% (wt) (on average) of the total water has frozen whereas only 35% (wt) on average has frozen in the case of the 45% (wt) dry matter sample. (2) The increase in the quantity of the dry matter (lactose + PVP) led to an important decrease in the amount of freezable water. For the 10% (wt) dry matter samples, 88% (wt) (on average) of the total water has frozen whereas only 35% (wt) on average has frozen in the case of the 45% (wt) dry matter sample. (3) The influence of polymer (PVP) on the unfreezable water content was more important than the influence of lactose. (4) At a constant dry matter upper concentration of 15% (wt), increasing the amount of PVP (or decreasing the amount of lactose) led to an increase in the amount of unfreezable water. This amount of water will contribute to the modification of the glass transition.31 Indeed, as mentioned by Drake et al.,32 one of the important requirements of freeze-drying in different systems is the ability to form a glass phase. Thus, this important point has to be taken into consideration in order to propose a formulation mixture that produces a sufficient amount of vitreous phase. In our case, using low quantities of both solutes could be an efficient way to form an adequate glassy phase to obtain the convenient quality attributes of the final freeze-dried product. (5) At a constant dry matter upper concentration of 15% (wt), increasing the amount of PVP (or decreasing the amount of lactose) led to an increase in the amount of unfreezable water. This amount of water will contribute to the modification of the glass transition.31 Indeed, as mentioned by Drake et al.,32 one of the important requirements of freeze-drying in different systems is the ability to form a glass phase. Thus, this important point has to be taken into consideration in order to propose a formulation mixture that produces a sufficient amount of vitreous phase. In our case, using small quantities of both solutes could be an efficient way to form an adequate glassy phase to obtain the convenient quality attributes of the final freeze-dried product. Ternary System (PVP + Lactose + Water): Influence of Lactose on Tg. Knowledge of the glass-transition temperature is necessary to define the most interesting storage temperature. As the PVP was chosen because of its high Tg values (162 °C)33 due to its high molecular weight, on the contrary, lactose

Table 4. Experimental Glass-Transition Temperatures Measured in Our Laboratory by DSCa wPVP (wt)

wW (wt)

wL (wt)

Tg (°C)

0.05 0.1 0.15 0.2 0.25 0.05 0.1 0.15 0.2 0.25 0.05 0.1 0.15 0.2 0.25 0.05 0.1 0.15 0.2 0.25

0.95 0.90 0.85 0.80 0.75 0.90 0.85 0.80 0.75 0.70 0.85 0.80 0.75 0.70 0.65 0.80 0.75 0.70 0.65 0.60

0.00 0.00 0.00 0.00 0.00 0.05 0.05 0.05 0.05 0.05 0.10 0.10 0.10 0.10 0.10 0.15 0.15 0.15 0.15 0.15

−22.6 −22.6 −22.6 −21.9 −22.1 −24.9 −25.1 −24.8 −24.2 −24.1 −25.6 −25.9 −25.4 −25.1 −25.1 −26.4 −26.3 −26.0 −25.6 −26.6

a

Standard uncertainties (u) are ur(wPVP) = 0.001 and u(T) = 0.5 K.

influence of the lactose in the aqueous solution of PVP, we analyzed by DSC different samples with three different lactose concentrations, namely, 5% (wt), 10% (wt), and 15% (wt). The melting temperatures, the glass-transition temperatures, and the enthalpy of fusion values were extracted from DSC thermograms. The enthalpies of fusion of the different solutions are presented herewith in Figure 5 and Table 3. As expected, generally an increase in the solute concentrations in the system led to a reduced fraction of ice formation as described by Singh and Roos.30 However, at the same total solute concentration, it is difficult to see the influence of the type of solute (lactose or PVP) on the amount of frozen water. That is why we studied the amounts of freezable water and liquid water remaining in the vitreous phase. These quantities were calculated with eq 2, where the enthalpy calculated from the integration of the area of the melting pick ΔHm was reported to be the enthalpy of melting of pure ice (ΔHice = 333.5 J·g−1),

wfw =

ΔHm ΔHice

(2)

The unfreezable water is the fraction of water that does not crystallize during freezing. It is the difference between the

Table 5. Fitting Polynomial Coefficients of Melting Temperatures (°C) from Equation 3 0% lactose A0 A1 A2 A3 A4 A5 A6 R2

−6.448 −9.628 −2.771 3.282 −1.560 3.259 −2.542 0.993

× × × × × × ×

10−3 10−1 102 103 104 104 104

5% lactose −7.203 −1.485 3.353 −3.540 1.605 −3.333 2.540 0.997

× × × × × × ×

10−1 101 102 103 104 104 104

G

10% lactose −1.166 2.813 −2.787 2.920 −1.341 2.741 −2.131 0.998

× × × × ×

102 103 104 104 104

15% lactose −1.812 4.523 −3.749 4.235 −2.120 4.681 −3.833 0.999

× × × × ×

102 103 104 104 104



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Figure 8. Solid/liquid phase diagram of the PVP/water/lactose system (in weight fraction). Colored solid lines show the melting temperature in °C. The blue points represent the concentrations of samples studied in our laboratory. (The red point is just an indication to help in the comprehension of the diagram: its composition is 20% (wt) PVP, 15% (wt) lactose, and 65% (wt) water. The melting temperature of this sample is between −3 °C and −4 °C.)

had a lower Tg (101 °C),34 which may influence the mixture properties in an unfavorable way. That is why we investigated the effect of the lactose concentration on the glass-transition temperatures of the frozen ternary system. Figure 7 and Table 4 represent the Tg values obtained from the MDSC data. Yellow rounds represent the Tg values for the water + PVP binary system between 0 and 25% weight concentrations of PVP, blue crosses represent the influence of an additional 5% (wt) lactose as a function of the PVP concentration, orange triangles represent the influence of 10% (wt) lactose, and gray squares represent the influence of 15% (wt) lactose. Thus, as expected, these data indicated that the addition of lactose decreased the Tg values of the system so that lactose played a plasticizer role. In fact, the hydroxyl polar groups of the lactose could establish hydrogen bonds by replacing the polymer− polymer interactions. However, these observed Tg values did not decrease proportionally with the concentration of lactose and seemed to tend toward a limit. These data show that the product temperature during freeze-drying needs to be fixed in the range between −22 and −30 °C for the investigated formulations. We should be precise that in Figure 7 the glass-transition temperature, Tg, has not been reported for concentrations higher than 25% (wt). Indeed, at these important concentrations, the DSC profiles are not very clear and the system probably has two glass transitions temperatures, Tg and Tg′, as mentioned in Nakagawa et al.,35 but the values are too close to be precisely assigned to Tg or Tg′.

Melting Temperature of a Ternary System (PVP/ Lactose/Water). The experimental melting temperatures of the ternary system PVP/lactose/water were determined from the DSC thermogram data obtained with the method described in the Section 2.2. We have fitted the melting-temperature values as a function of PVP concentration while holding the lactose concentration constant with the following polynomial equation (eq 3) Tm = A 0 + A1wPVP + A 2 wPVP 2 + A3wPVP3 + A4 wPVP 4 + A5wPVP5 + A 6wPVP6

(3)

where Tm is the melting temperature in °C and wPVP is the weight fraction of PVP. Coefficient values for the fitting and standard deviation of the correlation are reported in Table 5. The correlations are valid for a PVP concentration range of [0−45 wt %]. Equation 3 was solved at each respective lactose concentration at a specific temperature (272 K, 270 K...) to determine the PVP concentration and thus to build an isotherm on the ternary phase diagram. Figure 8 shows the water/PVP/lactose phase diagram with the calculated isotherms (colored lines in the figure with corresponding temperatures indicated on the plot). To set up this diagram, we studied 43 samples at different concentrations represented by the blue circles in Figure 8 (with values presented in Table 6). Nevertheless, it was not possible to investigate the region of high PVP and lactose concentrations because the solutes were quite difficult to dissolve in the water in this domain. H



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involved are not still completely understood, but these new experimental results allows us to fix the critical temperatures of the mixtures to optimize freeze-drying cycles of fragile and thermosensitive formulations with microorganisms. Nevertheless, these types of experiments have to be carried out with different formulations because each of them have different thermodynamic properties (vitreous transition temperatures) and physicochemical properties (states of water). Because of our large and new experimental investigations, we concluded that the mixtures based on PVP and lactose offer an adequate protection and stabilization effect for the freeze-drying of fragile bacterial formulations such as probiotics formulations.36 In future work, it could be interesting to extend these investigations by mixing PVP with other crystalline disaccharides such as mannitol.

Table 6. Experimental Melting Temperatures Measured in Our Laboratory by DSCa wPVP (wt)

WW (wt)

WL (wt)

Tm (°C)

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0 0.05 0.05

1.00 0.95 0.90 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.95 0.90 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.90 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 0.40 0.7 0.7 0.65

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.3 0.25 0.3

0 −0.5 −0.8 −1.1 −1.2 −1.5 −2.3 −3.2 −4.3 −6.7 −0.7 −0.9 −1.2 −1.4 −1.9 −2.4 −3.1 −3.9 −5.9 −7.9 −1.1 −1.5 −1.7 −2.2 −2.6 −2.9 −4.2 −5.6 −7.6 −11.3 −1.8 −2.2 −2.5 −2.9 −3.5 −4.4 −5.6 −7.5 −9.2 −13.6 −2.5 −3.4 −3.9

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Claudia Cogné: 0000-0002-5545-884X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the Ingelyse Federation for the financial support received during this work to improve the data acquisition system of the cryomicroscope.

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NOMENCLATURE Ai fitting parameters m mass, g ΔHm melting enthalpy, J·g−1 Tg glass-transition temperature, °C Tm melting temperature, °C w weight fraction

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SUBSCRIPTS fw freezable water L lactose PVP poly(vinylpyrrolidone) unfw unfreezable water W water

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a

Standard uncertainties (u) are ur(wPVP) = 0.001 and u(T) = 0.5 K.

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REFERENCES

(1) Nail, S. L.; Jiang, S.; Chongprasert, S.; Knopp, S. A. Fundamentals of Freeze-Drying. Pharm. Biotechnol. 2002, 14, 281− 360. (2) Patel, S. M.; Pikal, M. J. Emerging Freeze-Drying Process Development and Scale-up Issues. AAPS PharmSciTech 2011, 12, 372−378. (3) Barbosa, J.; Borges, S.; Amorim, M.; Pereira, M. J.; Oliveira, A.; Pintado, M. E.; Teixeira, P. Comparison of Spray Drying, Freeze Drying and Convective Hot Air Drying for the Production of a Probiotic Orange Powder. J. Funct. Foods 2015, 17, 340−351. (4) Andrieu, J.; Vessot, S. A Review on Experimental Determination and Optimization of Physical Quality Factors during Pharmaceuticals Freeze-Drying Cycles. Drying Technol. 2018, 36, 129−145. (5) Broeckx, G.; Vandenheuvel, D.; Claes, I. J. J.; Lebeer, S.; Kiekens, F. Drying Techniques of Probiotic Bacteria as an Important Step towards the Development of Novel Pharmabiotics. Int. J. Pharm. 2016, 505, 303−318.

CONCLUSIONS In this work, we were able to highlight the influence of the lactose concentrations in frozen PVP + water solutions as concerns the vitreous phase domain when cooling the system to below 0 °C. Crystallization during cooling does not operate at high PVP concentration as a result of high viscosity in the cryoconcentrated phase; nevertheless, ice recrystallization during warming is observed. The effects of PVP and lactose on the water states are quantified. The melting temperature of the ternary system (PVP/water/lactose) was also found to be significantly affected by the composition, and the depression of Tm is a function of increasing solids concentration. These excipients are bioprotectants which led to amorphous state diagrams. All of the different mechanisms and phenomena I



Article

(6) Celik, O. F.; O’Sullivan, D. J. Factors Influencing the Stability of Freeze-Dried Stress-Resilient and Stress-Sensitive Strains of Bifidobacteria. J. Dairy Sci. 2013, 96, 3506−3516. (7) Rijkers, G. T.; Bengmark, S.; Enck, P.; Haller, D.; Herz, U.; Kalliomaki, M.; Kudo, S.; Lenoir-Wijnkoop, I.; Mercenier, A.; Myllyluoma, E.; Rabot, S.; Rafter, J.; Szajewska, H.; Watzl, B.; Wells, J.; Wolvers, D.; Antoine, J. M. Guidance for Substantiating the Evidence for Beneficial Effects of Probiotics: Current Status and Recommendations for Future Research. J. Nutr. 2010, 140, 671S−6S. (8) Broadbent, J. R.; Lin, C. Effect of Heat Shock or Cold Shock Treatment on the Resistance of Lactococcus Lactis to Freezing and Lyophilization. Cryobiology 1999, 39, 88−102. (9) Crowe, J. H.; Carpenter, J. F.; Crowe, L. M.; Anchordoguy, T. J. Are Freezing and Dehydration Similar Stress Vectors? A Comparison of Modes of Interaction of Stabilizing Solutes with Biomolecules. Cryobiology 1990, 27, 219−231. (10) Dumont, F. Etude de La Cinétique et de L’amplitude de Stress Thermique Froid Sur Différents Modèles Cellulaires: Influence de La Cristallisation Sur La Viabilité Cellulaire. Ph.D. Thesis, Université de Dijon, 2005. (11) Simonin, H.; Winckler, P.; Gros, A.; Perrier-Cornet, J. M.; Gervais, P. How Dehydration with Glycerol Protects Bacteria Cell Membranes against Deleterious Effects of Supercooling. Cryobiology 2015, 71, 549. (12) Jofré, A.; Aymerich, T.; Garriga, M. Impact of Different Cryoprotectants on the Survival of Freeze-Dried Lactobacillus Rhamnosus and Lactobacillus Casei/paracasei during Long-Term Storage. Benefic. Microbes 2015, 6, 381−386. (13) Franks, F.; Asquith, M. H.; Hammond, C. C.; Skaer, H. B.; Echlin, P. Polymeric Cryoproteetants in the Preservation of Biological Ultrastructure. J. Microsc. 1977, 110, 223−238. (14) Champagne, C. P.; Mondou, F.; Raymond, Y.; Roy, D. Effect of Polymers and Storage Temperature on the Stability of Freeze-Dried Lactic Acid Bacteria. Food Res. Int. 1996, 29, 555−562. (15) Meryman, H. T. Cryoprotective Agents. Cryobiology 1971, 8, 173−183. (16) Reddy, K. B.; Awasthi, S. P.; Madhu, A. N.; Prapulla, S. G. Role of Cryoprotectants on the Viability and Functional Properties of Probiotic Lactic Acid Bacteria during Freeze Drying. Food Biotechnol. 2009, 23, 243−265. (17) Nag, A.; Das, S. Effect of Trehalose and Lactose as Cryoprotectant during Freeze-Drying, in Vitro Gastro-Intestinal Transit and Survival of Microencapsulated Freeze-Dried Lactobacillus Casei 431 Cells. Int. J. Dairy Technol. 2013, 66, 162−169. (18) Fan, F.; Roos, Y. H. X-Ray Diffraction Analysis of Lactose Crystallization in Freeze-Dried Lactose−whey Protein Systems. Food Res. Int. 2015, 67, 1−11. (19) Damjanovic, V.; Thomas, D. The Use of Polyvinylpyrrolidone as a Cryoprotectant in the Freezing of Human Lymphocytes. Cryobiology 1974, 11, 312−316. (20) Thirumala, S.; Wu, X.; Gimble, J. M.; Devireddy, R. V. Evaluation of Polyvinylpyrrolidone as a Cryoprotectant for Adipose Tissue-Derived Adult Stem Cells. Tissue Eng., Part C 2010, 16, 783− 792. (21) Santivarangkna, C.; Aschenbrenner, M.; Kulozik, U.; Foerst, P. Role of Glassy State on Stabilities of Freeze-Dried Probiotics. J. Food Sci. 2011, 76, R152−R156. (22) Furushima, Y.; Ishikiriyama, K.; Ueno, Y.; Sugaya, H. Analysis of the State of Water in Polyvinylpyrrolidone Aqueous Solutions Using DSC Method. Thermochim. Acta 2012, 538, 43−47. (23) Amorij, J. P.; Huckriede, A.; Wilschut, J.; Frijlink, H. W.; Hinrichs, W. L. J. Development of Stable Influenza Vaccine Powder Formulations: Challenges and Possibilities. Pharm. Res. 2008, 25, 1256−1273. (24) He, X. Methodological Advances in the Culture, Manipulation and Utilization of Embryonic Stem Cells for Basic and Practical Applications; InTech: Atwood, 2011.

(25) Levine, H.; Slade, L. A Polymer Physico-Chemical Approach to the Study of Commercial Starch Hydrolysis Products (SHPs). Carbohydr. Polym. 1986, 6, 213−244. (26) Roos, Y.; Karel, M. Amorphous State and Delayed Ice Formation in Sucrose Solutions. Int. J. Food Sci. Technol. 1991, 26, 553−566. (27) Roos, Y. Characterization of Food Polymers Using State Diagrams. J. Food Eng. 1995, 24, 339−360. (28) Listiohadi, Y.; Hourigan, J. A.; Sleigh, R. W.; Steele, R. J. Thermal Analysis of Amorphous Lactose and α-Lactose Monohydrate. Dairy Sci. Technol. 2009, 89, 43−67. (29) Zhivotova, E. N.; Zinchenko, A. V.; Kuleshova, L. G.; Chekanova, V. V.; Kompaniets, A. M. Physical States of Aqueous Solutions of Oxyethylated Glycerol with Polymerization Degree of n = 30 at Temperatures Lower than 283 K. CryoLetters 2007, 28, 261− 270. (30) Singh, K. J.; Roos, Y. H. Physical State Study of (Sugar Mixture)-Polymer Model Systems. Int. J. Food Prop. 2010, 13, 184− 197. (31) Ogawa, S.; Osanai, S. Glass Transition Behavior of Aqueous Solution of Sugar-Based Surfactants Supercooling; InTech: Wilson, 2012. (32) Drake, A. C.; Lee, Y.; Burgess, E. M.; Karlsson, J. O. M.; Eroglu, A.; Higgins, A. Z. Effect of Water Content on the Glass Transition Temperature of Mixtures of Sugars, Polymers, and Penetrating Cryoprotectants in Physiological Buffer. PLoS One 2018, 13, e0190713. (33) Chokshi, R. J.; Shah, N. H.; Sandhu, H. K.; Malick, A. W.; Zia, H. Stabilization of Low Glass Transition Temperature Indomethacin Formulations: Impact of Polymer-Type and Its Concentration. J. Pharm. Sci. 2008, 97, 2286−2298. (34) Lloyd, R. J.; Chen, X. D.; Hargreaves, J. B. Glass Transition and Caking of Spray-Dried Lactose. Int. J. Food Sci. Technol. 1996, 31, 305−311. (35) Nakagawa, K.; Tamiya, S.; Do, G.; Kono, S.; Ochiai, T. Observation of glassy state relaxation during annealing of frozen sugar solutions by X-ray computed tomography. Eur. J. Pharm. Biopharm. 2018, 127, 279−287. (36) Verlhac, P.; Vessot, S.; Degobert, G.; Cogné, C.; Andrieu, J.; Beney, L.; Gervais, P. Study and Optimization of Freeze-Drying Cycles of a Model Probiotic Strain; 21st International Drying Symposium, Valencia, Spain, September 11−14, 2018.

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