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Freeze drying of nanosuspensions, 2: the role of the critical formulation temperature on stability of drug nanosuspensions and its practical implication on process design
Freeze Drying of Nanosuspensions, 2: the Role of the Critical Formulation Temperature on Stability of Drug Nanosuspensions and Its Practical Implication on Process Design JAKOB BEIROWSKI,1 SABINE INGHELBRECHT,2 ALBERTINA ARIEN,2 HENNING GIESELER1 1
University of Erlangen, Division of Pharmaceutics, Freeze Drying Focus Group, 91058 Erlangen, Germany
2 Pharmaceutical Development & Manufacturing Sciences, Janssen Research & Development, A Division of Janssen Pharmaceutical NV, 2340 Beerse, Belgium
Received 14 January 2011; revised 28 February 2011; accepted 5 May 2011 Published online 23 May 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22634 ABSTRACT: The present study investigates whether controlling the product temperature below the critical formulation temperature (CFT) during primary drying in a freeze drying cycle is a prerequisite for the stabilization of drug nanoparticles. For that purpose, the CFT of four drug nanosuspensions stabilized with different types (amorphous and crystalline) and concentrations of steric stabilizers and either of the disaccharides, trehalose and sucrose, was determined by differential scanning calorimetry and freeze-dry microscopy. Freeze-drying experiments were performed such that product temperatures during primary drying remained either below or well above the CFT of individual mixtures. It was found that glass formation did not influence the stability of the nanoparticles, suggesting that an adequate type of steric stabilizer and lyoprotectant concentration is present. Freeze drying could also be performed above the eutectic temperature without compromising on the final product quality profile, such as nanoparticle size and structural preservation of the lyophilized cake. The high concentration of solid drug nanoparticles provided additional cake stability. The results of this study confirm for the first time that primary drying for drug nanosuspensions can be greatly shortened because induced viscous flow or even meltback is not a limitation for nanoparticle stability and cake elegancy. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4471–4481, 2011 Keywords: freeze drying; lyophilization; nanosuspensions; nanotechnology; vitrification; glass transition; collapse temperature; compounding; stabilization
INTRODUCTION It is well known that the underlying concept for the stabilization of proteins during freeze drying is glass formation by excipients. Such excipients are denoted as “cryoprotectants” in the event that they stabilize a protein structure primarily during freezing, and “lyoprotectants” in the event that stabilization can be achieved during the dehydration step.1–4 It has been reported that many excipients can serve as both cryo- and lyoprotectants. The term “vitrification” is defined as the transformation of an aqueous solution into a rigid, solid glass. Vitrification would be assumed when the product temperature of a formulaCorrespondence to: Henning Gieseler (Telephone: +49-91318529556; Fax: +49-9131-8529545; E-mail: gieseler@freeze-drying. eu) Journal of Pharmaceutical Sciences, Vol. 100, 4471–4481 (2011) © 2011 Wiley-Liss, Inc. and the American Pharmacists Association
tion is lower than the glass transition temperature of the freeze-concentrated solute, T g .2 Numerous studies have been presented addressing protein stability as a function of vitrification, and the validity of this rule has initially also been proposed for the stabilization of nanoparticulate formulations.5,6 Agglomeration of suspended nanoparticles is prevented by formation of a solid amorphous glass, where the colloidal particles are arrested in an infinite high viscous environment and are isolated from each other.7 However, even if glass formation of a stabilizer is the preferred mechanism to preserve the original particle size distribution (PSD), some questions still need to be addressed. Allison et al.8 suggested that the separation of individual particles within the unfrozen fraction prevents aggregation during the freezing step, and this idea was called the particle isolation hypothesis. In this hypothesis, the relatively low surface tension of mono- and disaccharides plays an integral role
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that, in turn, allows phase-separated particles to remain dispersed within the unfrozen solute. In addition, Armstrong and Anchordoquy9 modeled diffusion of particulate nonviral vectors in the frozen matrix and argued that these formulations could be dried above T g as long as aggregation was avoided. Drug nanoparticles are also referred to as particulate colloids, but they are clearly different in their structure than, for example, liposomal systems. It seems, therefore, of great interest to study whether drying of such systems above T g without negative impact on product stability attributes would be possible as well. Drug nanoparticles are commonly stabilized using a steric stabilizing mechanism. The relevance of this class of stabilizers in combination with the frequently used disaccharides sucrose and trehalose (both are known to serve as lyoprotectants during primary drying) is the goal of the present study. Importantly, it is not clear until today how crystalline steric stabilizers and amorphous lyoprotectants will affect each other in terms of stabilization capacity during freeze drying because principally, glass formation is considered to provide stabilization for amorphous structures.10 In the event that T g does not directly limit the product temperature profile during primary drying, a subsequent question would target the maximum allowable product temperature that still assures final product stability. Freeze-dry microscopy (FDM) has been used in the course of the present investigations to determine the (onset) collapse temperature (Toc ) of the mixtures. It is well known that T g and Toc are not identical for many drug formulations due to the different measurement principle.11 As a rule of thumb, Toc is found about 2◦ C–5◦ C higher than the corresponding T g . It has already been demonstrated that Toc is more indicative for the critical formulation temperature (CFT) than the corresponding glass transition.11 Maintaining the product temperature above Toc induces changes in the inner cake morphology due to viscous flow, which is denoted as either “microcollapse,” “shrinkage,” or “collapse”. Such structural changes are demonstrated to affect the target quality aspects of the final product (e.g., appearance, reconstitution time, etc.).12 However, although recent FDM reports illustrated that Toc is, among other things, a function of total solid content of the formulation,11 it is also of great interest to address the question that at which solid nanoparticle concentration level additional cake stability is provided. Another aspect that deserves consideration in the present discussion is the introduction of crystallizing materials (i.e., steric stabilizers). Assuming that nanoparticle stability is not a function of immobilization in the excipient matrix, freeze drying at or above the eutectic temperature (Teut ) might be feasible as well. As mentioned above, high concentrations of solid drug nanoparticles might also compensate for JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
meltback. PSD is not automatically expected to be influenced because the steric barrier surrounding the particles can avoid physical instabilities in a highly mobile environment. It has already been pointed out in the literature that the key role of the steric stabilizer is to impart the initial stability in the liquid phase right after milling.13,14 The concept of steric stabilization is possible for those materials that can be attached or adsorbed onto the nanoparticle surface and provide a large and dense steric barrier, which is mandatory to overcome the attractive van der Waals forces (e.g., polyethylene glycol, cellulosics, pluronics, polysorbates, etc.).13–15 In addition, previous studies have already depicted that the selection of an adequate steric stabilizer is important to preserve the original PSD. This observation might then mitigate the relevance of vitrification.16–18 An elevated mobility in the excipient matrix must not inevitably lead to aggregation or particle fusion during lyophilization. Besides the science-related aspect of this study, another practical consideration is grounded in process economics. The possibility to process drug nanosuspensions well above their CFT allows rather unconventional, aggressive cycle conditions, which could significantly decrease freeze-drying process times and therefore, turnover.19 Using drug nanoparticles as a model system might offer an opportunity to learn whether other nanopartiulate formulations, for example, nanospheres or nanocapsules, can be processed above T g as well. To get down to the essence of the matter experimentally, four drug nanosuspensions stabilized with two different steric stabilizers were freeze dried conservatively, moderately, or aggressively in the presence of two different commonly used lyoprotectants, namely, sucrose and trehalose. The original suspension of the nanoparticles right after milling was found unstable in terms of complete preservation of the original PSD during freeze thawing experiments. To investigate the influence of the steric stabilizer and disaccharide concentration, both excipients were added in varying concentrations.
MATERIAL A poorly water-soluble, crystalline active pharmaceutical ingredient (API) and 0.3-mm yttrium-stabilized zirconia beads were kindly provided by Johnson & Johnson Pharmaceutical Research & Development (Beerse, Belgium). Lutrol F108 Prill (Poloxamer 338) and Cremophor EL were donated by BASF (Ludwigshafen, Germany). Trehalose and sucrose were of analytical grade and were purchased from Sigma (Sigma Chemical Company, Munich, Germany). All excipients were used as received. Either water for injection (B.Braun Melsungen AG, Melsungen, Germany) or water distilled from an allglass apparatus was used throughout this study. As DOI 10.1002/jps
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primary packing material for freeze drying, 15R Vials from Lutz GmbH (Wertheim, Germany) and 20-mm R freeze drying stoppers from West PharmaFlurotec ceutical Services, Inc. (Lionville, Pennsylvania) were used.
METHODS Production of Nanosuspensions Four nanosuspensions stabilized with either 20 or 33 mg/mL of Poloxamer 338, or 33 or 50 mg/mL Cremophor EL were prepared by wet bead milling in R , Selb, a high shear media mill (Netzsch MiniCer Germany) using 0.3-mm yttrium-stabilized zirconia beads. Note that water was used as dispersion medium and milling time was adapted to achieve the desired nanoparticle size range. Because these surfactants are nonionic, their mechanism of stabilization is considered steric stabilization. API concentration was kept constant at 100 mg/mL in the stock nanosuspensions. The stock nanosuspensions were used as received, without any protectants, or mixed with 25, 50, or 75 mg/mL trehalose or sucrose. Particle Size Measurements To investigate the PSD after milling and freeze drying over a wide particle size range, laser diffraction analR ysis was performed on a Malvern MasterSizer (Malvern Instruments GmbH, Herrenberg, Germany), which is capable of detecting even larger particle aggregates. Dependent on the steric stabilizer present in the formulation, an aqueous solution of 5 mg/mL Poloxamer 338 or Cremophor EL were used as a medium because particles were not sufficiently stable in pure water during the analysis. Basis for particle size calculation was the Mie theory, with a product refractive index of 1.65 and product absorption of 0.001 (dimensionless parameter). The obtained fitting curve achieved by the MasterSizer 2000 software indicated the validity of the optical model. Background and measurement integration time were kept at 60 s and three measurement runs per sample were performed. Freeze Drying: Experimental Design Each formulation mixture (2 mL) was filled in 15R vials (0.52 cm fill depth). Lyophilization was performed on a laboratory scale freeze dryer (VirTis Advantage Plus, SP Scientific, Gardiner, NY, USA). Vials were loaded using a stainless steel tray and the bottom of the tray was removed to allow a direct placement of the vials on the shelf surface during the cycle. Product temperatures were measured using calibrated 30 gauge T-Type copper/constantan thermocouples from Omega (Omega Engineering, Stamford, Connecticut). Each thermocouple was DOI 10.1002/jps
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introduced through a stopper and positioned at the bottom center of the vial to achieve both representative temperature monitoring as well as accurate endpoint detection of the time point when no ice is left in the product. The applied cycle conditions were as follows: Freezing was performed at –40◦ C (shelf inlet temperature) for 60 min, including equilibrating steps at +5◦ C and–5◦ C for 15 min. The shelf temperature ramp rates from the freezing step in to the primary drying step were 1◦ C/min for all cycles performed. Three different primary drying conditions were employed, further denoted as “conservative” run (shelf inlet temperature: –35◦ C), “moderate” run (shelf inlet temperature: 0◦ C), and “highly aggressive” run (shelf inlet temperature: +40◦ C, 8 h). The shelf heating rate from the primary drying shelf setpoint to the secondary drying setpoint at +40◦ C was 0.15◦ C/min for the “conservative” and “moderate” cycles. Secondary drying for these cycles were performed over 360 min. The chamber pressure during primary and secondary drying was controlled at 75 mTorr throughout all experiments. Differential Scanning Calorimetry (DSC) Glass transitions of the maximally freeze concentrated solute (T g ) and other thermal events were determined using a Mettler DSC822e (Mettler-Toledo, Greifensee, Switzerland). Data acquisition for the liquid formulations was performed in temperature ranging from–80◦ C to 0◦ C, applying cooling rates of 20◦ C/ min and heating rates of 5◦ C/min. For those formulations where Poloxamer 338 was present, slow cooling rates were used (1◦ C/min) because these block polymers are known to remain semicrystalline after a fast freezing regime. Thus, an annealing step was not mandatory to facilitate crystallization. Freeze-dry Microscopy Freeze-dry microscopy was performed on a freezedrying stage (FDCS-196) from Linkam Scientific (Linkam Scientific Instruments, Surrey, UK) and a Zeiss (Carl Zeiss MicroImaging GmbH, G¨ottingen, Germany) optical microscope equipped with a 1.2 MP digital camera. Approximately 2 :L of the corresponding formulations were used to detect the Toc . The sample was frozen at a rate of 1◦ C/min to −40◦ C and held for 10 min. Vacuum was initiated and the temperature was slowly increased at a 1◦ C/min heating rate up to 0◦ C. Toc was determined from images displaying the first signs of structural changes within the dried layer. Scanning Electron Microscopy (SEM) Broken pieces of the freeze-dried samples were fixed on Al stubs and subsequently gold-sputtered at 20 mA/5 kV (Hummer JR Technics, Union City, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
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CA, USA). Cake Morphology was then examined using Amray 1810 T scanning electron microscope (Amray, Rochester, NY, USA) at 20 kV with magnifications between 6× and 500×. Karl Fischer Residual Moisture Measurements The residual moisture content of the freeze-dried samples was measured using a Mitsubishi moisture meter CA-06 coulometric connected to a Mitsubishi vaporizer VA-06 (Mitsubishi Chemical Company, Tokyo, Japan). About 50 mg of the sample was weighed into a sample glass holder and then transferred into the oven unit. The sample vessel was continuously purged with dry nitrogen. Then, the sample was heated at a predefined temperature (140◦ C, setpoint) for a defined time period and the water vapour accumulated in the titration cell. Depending on the sample weight, residual moisture content was calculated in percent.
RESULTS AND DISCUSSION Physical Stability of Initial Nanosuspensions After Milling and Storage All four stock nanosuspensions (denoted as “first order formulations”) were found stable with regard to their PSD directly after the milling process. As illustrated in Figure 1, for the stock nanosuspension stabilized with Poloxamer 338, a unimodal PSD could be readily obtained. A small tailing adjacent to the unimodal distribution was observed in every single case, which indicates that a few larger particles in the 1 :m
Figure 2. Picture of a stock nanosuspension indicating settling after 6 months of storage at ambient conditions.
range are still present. The absence of particles larger than 5 :m could be confirmed, which would be a prerequisite for the formulation to comply with the specifications for a parenteral route of administration.20 Extended milling could not eliminate this tailing, but it greatly deteriorated particle size stability. On the basis of theoretical considerations, the observation of larger particles may be explained by accelerated Ostwald ripening during storage.21 It might, therefore, be expected to obtain a close to optimum unimodal PSD while stabilizing drug nanosuspensions by freeze drying. Note that the effect of Ostwald ripening became even more pronounced after 3 months of storage when the tailing increased and the particle sizes exceeded the upper 5 :m particle size limit (cf. Fig. 1). Moreover, a decrease of the unimodal shape occurred, which seems to be in good agreement with the above mentioned phenomenon of particle size growth. Besides the tailing phenomenon, storage of the nanosuspensions led to settling (cf. Fig. 2), and the observed long-term physical stability of the liquid drug nanosuspensions was found poor. To preserve the original PSD, freeze drying was used as the method of choice to improve the stability of the system.5 It should be underlined that neither the addition of trehalose nor sucrose to the drug nanosuspensions (then denoted as “second order formulation”) prior to the freeze-drying process did affect the nanoparticle stability. Observations by Thermal Analysis, Part 1: Differential Scanning Calorimetry
Figure 1. Comparison of the PSDs of the stock nanosuspension stabilized with 33 mg/mL Poloxamer 338 right after milling (solid line) and 3 months of storage at ambient conditions (dashed line). As observable from the figure, the particle growth exceeding the 5-:m boundary makes a parenteral application questionable. Note that the other formulations showed similar particle growth characteristics. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
The concept of rational freeze-drying cycle design and optimization is based on the evaluation of the CFT.22 The CFT has been defined as the product temperature during primary drying when the inner product morphology will start undergoing structural changes, comprising significant changes in specific surface area DOI 10.1002/jps
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Table 1. T g and/or Teut Values of the Formulations Stabilized with 50 mg/mL Cremophor EL or 33 mg/mL Poloxamer 338 Mixed with Either 25, 50, or 75 mg/mL of the Disaccharides Sucrose or Trehalose Steric Stabilizer Cremophor EL
Poloxamer 338
Disaccharide Concentration – 25 mg/mL trehalose 50 mg/mL trehalose 75 mg/mL trehalose 25 mg/mL sucrose 50 mg/mL sucrose 75 mg/mL sucrose – 25 mg/mL trehalose 50 mg/mL trehalose 75 mg/mL trehalose 25 mg/mL sucrose 50 mg/mL sucrose 75 mg/mL sucrose
T g
Teut
–73◦ C
– – – – – – – –16◦ C –16◦ C –16◦ C –16◦ C – – –
–41◦ C –39◦ C –36◦ C –42◦ C –39◦ C –36◦ C – –31◦ C –31◦ C –31◦ C –42◦ C –39◦ C –37◦ C
Note that for the lower concentrated Cremophor EL or Poloxamer 338 formulations, similar T g and/or Teut values were found, indicating the same trend.
of the cake structure or even (local or global) structural loss. A routinely used technology to determine the CFT is differential scanning calorimetry (DSC). The DSC measurements revealed a T g at –73◦ C for the stock nanosuspensions with Cremophor EL (cf. Table 1 and Fig. 3). This finding would suggest that the product temperature at the ice sublimation interface must be maintained below–73◦ C during primary drying to arrest the PSD, which, in turn, is impossible. The stock nanosuspensions stabilized with Poloxamer 338 as a steric stabilizer revealed several thermal transitions while using a fast freezing rate (cf. Fig. 4). Fast freezing for these mixtures even led to an artificial T g at about–65◦ C, followed by a devitrification peak indicating recrystallization of the Poloxamer 338
Figure 3. DSC thermogram of Cremophor EL indicating a glass transition temperature of the maximally freezeconcentrated solute (T g ) at –73◦ C. DOI 10.1002/jps
Figure 4. DSC thermograms revealing the physicochemical state of the Poloxamer 338 in the stock nanosuspension after fast freezing (dash dotted line) and slow freezing (dotted line). The solid line shows that sucrose inhibited the crystallization of Poloxamer 338 because no eutectic temperature is detectable.
between–50◦ C and–30◦ C. An endothermic melting signal at about–16◦ C indicated that a fraction of Poloxamer 338 remained in the crystalline state while applying a fast freezing regime. In order to facilitate crystallization of Poloxamer 338, a slow freezing rate (1◦ C/min) was used. As a result, the T g at –65◦ C disappeared and the Teut at –15◦ C remained as the only thermal transition (Fig. 3). In this specific event, Teut must be considered as CFT. Note that slow freezing or even annealing (–20◦ C, 1 h) of the stock nanosuspensions stabilized with Cremophor EL did not result in crystallization at all. To stabilize the drug nanoparticles in the glassy state, the disaccharides trehalose or sucrose were added to the stock nanosuspensions with a stepwise increase in concentration. The measured T g and/or Teut values for the corresponding mixtures are given in Table 1. Mixing of the disaccharides with the Cremophor EL stock nanosuspension led to a massive decrease of T g relative to the T g of the pure disaccharide solutions (sucrose: –32◦ C, trehalose: –29◦ C).10 As expected, the T g of pure Cremophor EL at –73◦ C disappeared and a new T g is formed between the extremes of pure Chremophor EL and the pure disaccharide solution as a function of a binary (and homogenous) mixture of the two substances. As a side note, the analytical data obtained by DSC could be confirmed by theoretical calculations using the Gordon– Taylor/Kelley–Bueche relationship (data not shown in Table 1).23 In addition, Table 1 illustrates all transitions of the nanosuspensions stabilized with Poloxamer 338 and mixed with the corresponding disaccharide. As mentioned above, it is recommendable for such mixtures to apply slow cooling rates JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
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(i.e., 1◦ C/min) during the DSC experiment to avoid the formation of an artificial T g in the low temperature region. In this event, slow freezing was not even sufficient to facilitate crystallization of Poloxamer 338 when sucrose was present. Figure 4 clearly illustrates that no Teut can be detected in the total heat flow signal, which supports the hypothesis that sucrose inhibited crystallization of Poloxamer 338. Moreover, even annealing at –20◦ C for 120 min could not initiate crystallization in this mixture. As a consequence, the measured T g values of the corresponding formulations were considered as the maximum allowable temperature during primary drying. In contrast, a distinct T g at about–31◦ C was found when trehalose was added to the Poloxamer 338 nanosuspension. Note that Poloxamer 338 showed a Teut at –16◦ C, which is comparable to the pure substance. Intuitively, the critical product temperature during primary drying for the Poloxamer 338 nanosuspension mixed with trehalose was defined at –31◦ C because physical instabilities are expected to be avoided by a complete immobilization of the nanoparticles in the solid state. Observations by Thermal Analysis, Part 2: Freeze-dry Microscopy Freeze-dry microscopy measurements were performed to assure that the product temperature profiles during primary drying are sufficiently high to initialize structural deterioration within the inner cake morphology. As mentioned above, both glass transition and collapse temperature information are routinely used for freeze-drying cycle optimization. However, the data obtained from these two analytical procedures for the CFT is not necessarily the same. Differences up to 5◦ C (and more) have been presented in the literature,12 based on the different concepts in the analytical procedure. Freeze-dry microscopy experiments revealed that determination of a collapse temperature was massively impeded by the high total solid content of the individual mixtures (Fig. 5). Removal of the polarization filter facilitated the measurements, and the progressing sublimation front during the measurement could be pinpointed. The results obtained suggest that the Toc values, if detectable, did not always correlate with the T g values. For example, the pure nanosuspension stabilized with Poloxamer 338 showed no distinct melting of the product matrix, although DSC revealed a melting event at –16◦ C. FDM of the stock nanosuspension stabilized with Cremophor EL (T g =–73◦ C) showed no abnormality during heating, but at some point, the whole structure appeared to flow, which limited the accuracy of the analysis. When a disaccharide was added to the Cremophor EL nanosuspension, the observed Toc values were slightly higher than the corresponding T g data. In contrast to this, a mixture of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
Figure 5. (a and b) FDM pictures of the drug nanosuspensions stabilized with 33 mg/mL Cremophor EL and 25 mg/ mL sucrose. Note that using the polarization filter makes measurement almost impossible (a). In contrast, the sublimation interface can be barely followed up when the filter is removed (b). Panel (c) (without polarization filter) shows the formulation containing 33 mg/mL Poloxamer 338 and 75 mg/mL trehalose underlining the deviation between DSC and FDM analytics. The bright spots (arrows) indicate rupture of the dried structure, which was first detectable at –18◦ C. In comparison, a T g for this formulation was found at –31◦ C and a Teut at –16◦ C.
the Poloxamer 338 nanosuspension with either disaccharide revealed a strong bias in the obtained results. Addition of trehalose led to a detectable onset of collapse at –18◦ C (cf. Fig. 5), which is in clear contrast to T g measurements (–31◦ C, Table 1). One may speculate that the collapse of the dried structure indeed occurred at lower temperatures, but the DOI 10.1002/jps
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high nanoparticle concentration impeded the measurement significantly enough to blur the visual detectability. A complementary investigation by scanning electron microscopy (SEM) was performed to reveal possible changes in the inner product morphology and to compensate the limitations encountered by FDM (see further below). Freeze Drying Experiments, Part 1: Conservative Cycle Conditions First experiments indicated that all formulations containing sucrose or trehalose preserved the original PSD after a freeze thaw cycle (data not shown). Stock nanosuspensions without any disaccharide were found unstable in terms of aggregation, whereas the higher concentrated Cremophor EL nanosuspension revealed only a slight shift in the PSD. An increase in the steric stabilizer concentration led to less aggregation tendency of the particles. This observation is in good agreement with a previous hypothesis that states that an increase in the steric stabilizer concentration generates a denser steric layer on top of the nanoparticle, which keeps them apart in the freeze concentrated state.24 In addition, surplus of a steric stabilizer could also act as a cryo-/ lyoprotectant when the stabilizer is not fully adsorbed onto the surface of the drug particles. In a previous report, the combination of the two factors “steric stabilizer concentration” and “cryoprotectant concentration” were revealed to influence each other during the freezing step.17,18,24 Table 2 depicts all d95 values of the formulations after a conservative freeze-drying cycle, resulting in product temperatures below the determined CFT when a lyoprotectant was present (cf. Table 1). Note that the parameter d95 states that Table 2.
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95% of the particles are equal or less in size than the reported value. The results obtained prove that an increase in the steric stabilizer concentration led to an improved nanoparticle stability, whereas an increase in the disaccharide concentration up to 75 mg/ mL correlated with a better particle size particularly for those nanosuspensions containing a higher steric stabilizer concentration. However, a smaller amount of 25 mg/mL was found beneficial for cake stability regardless of the disaccharide or the steric stabilizer used. Cake appearance suffered from crack formation while using a high disaccharide concentration. Although this is only a cosmetic defect, it seems recommendable to use as little as possible of the disaccharide in combination with a high steric stabilizer concentration if cracking of the cake must be avoided. As a general observation, trehalose was always found superior to sucrose as a protectant. Freeze Drying Experiments, Part 2: Moderate Cycle Conditions To investigate the need of immobilization of the drug nanoparticles for particle size stabilization, a second run was performed, and the shelf (inlet) temperature was elevated to 0◦ C to obtain product temperatures above T g . Data of the maximum product temperatures achieved during the primary drying phase are provided in Table 3. Note that the authors simply denote these cycle conditions as “moderate” to allow a differentiation between the sets of experiments. Product temperature (Tp ) measurements using thin wire thermocouples revealed that Tp for the nanosuspensions stabilized with the crystallizing Poloxamer 338 was always found below Teut (Table 3). Most importantly, determination of PSD for the
d95 Values Obtained After Various Freeze-Drying Conditions
Concentration Steric Stabilizer (Type; mg/mL) Cr EL; 33 Cr EL; 33 Cr EL; 33 Cr EL; 33 Cr EL; 50 Cr EL; 50 Cr EL; 50 Cr EL; 50 P338; 20 P338; 20 P338; 20 P338; 20 P338; 33 P338; 33 P338; 33 P338; 33
Concentration Protectant (Type; mg/mL)
d95 Values, Conservative Run (:m)
d95 Values, Moderate Run (:m)
d95 Values, Highly Aggressive Run (:m)
T; 25 T; 75 S; 25 S; 75 T; 25 T; 75 S; 25 S; 75 T; 25 T; 75 S; 25 S; 75 T; 25 T; 75 S; 25 S; 75
2.41 0.67 1.73 1.36 0.63 0.51 0.91 0.88 7.44 3.96 16.79 6.52 0.80 0.57 1.05 0.84
2.20 0.48 1.56 1.57 0.57 0.43 0.82 0.85 6.04 2.88 16.93 5.12 0.87 0.40 1.38 0.90
10.44 19.78 23.61 39.31 0.43 0.66 1.03 1.12 18.63 44.96 53.45 99.97 0.91 0.68 1.49 1.01
The nanoparticle concentration was maintained at 100 mg/mL. Note that the calculated standard deviations were small, indicating very consistent d95 values (n = 4 for each freeze-drying run). Cr EL, Cremophor EL; P338, Poloxamer 338; T, trehalose; S, sucrose.
DOI 10.1002/jps
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Table 3. Comparison of Product Temperatures (Steady State Phase During Primary Drying) Obtained During the “Moderate” Cycle Conditions Steric Stabilizer (Type; mg/mL) Cr EL; 50 Cr EL; 50 Cr EL; 50 P338; 33 P338; 33 P338; 33
Concentration Protectant (Type; mg/mL)
T g [◦ C]
Teut [◦ C]
Effective Product Temperature [◦ C]
T; 25 T; 50 T; 75 T; 25 T; 50 T; 75
–41 –39 –36 –31 –31 –31
– – – –16 –16 –16
–25 –25 –23 –23 –22 –21
The corresponding CFTs are provided for convenience purpose. Cr EL, Cremophor EL; P338, Poloxamer 338; T, trehalose.
moderately processed formulations was similar to those obtained from the conservative run. As illustrated in Table 2, the d95 values indicate that particle size stability is not a function of glass formation of the disaccharides used, that is, the drug nanoparticles must not be arrested in a rigid glass throughout the process. Moreover, the nanosuspensions stabilized with Cremophor EL and mixed with a disaccharide revealed a much better PSD, although product temperature was about 15◦ C (according to Table 3) above the measured T g . It is important to note that the nanosuspensions consisting of lower concentrations of any of the disaccharides did not suffer from any visual cake defects. Cake appearance was not affected as long as high nanoparticle and low disaccharide concentrations were used. Similar results were also found for formulations containing Poloxamer 338. Although product temperatures were below Teut of Poloxamer 338 (but above T g ), cake appearance was quite elegant. As mentioned above, sucrose inhibited crystallization of Poloxamer 338 in the mixture, but no difference in cake appearance or PSD could be found. The data suggest that the physicochemical state of the formulation does not affect the PSD and cake stability. In fact, the formation of an elegant cake appears to be dependent on the nanoparticle and disaccharide concentration in the formulation. The solid drug nanoparticles provide cake stability and the disaccharides are not required to form a product structure, but it may act as a separating medium in which the drug nanoparticles are diluted. The outlined observations could be confirmed in a parallel investigation that involved other mono- and disaccharides (e.g., lactose, glucose, and raffinose).
both primary and secondary drying can be performed in a single step. The time of the freeze drying cycle was compared with the effective time of the conservative cycle. The completion of the experiment was defined once the residual moisture content was found below 1% in the freeze-dried product. The highly aggressive freeze-drying run was completed after about 12 h for both Cremophor EL and Poloxamer 338 formulations. The product temperature profile for this cycle is illustrated in Figure 6. It is obvious from Figure 6 that the product temperature during primary drying is well above the investigated Teut of Poloxamer 338. Nevertheless, no meltback could be observed. It appears that the high total solid content of nanoparticles in the formulation counteracts structural deterioration. Considering the T g values of the Cremophor EL (–36◦ C to–42◦ C) and the Poloxamer 338 (–31◦ C to–42◦ C) mixtures, the conservative cycle was completed between 48 and 72 h, respectively. This would, as a rough estimate,
Freeze Drying Experiments, Part 3: Highly Aggressive Cycle Conditions In a final set of experiments, all formulations were freeze dried using “highly aggressive” primary drying conditions (shelf inlet temperature: +40◦ C; chamber pressure: 75 mTorr). If the formulation is not a limitation for the process, only the equipment design will impose restrictions to the cycle conditions.25 The benefit of using 40◦ C during primary drying is that JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
Figure 6. Product temperature over time profiles obtained while using the highly aggressive freeze-drying cycle recipe. Solid line represents shelf inlet temperature, dashed line represents product temperature over time profile for the Poloxamer 338 and 25 mg/mL trehalose formulation, and dotted line represents product temperature over time profile for the Poloxamer 338 and 75 mg/mL trehalose formulation. DOI 10.1002/jps
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Figure 7. Comparison of the cake appearance for the stock nanosuspension stabilized with 33 mg/mL Poloxamer 338 and addition of 25, 50, and 75 mg/mL sucrose after the highly aggressive run. Note that particle size stability was comparable for the shown products even though the formulation with 75 mg/mL sucrose indicated collapse of the porous structure at the bottom of the cake.
mean that freeze drying can be shortened up to 85%. Although just stating an arbitrary percentage does not seem perfectly reliable, it does underline a true potential for cycle optimization. As an important observation, all formulations containing a lower amount of either Poloxamer 338 or Cremophor EL, and processed using the highly aggressive cycle revealed a formation of aggregates, which was not the case with very high concentrations of the steric stabilizers. The results clearly demonstrate that the steric stabilizer plays a key role in the preservation of the PSD during the freeze-drying process. Interpretation of cake appearance and corresponding particle stability highlighted that the highly aggressive freezedrying run did not affect the nano-PSD at all for those cases where the formulation of the drug nanosuspension contained high steric stabilizer concentrations (Table 2). As depicted in Figures 7 and 8, increasing the disaccharide concentration up to 75 mg/mL caused collapse at the bottom of the freeze-dried cake, which confirms that the product temperature exceeded the collapse temperature. In the course of the highly aggressive freeze-drying run, the product temperature exceeded even the Teut of the Poloxamer 338 formulation (Fig. 6). Nevertheless, neither meltback DOI 10.1002/jps
nor particle size instability was observed, presumably due to the large amount of solid suspended nanoparticles. Another practical implication of this observation is that typical bulking agents such as mannitol are not compulsory while freeze drying highly concentrated drug nanosuspensions.10
CONCLUSIONS The present study shows that freeze drying is the method of choice to preserve the original PSD of drug nanoparticles. However, it was also illustrated that in contrast to freeze drying of biological, drug nanoparticles do not require immobilization by glass-forming excipients to suppress the mobility of the colloidal particles and to inhibit aggregation. Even the collapse temperature, Toc , must not be considered as the upper product temperature boundary to stabilize colloidal-related systems, such as drug nanoparticles, during the entire freeze-drying process unless an appropriate type of steric stabilizer is present in a suitable concentration. In this context, the stability of the drug nanoparticles in this study was not only a function of the physicochemical characteristics of the lyoprotectants, as described by the particle JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
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stability is governed by the surface tension of the dispersion medium. In line with this argumentation, the rubber-like carbohydrate solution serves as the dispersion medium in which the nanoparticles are assumed to remain well dispersed, but immobilization is not a prerequisite. As a consequence, DSC and FDM experiments cannot be considered indispensable to evaluate the physicochemical properties of a mixture or the drying behavior in microscale, but data seem less important for process design and optimization. This, in turn, underlines the argument that more attention must be paid to the formulation design while freeze drying drug nanosuspensions.
REFERENCES
Figure 8. Comparison of SEM pictures for the stock nanosuspension stabilized with 33 mg/mL Poloxamer 338 and addition of 25 (top), 50, (middle) and 75 mg/mL (below) sucrose after the highly aggressive run (all cake bottom side). Regardless of the collapse dimension, a unimodal distribution was preserved in all cases.
isolation hypothesis, but also dependent on the functionality of a both dense and large steric barrier surrounding the nanoparticles. In coincidence with this hypothesis, the whole system could be considered as an emulsion-like system during primary drying, where particles have the freedom of mobility and the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
1. Levine H, Slade L. 1992. Another view of trehalose for drying and stabilizing biological materials. BioPharm 5:36–40. 2. Pikal MJ. 1998. Mechanisms of protein stabilization during freeze-drying and storage: The relative importance of thermodynamic relaxation and glassy state relaxation dynamics. In Freeze-drying/lyophilization of pharmaceuticals and biological products 96; Rey L, May JC, Eds. New York: Marcel Dekker, pp 161–198. 3. Tang X, Pikal MJ. 2004. Design of freeze-drying processes for pharmaceuticals: Practical advice. Pharm Res 21(2):191–200. 4. Franks F. 1994. Long-term stabilization of biologicals. Biotechnology 12:253–256. 5. Abdelwahed W, Degobert G, Stainmesse S, Fessi H. 2006. Freeze-drying of nanoparticles: Formulation, process and storage considerations. Adv Drug Deliv Rev 58(15):1688–1713. 6. Abdelwahed W, Degobert G, Fessi H. 2006. Investigation of nanocapsules stabilization by amorphous excipients during freeze-drying and storage. Eur J Pharm Biopharm 63:87–94. 7. Angell C. 1995. Formation of glasses from liquids and biopolymers. Science 267:1924–1935. 8. Allison SD, Molina MDC, Anchordoquy TJ. 2000.Stabilization of lipid/DNA complexes during the freezing step of the lyophilization process: The particle isolation hypothesis. Biochim Biophys Acta 1468:127–138. 9. Armstrong TK, Anchordoquy TJ. 2004. Immobilization of nonviral vectors during the freezing step of lyophilization. J Pharm Sci 93:2698–2709. 10. Wang W. 2000. Lyophilization and development of solid protein pharmaceuticals. Int J Pharm 203:1–60. 11. Meister E, Gieseler H. 2009. Freeze-dry microscopy of protein/ sugar mixtures: Drying behavior, interpretation of collapse temperatures and a comparison to corresponding glass transition data. J Pharm Sci 98(9):3072–3087. 12. Pikal MJ, Shah S. 1990. The collapse temperature in freezedrying: Dependence on measurement methodology and rate of water removal from the glassy phase. Int J Pharm 62:165– 186. 13. Merisko-Liversidge E, Liversidge GG, Cooper ER. 2002. Nanosizing: A formulation approach for poorly-water-soluble compounds. Eur J Pharm Sci 18:113–120. 14. Kesisoglou F, Panmai S, Wu Y. 2007. Nanosizing—Oral formulation development and biopharmaceutical evaluation. Adv Drug Del Rev 59:631–644. 15. Lourenco C, Teixeira M, Simoes S, Gaspar R. 1996. Steric stabilization of nanoparticles: Size and surface properties. Int J Pharm 138:1–12. 16. Beirowski J, Gieseler H, Inghelbrecht S, Arien T, van Asche I. 2009. Investigation of various Poloxamers as freeze-drying stabilizers in freeze-drying of nanosuspensions. Proc. 2009 PDA DOI 10.1002/jps
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Pharmaceutical Freeze Drying Technology; September 29–30, 2009; Frankfurt, Germany. 17. Beirowski J, Inghelbrecht S, Arien T, van Assche I, Gieseler H. 2010. Stabilization of nanosuspensions during freeze-drying: The role of vitrification (part 1). Proc. 7th World Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology; March 8–11, 2010; Valetta, Malta (published online: http://www.freeze-drying.eu/html/publications.html). 18. Beirowski J, Inghelbrecht S, Arien T, van Assche I, Gieseler H. 2010. Stabilization of nanosuspensions during freeze-drying: The role of vitrification and its practical implications (part 2). Proc. 7th World Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology; March 8–11, 2010; Valetta, Malta (published online: http://www.freeze-drying.eu/ html/publications.html). 19. FDA/ORA Compliance Policy Guide. 2006. Process validation requirements for drug products and active pharmaceutical ingredients subject to pre market approval. Sub chapter 490.100. Accessed November 3, 2010 at: http://www.fda.gov/ ICECI/compliancemanuals/compliancepolicyguidancemanual/ default.htm.
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20. Wong J, Brugger A, Khare A, Chaubal M, Papadopoulos P, Rabinow B, Kipp J, Ning J. 2008. Suspensions for intravenous (IV) injection: A review of development, preclinical and clinical aspects. Adv Drug Deliv Rev 60(8):939–954. 21. Grant DJW, Brittain HG. 1995. Solubility of pharmaceutical solids. In Physical characterization of pharmaceutical solids; Brittain HG, Ed. New York: Marcel Dekker, pp 321–386. 22. Beirowski J, Gieseler H. 2008. Application of DSC and MDSC in the development of freeze-dried pharmaceuticals. Eur Pharm Rev 6:63–70. 23. Gordon M, Taylor JS. 1952. Ideal copolymers and the secondorder transitions of synthetic rubbers. I. Noncrystalline copolymers. J Appl Chem 2:493–500. 24. Beirowski J, Inghelbrecht S, Arien A, Gieseler H. 2011. Freezedrying of nanosuspensions, 1: Freezing rate versus formulation design as critical factors to preserve the original particle size distribution. J Pharm Sci 100(5):1958–1968. 25. Patel SM. 2008. Choked flow and importance of Mach 1 in freeze-drying process design. Proc. CPPR Freeze-Drying of Pharmaceuticals and Biologicals; August 6–9, 2009; Breckenridge, Colorado.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
University of Erlangen, Division of Pharmaceutics, Freeze Drying Focus Group, 91058 Erlangen, Germany
2 Pharmaceutical Development & Manufacturing Sciences, Janssen Research & Development, A Division of Janssen Pharmaceutical NV, 2340 Beerse, Belgium
Received 14 January 2011; revised 28 February 2011; accepted 5 May 2011 Published online 23 May 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22634 ABSTRACT: The present study investigates whether controlling the product temperature below the critical formulation temperature (CFT) during primary drying in a freeze drying cycle is a prerequisite for the stabilization of drug nanoparticles. For that purpose, the CFT of four drug nanosuspensions stabilized with different types (amorphous and crystalline) and concentrations of steric stabilizers and either of the disaccharides, trehalose and sucrose, was determined by differential scanning calorimetry and freeze-dry microscopy. Freeze-drying experiments were performed such that product temperatures during primary drying remained either below or well above the CFT of individual mixtures. It was found that glass formation did not influence the stability of the nanoparticles, suggesting that an adequate type of steric stabilizer and lyoprotectant concentration is present. Freeze drying could also be performed above the eutectic temperature without compromising on the final product quality profile, such as nanoparticle size and structural preservation of the lyophilized cake. The high concentration of solid drug nanoparticles provided additional cake stability. The results of this study confirm for the first time that primary drying for drug nanosuspensions can be greatly shortened because induced viscous flow or even meltback is not a limitation for nanoparticle stability and cake elegancy. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4471–4481, 2011 Keywords: freeze drying; lyophilization; nanosuspensions; nanotechnology; vitrification; glass transition; collapse temperature; compounding; stabilization
INTRODUCTION It is well known that the underlying concept for the stabilization of proteins during freeze drying is glass formation by excipients. Such excipients are denoted as “cryoprotectants” in the event that they stabilize a protein structure primarily during freezing, and “lyoprotectants” in the event that stabilization can be achieved during the dehydration step.1–4 It has been reported that many excipients can serve as both cryo- and lyoprotectants. The term “vitrification” is defined as the transformation of an aqueous solution into a rigid, solid glass. Vitrification would be assumed when the product temperature of a formulaCorrespondence to: Henning Gieseler (Telephone: +49-91318529556; Fax: +49-9131-8529545; E-mail: gieseler@freeze-drying. eu) Journal of Pharmaceutical Sciences, Vol. 100, 4471–4481 (2011) © 2011 Wiley-Liss, Inc. and the American Pharmacists Association
tion is lower than the glass transition temperature of the freeze-concentrated solute, T g .2 Numerous studies have been presented addressing protein stability as a function of vitrification, and the validity of this rule has initially also been proposed for the stabilization of nanoparticulate formulations.5,6 Agglomeration of suspended nanoparticles is prevented by formation of a solid amorphous glass, where the colloidal particles are arrested in an infinite high viscous environment and are isolated from each other.7 However, even if glass formation of a stabilizer is the preferred mechanism to preserve the original particle size distribution (PSD), some questions still need to be addressed. Allison et al.8 suggested that the separation of individual particles within the unfrozen fraction prevents aggregation during the freezing step, and this idea was called the particle isolation hypothesis. In this hypothesis, the relatively low surface tension of mono- and disaccharides plays an integral role
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that, in turn, allows phase-separated particles to remain dispersed within the unfrozen solute. In addition, Armstrong and Anchordoquy9 modeled diffusion of particulate nonviral vectors in the frozen matrix and argued that these formulations could be dried above T g as long as aggregation was avoided. Drug nanoparticles are also referred to as particulate colloids, but they are clearly different in their structure than, for example, liposomal systems. It seems, therefore, of great interest to study whether drying of such systems above T g without negative impact on product stability attributes would be possible as well. Drug nanoparticles are commonly stabilized using a steric stabilizing mechanism. The relevance of this class of stabilizers in combination with the frequently used disaccharides sucrose and trehalose (both are known to serve as lyoprotectants during primary drying) is the goal of the present study. Importantly, it is not clear until today how crystalline steric stabilizers and amorphous lyoprotectants will affect each other in terms of stabilization capacity during freeze drying because principally, glass formation is considered to provide stabilization for amorphous structures.10 In the event that T g does not directly limit the product temperature profile during primary drying, a subsequent question would target the maximum allowable product temperature that still assures final product stability. Freeze-dry microscopy (FDM) has been used in the course of the present investigations to determine the (onset) collapse temperature (Toc ) of the mixtures. It is well known that T g and Toc are not identical for many drug formulations due to the different measurement principle.11 As a rule of thumb, Toc is found about 2◦ C–5◦ C higher than the corresponding T g . It has already been demonstrated that Toc is more indicative for the critical formulation temperature (CFT) than the corresponding glass transition.11 Maintaining the product temperature above Toc induces changes in the inner cake morphology due to viscous flow, which is denoted as either “microcollapse,” “shrinkage,” or “collapse”. Such structural changes are demonstrated to affect the target quality aspects of the final product (e.g., appearance, reconstitution time, etc.).12 However, although recent FDM reports illustrated that Toc is, among other things, a function of total solid content of the formulation,11 it is also of great interest to address the question that at which solid nanoparticle concentration level additional cake stability is provided. Another aspect that deserves consideration in the present discussion is the introduction of crystallizing materials (i.e., steric stabilizers). Assuming that nanoparticle stability is not a function of immobilization in the excipient matrix, freeze drying at or above the eutectic temperature (Teut ) might be feasible as well. As mentioned above, high concentrations of solid drug nanoparticles might also compensate for JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
meltback. PSD is not automatically expected to be influenced because the steric barrier surrounding the particles can avoid physical instabilities in a highly mobile environment. It has already been pointed out in the literature that the key role of the steric stabilizer is to impart the initial stability in the liquid phase right after milling.13,14 The concept of steric stabilization is possible for those materials that can be attached or adsorbed onto the nanoparticle surface and provide a large and dense steric barrier, which is mandatory to overcome the attractive van der Waals forces (e.g., polyethylene glycol, cellulosics, pluronics, polysorbates, etc.).13–15 In addition, previous studies have already depicted that the selection of an adequate steric stabilizer is important to preserve the original PSD. This observation might then mitigate the relevance of vitrification.16–18 An elevated mobility in the excipient matrix must not inevitably lead to aggregation or particle fusion during lyophilization. Besides the science-related aspect of this study, another practical consideration is grounded in process economics. The possibility to process drug nanosuspensions well above their CFT allows rather unconventional, aggressive cycle conditions, which could significantly decrease freeze-drying process times and therefore, turnover.19 Using drug nanoparticles as a model system might offer an opportunity to learn whether other nanopartiulate formulations, for example, nanospheres or nanocapsules, can be processed above T g as well. To get down to the essence of the matter experimentally, four drug nanosuspensions stabilized with two different steric stabilizers were freeze dried conservatively, moderately, or aggressively in the presence of two different commonly used lyoprotectants, namely, sucrose and trehalose. The original suspension of the nanoparticles right after milling was found unstable in terms of complete preservation of the original PSD during freeze thawing experiments. To investigate the influence of the steric stabilizer and disaccharide concentration, both excipients were added in varying concentrations.
MATERIAL A poorly water-soluble, crystalline active pharmaceutical ingredient (API) and 0.3-mm yttrium-stabilized zirconia beads were kindly provided by Johnson & Johnson Pharmaceutical Research & Development (Beerse, Belgium). Lutrol F108 Prill (Poloxamer 338) and Cremophor EL were donated by BASF (Ludwigshafen, Germany). Trehalose and sucrose were of analytical grade and were purchased from Sigma (Sigma Chemical Company, Munich, Germany). All excipients were used as received. Either water for injection (B.Braun Melsungen AG, Melsungen, Germany) or water distilled from an allglass apparatus was used throughout this study. As DOI 10.1002/jps
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primary packing material for freeze drying, 15R Vials from Lutz GmbH (Wertheim, Germany) and 20-mm R freeze drying stoppers from West PharmaFlurotec ceutical Services, Inc. (Lionville, Pennsylvania) were used.
METHODS Production of Nanosuspensions Four nanosuspensions stabilized with either 20 or 33 mg/mL of Poloxamer 338, or 33 or 50 mg/mL Cremophor EL were prepared by wet bead milling in R , Selb, a high shear media mill (Netzsch MiniCer Germany) using 0.3-mm yttrium-stabilized zirconia beads. Note that water was used as dispersion medium and milling time was adapted to achieve the desired nanoparticle size range. Because these surfactants are nonionic, their mechanism of stabilization is considered steric stabilization. API concentration was kept constant at 100 mg/mL in the stock nanosuspensions. The stock nanosuspensions were used as received, without any protectants, or mixed with 25, 50, or 75 mg/mL trehalose or sucrose. Particle Size Measurements To investigate the PSD after milling and freeze drying over a wide particle size range, laser diffraction analR ysis was performed on a Malvern MasterSizer (Malvern Instruments GmbH, Herrenberg, Germany), which is capable of detecting even larger particle aggregates. Dependent on the steric stabilizer present in the formulation, an aqueous solution of 5 mg/mL Poloxamer 338 or Cremophor EL were used as a medium because particles were not sufficiently stable in pure water during the analysis. Basis for particle size calculation was the Mie theory, with a product refractive index of 1.65 and product absorption of 0.001 (dimensionless parameter). The obtained fitting curve achieved by the MasterSizer 2000 software indicated the validity of the optical model. Background and measurement integration time were kept at 60 s and three measurement runs per sample were performed. Freeze Drying: Experimental Design Each formulation mixture (2 mL) was filled in 15R vials (0.52 cm fill depth). Lyophilization was performed on a laboratory scale freeze dryer (VirTis Advantage Plus, SP Scientific, Gardiner, NY, USA). Vials were loaded using a stainless steel tray and the bottom of the tray was removed to allow a direct placement of the vials on the shelf surface during the cycle. Product temperatures were measured using calibrated 30 gauge T-Type copper/constantan thermocouples from Omega (Omega Engineering, Stamford, Connecticut). Each thermocouple was DOI 10.1002/jps
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introduced through a stopper and positioned at the bottom center of the vial to achieve both representative temperature monitoring as well as accurate endpoint detection of the time point when no ice is left in the product. The applied cycle conditions were as follows: Freezing was performed at –40◦ C (shelf inlet temperature) for 60 min, including equilibrating steps at +5◦ C and–5◦ C for 15 min. The shelf temperature ramp rates from the freezing step in to the primary drying step were 1◦ C/min for all cycles performed. Three different primary drying conditions were employed, further denoted as “conservative” run (shelf inlet temperature: –35◦ C), “moderate” run (shelf inlet temperature: 0◦ C), and “highly aggressive” run (shelf inlet temperature: +40◦ C, 8 h). The shelf heating rate from the primary drying shelf setpoint to the secondary drying setpoint at +40◦ C was 0.15◦ C/min for the “conservative” and “moderate” cycles. Secondary drying for these cycles were performed over 360 min. The chamber pressure during primary and secondary drying was controlled at 75 mTorr throughout all experiments. Differential Scanning Calorimetry (DSC) Glass transitions of the maximally freeze concentrated solute (T g ) and other thermal events were determined using a Mettler DSC822e (Mettler-Toledo, Greifensee, Switzerland). Data acquisition for the liquid formulations was performed in temperature ranging from–80◦ C to 0◦ C, applying cooling rates of 20◦ C/ min and heating rates of 5◦ C/min. For those formulations where Poloxamer 338 was present, slow cooling rates were used (1◦ C/min) because these block polymers are known to remain semicrystalline after a fast freezing regime. Thus, an annealing step was not mandatory to facilitate crystallization. Freeze-dry Microscopy Freeze-dry microscopy was performed on a freezedrying stage (FDCS-196) from Linkam Scientific (Linkam Scientific Instruments, Surrey, UK) and a Zeiss (Carl Zeiss MicroImaging GmbH, G¨ottingen, Germany) optical microscope equipped with a 1.2 MP digital camera. Approximately 2 :L of the corresponding formulations were used to detect the Toc . The sample was frozen at a rate of 1◦ C/min to −40◦ C and held for 10 min. Vacuum was initiated and the temperature was slowly increased at a 1◦ C/min heating rate up to 0◦ C. Toc was determined from images displaying the first signs of structural changes within the dried layer. Scanning Electron Microscopy (SEM) Broken pieces of the freeze-dried samples were fixed on Al stubs and subsequently gold-sputtered at 20 mA/5 kV (Hummer JR Technics, Union City, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
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CA, USA). Cake Morphology was then examined using Amray 1810 T scanning electron microscope (Amray, Rochester, NY, USA) at 20 kV with magnifications between 6× and 500×. Karl Fischer Residual Moisture Measurements The residual moisture content of the freeze-dried samples was measured using a Mitsubishi moisture meter CA-06 coulometric connected to a Mitsubishi vaporizer VA-06 (Mitsubishi Chemical Company, Tokyo, Japan). About 50 mg of the sample was weighed into a sample glass holder and then transferred into the oven unit. The sample vessel was continuously purged with dry nitrogen. Then, the sample was heated at a predefined temperature (140◦ C, setpoint) for a defined time period and the water vapour accumulated in the titration cell. Depending on the sample weight, residual moisture content was calculated in percent.
RESULTS AND DISCUSSION Physical Stability of Initial Nanosuspensions After Milling and Storage All four stock nanosuspensions (denoted as “first order formulations”) were found stable with regard to their PSD directly after the milling process. As illustrated in Figure 1, for the stock nanosuspension stabilized with Poloxamer 338, a unimodal PSD could be readily obtained. A small tailing adjacent to the unimodal distribution was observed in every single case, which indicates that a few larger particles in the 1 :m
Figure 2. Picture of a stock nanosuspension indicating settling after 6 months of storage at ambient conditions.
range are still present. The absence of particles larger than 5 :m could be confirmed, which would be a prerequisite for the formulation to comply with the specifications for a parenteral route of administration.20 Extended milling could not eliminate this tailing, but it greatly deteriorated particle size stability. On the basis of theoretical considerations, the observation of larger particles may be explained by accelerated Ostwald ripening during storage.21 It might, therefore, be expected to obtain a close to optimum unimodal PSD while stabilizing drug nanosuspensions by freeze drying. Note that the effect of Ostwald ripening became even more pronounced after 3 months of storage when the tailing increased and the particle sizes exceeded the upper 5 :m particle size limit (cf. Fig. 1). Moreover, a decrease of the unimodal shape occurred, which seems to be in good agreement with the above mentioned phenomenon of particle size growth. Besides the tailing phenomenon, storage of the nanosuspensions led to settling (cf. Fig. 2), and the observed long-term physical stability of the liquid drug nanosuspensions was found poor. To preserve the original PSD, freeze drying was used as the method of choice to improve the stability of the system.5 It should be underlined that neither the addition of trehalose nor sucrose to the drug nanosuspensions (then denoted as “second order formulation”) prior to the freeze-drying process did affect the nanoparticle stability. Observations by Thermal Analysis, Part 1: Differential Scanning Calorimetry
Figure 1. Comparison of the PSDs of the stock nanosuspension stabilized with 33 mg/mL Poloxamer 338 right after milling (solid line) and 3 months of storage at ambient conditions (dashed line). As observable from the figure, the particle growth exceeding the 5-:m boundary makes a parenteral application questionable. Note that the other formulations showed similar particle growth characteristics. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
The concept of rational freeze-drying cycle design and optimization is based on the evaluation of the CFT.22 The CFT has been defined as the product temperature during primary drying when the inner product morphology will start undergoing structural changes, comprising significant changes in specific surface area DOI 10.1002/jps
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Table 1. T g and/or Teut Values of the Formulations Stabilized with 50 mg/mL Cremophor EL or 33 mg/mL Poloxamer 338 Mixed with Either 25, 50, or 75 mg/mL of the Disaccharides Sucrose or Trehalose Steric Stabilizer Cremophor EL
Poloxamer 338
Disaccharide Concentration – 25 mg/mL trehalose 50 mg/mL trehalose 75 mg/mL trehalose 25 mg/mL sucrose 50 mg/mL sucrose 75 mg/mL sucrose – 25 mg/mL trehalose 50 mg/mL trehalose 75 mg/mL trehalose 25 mg/mL sucrose 50 mg/mL sucrose 75 mg/mL sucrose
T g
Teut
–73◦ C
– – – – – – – –16◦ C –16◦ C –16◦ C –16◦ C – – –
–41◦ C –39◦ C –36◦ C –42◦ C –39◦ C –36◦ C – –31◦ C –31◦ C –31◦ C –42◦ C –39◦ C –37◦ C
Note that for the lower concentrated Cremophor EL or Poloxamer 338 formulations, similar T g and/or Teut values were found, indicating the same trend.
of the cake structure or even (local or global) structural loss. A routinely used technology to determine the CFT is differential scanning calorimetry (DSC). The DSC measurements revealed a T g at –73◦ C for the stock nanosuspensions with Cremophor EL (cf. Table 1 and Fig. 3). This finding would suggest that the product temperature at the ice sublimation interface must be maintained below–73◦ C during primary drying to arrest the PSD, which, in turn, is impossible. The stock nanosuspensions stabilized with Poloxamer 338 as a steric stabilizer revealed several thermal transitions while using a fast freezing rate (cf. Fig. 4). Fast freezing for these mixtures even led to an artificial T g at about–65◦ C, followed by a devitrification peak indicating recrystallization of the Poloxamer 338
Figure 3. DSC thermogram of Cremophor EL indicating a glass transition temperature of the maximally freezeconcentrated solute (T g ) at –73◦ C. DOI 10.1002/jps
Figure 4. DSC thermograms revealing the physicochemical state of the Poloxamer 338 in the stock nanosuspension after fast freezing (dash dotted line) and slow freezing (dotted line). The solid line shows that sucrose inhibited the crystallization of Poloxamer 338 because no eutectic temperature is detectable.
between–50◦ C and–30◦ C. An endothermic melting signal at about–16◦ C indicated that a fraction of Poloxamer 338 remained in the crystalline state while applying a fast freezing regime. In order to facilitate crystallization of Poloxamer 338, a slow freezing rate (1◦ C/min) was used. As a result, the T g at –65◦ C disappeared and the Teut at –15◦ C remained as the only thermal transition (Fig. 3). In this specific event, Teut must be considered as CFT. Note that slow freezing or even annealing (–20◦ C, 1 h) of the stock nanosuspensions stabilized with Cremophor EL did not result in crystallization at all. To stabilize the drug nanoparticles in the glassy state, the disaccharides trehalose or sucrose were added to the stock nanosuspensions with a stepwise increase in concentration. The measured T g and/or Teut values for the corresponding mixtures are given in Table 1. Mixing of the disaccharides with the Cremophor EL stock nanosuspension led to a massive decrease of T g relative to the T g of the pure disaccharide solutions (sucrose: –32◦ C, trehalose: –29◦ C).10 As expected, the T g of pure Cremophor EL at –73◦ C disappeared and a new T g is formed between the extremes of pure Chremophor EL and the pure disaccharide solution as a function of a binary (and homogenous) mixture of the two substances. As a side note, the analytical data obtained by DSC could be confirmed by theoretical calculations using the Gordon– Taylor/Kelley–Bueche relationship (data not shown in Table 1).23 In addition, Table 1 illustrates all transitions of the nanosuspensions stabilized with Poloxamer 338 and mixed with the corresponding disaccharide. As mentioned above, it is recommendable for such mixtures to apply slow cooling rates JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
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(i.e., 1◦ C/min) during the DSC experiment to avoid the formation of an artificial T g in the low temperature region. In this event, slow freezing was not even sufficient to facilitate crystallization of Poloxamer 338 when sucrose was present. Figure 4 clearly illustrates that no Teut can be detected in the total heat flow signal, which supports the hypothesis that sucrose inhibited crystallization of Poloxamer 338. Moreover, even annealing at –20◦ C for 120 min could not initiate crystallization in this mixture. As a consequence, the measured T g values of the corresponding formulations were considered as the maximum allowable temperature during primary drying. In contrast, a distinct T g at about–31◦ C was found when trehalose was added to the Poloxamer 338 nanosuspension. Note that Poloxamer 338 showed a Teut at –16◦ C, which is comparable to the pure substance. Intuitively, the critical product temperature during primary drying for the Poloxamer 338 nanosuspension mixed with trehalose was defined at –31◦ C because physical instabilities are expected to be avoided by a complete immobilization of the nanoparticles in the solid state. Observations by Thermal Analysis, Part 2: Freeze-dry Microscopy Freeze-dry microscopy measurements were performed to assure that the product temperature profiles during primary drying are sufficiently high to initialize structural deterioration within the inner cake morphology. As mentioned above, both glass transition and collapse temperature information are routinely used for freeze-drying cycle optimization. However, the data obtained from these two analytical procedures for the CFT is not necessarily the same. Differences up to 5◦ C (and more) have been presented in the literature,12 based on the different concepts in the analytical procedure. Freeze-dry microscopy experiments revealed that determination of a collapse temperature was massively impeded by the high total solid content of the individual mixtures (Fig. 5). Removal of the polarization filter facilitated the measurements, and the progressing sublimation front during the measurement could be pinpointed. The results obtained suggest that the Toc values, if detectable, did not always correlate with the T g values. For example, the pure nanosuspension stabilized with Poloxamer 338 showed no distinct melting of the product matrix, although DSC revealed a melting event at –16◦ C. FDM of the stock nanosuspension stabilized with Cremophor EL (T g =–73◦ C) showed no abnormality during heating, but at some point, the whole structure appeared to flow, which limited the accuracy of the analysis. When a disaccharide was added to the Cremophor EL nanosuspension, the observed Toc values were slightly higher than the corresponding T g data. In contrast to this, a mixture of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
Figure 5. (a and b) FDM pictures of the drug nanosuspensions stabilized with 33 mg/mL Cremophor EL and 25 mg/ mL sucrose. Note that using the polarization filter makes measurement almost impossible (a). In contrast, the sublimation interface can be barely followed up when the filter is removed (b). Panel (c) (without polarization filter) shows the formulation containing 33 mg/mL Poloxamer 338 and 75 mg/mL trehalose underlining the deviation between DSC and FDM analytics. The bright spots (arrows) indicate rupture of the dried structure, which was first detectable at –18◦ C. In comparison, a T g for this formulation was found at –31◦ C and a Teut at –16◦ C.
the Poloxamer 338 nanosuspension with either disaccharide revealed a strong bias in the obtained results. Addition of trehalose led to a detectable onset of collapse at –18◦ C (cf. Fig. 5), which is in clear contrast to T g measurements (–31◦ C, Table 1). One may speculate that the collapse of the dried structure indeed occurred at lower temperatures, but the DOI 10.1002/jps
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high nanoparticle concentration impeded the measurement significantly enough to blur the visual detectability. A complementary investigation by scanning electron microscopy (SEM) was performed to reveal possible changes in the inner product morphology and to compensate the limitations encountered by FDM (see further below). Freeze Drying Experiments, Part 1: Conservative Cycle Conditions First experiments indicated that all formulations containing sucrose or trehalose preserved the original PSD after a freeze thaw cycle (data not shown). Stock nanosuspensions without any disaccharide were found unstable in terms of aggregation, whereas the higher concentrated Cremophor EL nanosuspension revealed only a slight shift in the PSD. An increase in the steric stabilizer concentration led to less aggregation tendency of the particles. This observation is in good agreement with a previous hypothesis that states that an increase in the steric stabilizer concentration generates a denser steric layer on top of the nanoparticle, which keeps them apart in the freeze concentrated state.24 In addition, surplus of a steric stabilizer could also act as a cryo-/ lyoprotectant when the stabilizer is not fully adsorbed onto the surface of the drug particles. In a previous report, the combination of the two factors “steric stabilizer concentration” and “cryoprotectant concentration” were revealed to influence each other during the freezing step.17,18,24 Table 2 depicts all d95 values of the formulations after a conservative freeze-drying cycle, resulting in product temperatures below the determined CFT when a lyoprotectant was present (cf. Table 1). Note that the parameter d95 states that Table 2.
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95% of the particles are equal or less in size than the reported value. The results obtained prove that an increase in the steric stabilizer concentration led to an improved nanoparticle stability, whereas an increase in the disaccharide concentration up to 75 mg/ mL correlated with a better particle size particularly for those nanosuspensions containing a higher steric stabilizer concentration. However, a smaller amount of 25 mg/mL was found beneficial for cake stability regardless of the disaccharide or the steric stabilizer used. Cake appearance suffered from crack formation while using a high disaccharide concentration. Although this is only a cosmetic defect, it seems recommendable to use as little as possible of the disaccharide in combination with a high steric stabilizer concentration if cracking of the cake must be avoided. As a general observation, trehalose was always found superior to sucrose as a protectant. Freeze Drying Experiments, Part 2: Moderate Cycle Conditions To investigate the need of immobilization of the drug nanoparticles for particle size stabilization, a second run was performed, and the shelf (inlet) temperature was elevated to 0◦ C to obtain product temperatures above T g . Data of the maximum product temperatures achieved during the primary drying phase are provided in Table 3. Note that the authors simply denote these cycle conditions as “moderate” to allow a differentiation between the sets of experiments. Product temperature (Tp ) measurements using thin wire thermocouples revealed that Tp for the nanosuspensions stabilized with the crystallizing Poloxamer 338 was always found below Teut (Table 3). Most importantly, determination of PSD for the
d95 Values Obtained After Various Freeze-Drying Conditions
Concentration Steric Stabilizer (Type; mg/mL) Cr EL; 33 Cr EL; 33 Cr EL; 33 Cr EL; 33 Cr EL; 50 Cr EL; 50 Cr EL; 50 Cr EL; 50 P338; 20 P338; 20 P338; 20 P338; 20 P338; 33 P338; 33 P338; 33 P338; 33
Concentration Protectant (Type; mg/mL)
d95 Values, Conservative Run (:m)
d95 Values, Moderate Run (:m)
d95 Values, Highly Aggressive Run (:m)
T; 25 T; 75 S; 25 S; 75 T; 25 T; 75 S; 25 S; 75 T; 25 T; 75 S; 25 S; 75 T; 25 T; 75 S; 25 S; 75
2.41 0.67 1.73 1.36 0.63 0.51 0.91 0.88 7.44 3.96 16.79 6.52 0.80 0.57 1.05 0.84
2.20 0.48 1.56 1.57 0.57 0.43 0.82 0.85 6.04 2.88 16.93 5.12 0.87 0.40 1.38 0.90
10.44 19.78 23.61 39.31 0.43 0.66 1.03 1.12 18.63 44.96 53.45 99.97 0.91 0.68 1.49 1.01
The nanoparticle concentration was maintained at 100 mg/mL. Note that the calculated standard deviations were small, indicating very consistent d95 values (n = 4 for each freeze-drying run). Cr EL, Cremophor EL; P338, Poloxamer 338; T, trehalose; S, sucrose.
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Table 3. Comparison of Product Temperatures (Steady State Phase During Primary Drying) Obtained During the “Moderate” Cycle Conditions Steric Stabilizer (Type; mg/mL) Cr EL; 50 Cr EL; 50 Cr EL; 50 P338; 33 P338; 33 P338; 33
Concentration Protectant (Type; mg/mL)
T g [◦ C]
Teut [◦ C]
Effective Product Temperature [◦ C]
T; 25 T; 50 T; 75 T; 25 T; 50 T; 75
–41 –39 –36 –31 –31 –31
– – – –16 –16 –16
–25 –25 –23 –23 –22 –21
The corresponding CFTs are provided for convenience purpose. Cr EL, Cremophor EL; P338, Poloxamer 338; T, trehalose.
moderately processed formulations was similar to those obtained from the conservative run. As illustrated in Table 2, the d95 values indicate that particle size stability is not a function of glass formation of the disaccharides used, that is, the drug nanoparticles must not be arrested in a rigid glass throughout the process. Moreover, the nanosuspensions stabilized with Cremophor EL and mixed with a disaccharide revealed a much better PSD, although product temperature was about 15◦ C (according to Table 3) above the measured T g . It is important to note that the nanosuspensions consisting of lower concentrations of any of the disaccharides did not suffer from any visual cake defects. Cake appearance was not affected as long as high nanoparticle and low disaccharide concentrations were used. Similar results were also found for formulations containing Poloxamer 338. Although product temperatures were below Teut of Poloxamer 338 (but above T g ), cake appearance was quite elegant. As mentioned above, sucrose inhibited crystallization of Poloxamer 338 in the mixture, but no difference in cake appearance or PSD could be found. The data suggest that the physicochemical state of the formulation does not affect the PSD and cake stability. In fact, the formation of an elegant cake appears to be dependent on the nanoparticle and disaccharide concentration in the formulation. The solid drug nanoparticles provide cake stability and the disaccharides are not required to form a product structure, but it may act as a separating medium in which the drug nanoparticles are diluted. The outlined observations could be confirmed in a parallel investigation that involved other mono- and disaccharides (e.g., lactose, glucose, and raffinose).
both primary and secondary drying can be performed in a single step. The time of the freeze drying cycle was compared with the effective time of the conservative cycle. The completion of the experiment was defined once the residual moisture content was found below 1% in the freeze-dried product. The highly aggressive freeze-drying run was completed after about 12 h for both Cremophor EL and Poloxamer 338 formulations. The product temperature profile for this cycle is illustrated in Figure 6. It is obvious from Figure 6 that the product temperature during primary drying is well above the investigated Teut of Poloxamer 338. Nevertheless, no meltback could be observed. It appears that the high total solid content of nanoparticles in the formulation counteracts structural deterioration. Considering the T g values of the Cremophor EL (–36◦ C to–42◦ C) and the Poloxamer 338 (–31◦ C to–42◦ C) mixtures, the conservative cycle was completed between 48 and 72 h, respectively. This would, as a rough estimate,
Freeze Drying Experiments, Part 3: Highly Aggressive Cycle Conditions In a final set of experiments, all formulations were freeze dried using “highly aggressive” primary drying conditions (shelf inlet temperature: +40◦ C; chamber pressure: 75 mTorr). If the formulation is not a limitation for the process, only the equipment design will impose restrictions to the cycle conditions.25 The benefit of using 40◦ C during primary drying is that JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
Figure 6. Product temperature over time profiles obtained while using the highly aggressive freeze-drying cycle recipe. Solid line represents shelf inlet temperature, dashed line represents product temperature over time profile for the Poloxamer 338 and 25 mg/mL trehalose formulation, and dotted line represents product temperature over time profile for the Poloxamer 338 and 75 mg/mL trehalose formulation. DOI 10.1002/jps
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Figure 7. Comparison of the cake appearance for the stock nanosuspension stabilized with 33 mg/mL Poloxamer 338 and addition of 25, 50, and 75 mg/mL sucrose after the highly aggressive run. Note that particle size stability was comparable for the shown products even though the formulation with 75 mg/mL sucrose indicated collapse of the porous structure at the bottom of the cake.
mean that freeze drying can be shortened up to 85%. Although just stating an arbitrary percentage does not seem perfectly reliable, it does underline a true potential for cycle optimization. As an important observation, all formulations containing a lower amount of either Poloxamer 338 or Cremophor EL, and processed using the highly aggressive cycle revealed a formation of aggregates, which was not the case with very high concentrations of the steric stabilizers. The results clearly demonstrate that the steric stabilizer plays a key role in the preservation of the PSD during the freeze-drying process. Interpretation of cake appearance and corresponding particle stability highlighted that the highly aggressive freezedrying run did not affect the nano-PSD at all for those cases where the formulation of the drug nanosuspension contained high steric stabilizer concentrations (Table 2). As depicted in Figures 7 and 8, increasing the disaccharide concentration up to 75 mg/mL caused collapse at the bottom of the freeze-dried cake, which confirms that the product temperature exceeded the collapse temperature. In the course of the highly aggressive freeze-drying run, the product temperature exceeded even the Teut of the Poloxamer 338 formulation (Fig. 6). Nevertheless, neither meltback DOI 10.1002/jps
nor particle size instability was observed, presumably due to the large amount of solid suspended nanoparticles. Another practical implication of this observation is that typical bulking agents such as mannitol are not compulsory while freeze drying highly concentrated drug nanosuspensions.10
CONCLUSIONS The present study shows that freeze drying is the method of choice to preserve the original PSD of drug nanoparticles. However, it was also illustrated that in contrast to freeze drying of biological, drug nanoparticles do not require immobilization by glass-forming excipients to suppress the mobility of the colloidal particles and to inhibit aggregation. Even the collapse temperature, Toc , must not be considered as the upper product temperature boundary to stabilize colloidal-related systems, such as drug nanoparticles, during the entire freeze-drying process unless an appropriate type of steric stabilizer is present in a suitable concentration. In this context, the stability of the drug nanoparticles in this study was not only a function of the physicochemical characteristics of the lyoprotectants, as described by the particle JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
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stability is governed by the surface tension of the dispersion medium. In line with this argumentation, the rubber-like carbohydrate solution serves as the dispersion medium in which the nanoparticles are assumed to remain well dispersed, but immobilization is not a prerequisite. As a consequence, DSC and FDM experiments cannot be considered indispensable to evaluate the physicochemical properties of a mixture or the drying behavior in microscale, but data seem less important for process design and optimization. This, in turn, underlines the argument that more attention must be paid to the formulation design while freeze drying drug nanosuspensions.
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Figure 8. Comparison of SEM pictures for the stock nanosuspension stabilized with 33 mg/mL Poloxamer 338 and addition of 25 (top), 50, (middle) and 75 mg/mL (below) sucrose after the highly aggressive run (all cake bottom side). Regardless of the collapse dimension, a unimodal distribution was preserved in all cases.
isolation hypothesis, but also dependent on the functionality of a both dense and large steric barrier surrounding the nanoparticles. In coincidence with this hypothesis, the whole system could be considered as an emulsion-like system during primary drying, where particles have the freedom of mobility and the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
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Pharmaceutical Freeze Drying Technology; September 29–30, 2009; Frankfurt, Germany. 17. Beirowski J, Inghelbrecht S, Arien T, van Assche I, Gieseler H. 2010. Stabilization of nanosuspensions during freeze-drying: The role of vitrification (part 1). Proc. 7th World Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology; March 8–11, 2010; Valetta, Malta (published online: http://www.freeze-drying.eu/html/publications.html). 18. Beirowski J, Inghelbrecht S, Arien T, van Assche I, Gieseler H. 2010. Stabilization of nanosuspensions during freeze-drying: The role of vitrification and its practical implications (part 2). Proc. 7th World Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology; March 8–11, 2010; Valetta, Malta (published online: http://www.freeze-drying.eu/ html/publications.html). 19. FDA/ORA Compliance Policy Guide. 2006. Process validation requirements for drug products and active pharmaceutical ingredients subject to pre market approval. Sub chapter 490.100. Accessed November 3, 2010 at: http://www.fda.gov/ ICECI/compliancemanuals/compliancepolicyguidancemanual/ default.htm.
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