Optimization of storage stability of lyophilized actin using combinations of disaccharides and dextran

The storage stability of a dry protein depends on the structure of the dried protein, as well as on the storage temperature relative to the glass transition temperature of the dried preparation. Disaccharides are known to preserve the native conformation of a dried protein; however, the resulting T(g) of the sample may be too low ensure adequate storage stability. On the other hand, formulations dried with high molecular weight carbohydrates, such as dextran, have higher glass transition temperatures, but fail to preserve native protein conformation. We tested the hypothesis that optimizing both protein structure and T(g) by freeze-drying actin with mixtures of disaccharides and dextran would result in increased storage stability compared to actin dried with either disaccharide or dextran alone. Protein structure in the dried solid was analyzed immediately after lyophilization and after storage at elevated temperatures with infrared spectroscopy, and after rehydration by infrared and circular dichroism spectroscopy. Structural results were related to the polymerization activity recovered after rehydration. Degradation was noted with storage for formulations containing either sucrose, trehalose, or dextran alone. Slight increases in T(g) observed in trehalose formulations compared to sucrose formulations did not result in appreciable increases in storage stability. Addition of dextran to sucrose or trehalose increased formulation T(g) without affecting the capacity of the sugar to inhibit protein unfolding during lyophilization and resulted in improved storage stability. Also, dextran provides an excellent amorphous bulking agent, which can be lyophilized rapidly with formation of strong, elegant cake structure. These results suggest that the strategy of using a mixture of disaccharide and polymeric carbohydrates can optimize protein storage stability.     

Optimizing storage stability of lyophilized recombinant human interleukin-11 with disaccharide/hydroxyethyl starch mixtures

Optimal storage stability of a protein in a dry formulation depends on the storage temperature relative to the glass transition temperature (T(g)) of the dried formulation and the structure of the dried protein. We tested the hypothesis that optimizing both protein structure and T(g)--by freeze-drying recombinant human interleukin-11 (rhIL-11) with mixtures of disaccharides and hydroxyethyl starch (HES)--would result in increased storage stability compared with the protein lyophilized with either disaccharide or hydroxyethyl starch alone. The secondary structure of the protein in the dried solid was analyzed immediately after lyophilization and after storage at elevated temperatures by infrared spectroscopy. After rehydration, aggregation was monitored by size exclusion chromatography. Oxidation levels and cleavage products were quantified by reversed-phase chromatography. For the formulation with HES alone, which has a relatively high T(g), storage stability of rhIL-11 was poor, because HES failed to inhibit lyophilization-induced unfolding. The sugar formulations inhibited unfolding, and had intermediate T(g) values and storage stabilities. Addition of hydroxyethyl starch to sucrose or trehalose increased T(g) without affecting the capacity of the sugar to inhibit protein unfolding during lyophilization. Optimal storage stability of lyophilized rhIL-11 was achieved by using a mixture of disaccharide and polymeric carbohydrates.     

A specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody

The selection of the appropriate excipient and the amount of excipient required to achieve a 2-year shelf-life is often done by using iso-osmotic concentrations of excipients such as sugars (e.g., 275 mM sucrose or trehalose) and salts. Excipients used for freeze-dried protein formulations are selected for their ability to prevent protein denaturation during the freeze-drying process as well as during storage. Using a model recombinant humanized monoclonal antibody (rhuMAb HER2), we assessed the impact of lyoprotectants, sucrose, and trehalose, alone or in combination with mannitol, on the storage stability at 40 degrees C. Molar ratios of sugar to protein were used, and the stability of the resulting lyophilized formulations was determined by measuring aggregation, deamidation, and oxidation of the reconstituted protein and by infrared (IR) spectroscopy (secondary structure) of the dried protein. A 360:1 molar ratio of lyoprotectant to protein was required for storage stability of the protein, and the sugar concentration was 3-4-fold below the iso-osmotic concentration typically used in formulations. Formulations with combinations of sucrose (20 mM) or trehalose (20 mM) and mannitol (40 mM) had comparable stability to those with sucrose or trehalose alone at 60 mM concentration. A formulation with 60 mM mannitol alone provided slightly less protection during storage than 60 mM sucrose or trehalose. The disaccharide/mannitol formulations also inhibited deamidation during storage to a greater extent than the lyoprotectant formulations alone. The reduction in aggregation and deamidation during storage correlated directly with inhibition of unfolding during lyophilization, as assessed by IR spectroscopy. Thus, it appears that the protein must be retained in its native-like state during freeze-drying to assure storage stability in the dried solid. Long-term studies (23-54 months) performed at 40 degrees C revealed that the appropriate molar ratio of sugar to protein stabilized against aggregation and deamidation for up to 33 months. Therefore, long-term storage at room temperature or above may be achieved by proper selection of the molar ratio and sugar mixture. Overall, a specific sugar/protein molar ratio was sufficient to provide storage stability of rhuMAb HER2.     

Dehydration of trehalose dihydrate at low relative humidity and ambient temperature

The physico-chemical behaviour of trehalose dihydrate during storage at low relative humidity and ambient temperature was investigated, using a combination of techniques commonly employed in pharmaceutical research. Weight loss, water content determinations, differential scanning calorimetry and X-ray powder diffraction showed that at low relative humidity (0.1% RH) and ambient temperature (25 degrees C) trehalose dihydrate dehydrates forming the alpha-polymorph. Physical examination of trehalose particles by scanning electron microscopy and of the dominant growth faces of trehalose crystals by environmentally controlled atomic force microscopy revealed significant changes in surface morphology upon partial dehydration, in particular the formation of cracks. These changes were not fully reversible upon complete rehydration at 50% RH. These findings should be considered when trehalose dihydrate is used as a pharmaceutical excipient in situations where surface properties are key to behaviour, for example as a carrier in a dry powder inhalation formulations, as morphological changes under common processing or storage conditions may lead to variations in formulation performance.     

Stabilization of Lactate Dehydrogenase Following Freeze-Thawing and Vacuum-Drying in the Presence of Trehalose and Borate

Purpose. The purpose of this work was to investigate the effects of trehalose and trehalose/sodium tetraborate mixtures on the recovery of lactate dehydrogenase (LDH) activity following freeze-thawing and centrifugal vacuum-drying/rehydration. The storage stability of LDH under conditions of either high relative humidity or high temperature was also studied. Methods. LDH was prepared in buffered aqueous formulations containing trehalose alone and trehalose/'borate' mixtures. Enzymatic activity was measured immediately following freeze-thawing and vacuum-drying/rehydration processes, and also after vacuum-dried formulations were stored in either high humidity or high temperature environments. Also, glass transition temperatures (T_g) were measured for both freeze-dried and vacuum-dried formulations. Results. The T_gvalues of freeze-dried trehalose/borate mixtures are considerably higher than that of trehalose alone. Freezing and vacuum-drying LDH in the presence of 300 mM trehalose resulted in the recovery of 80% and 65% of the original activity, respectively. For vacuum-dried mixtures, boron concentrations below 1.2 mole boron/ mole trehalose had no effect on recovered LDH. After several weeks storage in either humid (100% relative humidity) or warm (45°C) environments, vacuum-dried formulations that included trehalose and borate showed greater enzymatic activities than those prepared with trehalose alone. We attribute this stability to the formation of a chemical complex between trehalose and borate. Conclusions. The high T_gvalues of trehalose/borate mixtures offer several advantages over the use of trehalose alone. Most notable is the storage stability under conditions of high temperature and high relative humidity. In these cases, formulations that contain trehalose and borate are superior to those containing trehalose alone. These results have practical implications for long-term storage of biological materials.     

Effects of sugar additives on protein stability of recombinant human serum albumin during lyophilization and storage

Few researches on the protein stabilization of recombinant human serum albumin (rHSA) have been done. In the present study, we assessed the impact of sugar lyoprotectants on the protein stability of lyophilized rHSA (65 KDa) in the solid state. For the assessment, rHSA was formulated with sucrose and trehalose, respectively, alone or in combination with mannitol, which were lyophilized and stored at 35 degrees C. Degradation and aggregation of the resulting lyophilized formulations was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Induction of amorphous state by the lyophilactants with rHSA was determined by differential scanning calorimetry (DSC). The protein secondary structure of the rHSA in the formulations was analyzed by Fourier transform infrared spectroscopy (FT-IR). Results from SDS-PAGE analysis displayed that mannitol formulation caused aggregation resulting in a few bands that were greater than 65 KDa, whereas sucrose and trehalose formulations revealed no such aggregation. However, the aggregation of the protein decreased when mannitol was combined with sucrose or trehalose. DSC measurement supported the electrophoresis data showing that sucrose and trehalose formed complete amorphous state, but mannitol induced a partial amorphous state. These data indicate during lyophilization the most effective protein protection against aggregation was provided by sucrose and trehalose. The protection lasted during 4 months storage at 35 degrees C. FT-IR analysis displayed that the sucrose formulation inhibited deamidation. In conclusion, our data suggest that sucrose and trehalose as additives seems to be sufficient to protect from lyophilization of rHSA protein and also maintain its stability in the solid state during storage.     

The Effect of Carbohydrate Additives in the Freeze-Drying of Alkaline Phosphatase

Abstract— Alkaline phosphatase was used as a model in studies to assess the effects of lyophilization on biological activity and molecular integrity in the presence or absence of added carbohydrate. The stability of the activity of alkaline phosphatase, lyophilized in Tris buffer alone or in the presence of the carbohydrates mannitol, lactose or trehalose was examined. Enzyme activity in formulations with Tris buffer alone or with mannitol was considerably reduced by freeze-drying and further storage at elevated temperatures; freeze-drying with mannitol failed to maintain activity at a temperature of 37°C over 21 days, whilst the loss of activity was more gradual when freeze-dried in buffer alone and stored at higher temperatures. Lactose and trehalose maintained the alkaline phosphatase activity after freeze-drying and, furthermore, preparations containing trehalose retained activity even when the material was subjected to temperatures of up to 45°C for up to 84 days. At 56°C the alkaline phosphatase activity did not show a significant drop until 14 days with the lactose formulation or until 21 days with trehalose. After 84 days at 56°C, 30% of the activity still remained in the formulation containing trehalose. In addition to the changes in the enzyme activity, FPLC chromatographic traces and SDS-PAGE gels demonstrated compositional differences between each formulation after storage.     

Prediction of the bitterness of single,binary- and multiple-component amino acid solutions using a taste sensor

Upon freeze-drying in the absence of lyoprotectants, Fourier transform infrared (FTIR) spectroscopy has detected changes in the secondary structures of proteins. Such FTIR studies have been typically conducted using protein/KBr pellets, where additional protein degradation could potentially occur due to pressure effects and partial dissolution of the chaotropic KBr. Diffuse reflectance FTIR spectroscopy, in which no sample preparation is necessary, was evaluated as an alternative spectroscopic method to examine protein structure upon freeze-drying. The therapeutic proteins recombinant human deoxyribonuclease I (rh-DNase) and recombinant human insulin like growth factor I (rh-IGF-I) were freeze-dried with mannitol, sucrose, trehalose, and two molecular weight dextrans (69 and 503 kDa) separately, at concentrations ranging from 0 to 100% (w/w). Upon freeze-drying, rh-DNase and rh-IGF-I underwent significant changes in their secondary structure. For both proteins, the presence of intermolecular beta-sheets due to aggregation was detected and the alpha-helix content decreased significantly. The addition of carbohydrates to the formulations inhibited the protein secondary structure rearrangement in a concentration-dependent manner. Sucrose and trehalose appeared to be the most efficient excipients in preventing secondary structure changes. The conformational changes observed for both proteins appeared to be reversible upon rehydration.     

Freeze drying of human serum albumin (HSA) nanoparticles with different excipients

Freeze drying is a suitable technique to improve the long-term storage stability of colloidal drug carrier systems such as nanoparticles. Aim of this study was to systematically evaluate excipients for the freeze drying and long-term stability of albumin-based nanoparticles. In our study, nanoparticles made of human serum albumin (HSA) were freeze dried in the presence of different cryoprotective agents and after reconstitution were evaluated with regard to their physico-chemical characteristics. Empty, doxorubicin-loaded, and PEGylated nanoparticles were prepared and were freeze dried in the presence of different concentrations of sucrose, trehalose, and mannitol, respectively. The samples were physico-chemically characterised with regard to lyophilisate appearance, particle size, and polydispersity using photon correlation spectroscopy. For evaluation of long-term stability, the samples were stored at 2-8, 25, and 40 degrees C over predetermined time intervals. In the absence of cryoprotectants, particle growth was observed in all freeze-dried formulations. In the presence of sucrose, mannitol, and trehalose aggregation of HSA nanoparticles during the freeze-drying procedure was prevented. Although all of the excipients were identified to be suitable stabilisers for freeze drying of HSA nanoparticles, sucrose and trehalose were superior to mannitol, especially with regard to the long-term storage stability results.     

Stabilizing Effect of Four Types of Disaccharide on the Enzymatic Activity of Freeze-dried Lactate Dehydrogenase: Step by Step Evaluation from Freezing to Storage

Purpose In order to understand the stabilizing effects of disaccharides on freeze-dried proteins, the enzymatic activity of lactate dehydrogenase (LDH) formulations containing four types of disaccharide (trehalose, sucrose, maltose, and lactose) at two relative humidity (RH) levels (about 0 and 32.8%) was investigated after three processes: freeze-thawing, freeze-drying, and storage at three temperatures (20, 40, and 60°C) above and/or below the glass transition temperature (T _g). Materials and Methods The enzymatic activity was determined from the absorbance at 340 nm, and T _g of the samples was investigated by differential scanning calorimetry. Results At each RH condition, T _g values of sucrose formulations were lower than those of other formulations. Although effects of the disaccharides on the process stability of LDH were comparable, storage stability was dependent on the type of disaccharide. All the formulations were destabilized significantly during storage at temperature above T _g. During storage at temperature below T _g, the LDH activity decreased with increases in the storage temperature and moisture. Maltose and lactose formulations showed significant destabilization with the change of color to browning. Conclusions Taking the storage stability of freeze-dried proteins under the various conditions (temperature and RH) into consideration, trehalose is better suited as the stabilizer than other disaccharides.     

Investigation of nanocapsules stabilization by amorphous excipients during freeze-drying and storage.

Freeze-drying was recently applied to improve the long-term storage stability of nanoparticles. Nanocapsules have a thin polymeric envelope that may not withstand the stresses of such process. So, cryoprotectants and lyoprotectants are usually added to the formulation to protect these vectors during freezing and desiccation steps. The aim of this paper was to investigate the importance of the vitrification of cryoprotectants on the stabilization of nanocapsules during freezing, desiccation, and storage steps. Furthermore, the effect of stabilizer crystallization on the conservation of nanocapsules properties was studied. Finally, the effect of temperature storage and relative humidity on the stability of nanocapsules was tested through an accelerated stability study. Results indicate that nanocapsules stabilization during the different steps of freeze-drying requires their dispersion within a vitrified matrix of amorphous excipient to protect them against the stress of freezing and dehydration. The crystallization of this stabilizer during the freezing, the desiccation or the storage steps can destabilize these fragile particles. Electron spectroscopy for chemical analysis revealed the adsorption of nanocapsules at the interface ice/liquid during the freezing step. Such adsorption must be avoided in the case of freeze-drying of immuno-nanoparticles to preserve the native structure of proteins attached to their surface.     

Investigation of the stabilisation of freeze-dried lysozyme and the physical properties of the formulations.

The long-term stability of a protein formulation requires that the glass transition temperature (Tg) of the formulation should be maximised and the perturbation of the protein native structure in the dried form after processing minimised. In the present study, the stabilisation of lysozyme structure conferred by excipients was monitored using second derivative Fourier transform infrared spectroscopy and the physical properties of protein formulations were investigated using differential scanning calorimetry. The results showed that the preservation of protein native structure during freeze-drying and the Tg of freeze-dried formulations were excipient- and excipient to enzyme mass ratio-dependent. The freeze-dried lysozyme appeared to be less effectively stabilised compared with the spray-dried enzyme when the excipients and the excipient to enzyme mass ratios were the same. In terms of the preservation of the secondary structure of lysozyme, glycerol and sucrose seemed to be more efficient than trehalose, although the Tg of trehalose-containing formulations were found to be higher than the Tg of the equivalent sucrose-based ones. With adding either trehalose or dextran to sucrose-containing formulations, the stabilisation of lysozyme native structure could be as effective as with sucrose alone, whilst the Tg could be enhanced. The results in this study suggested that lysozyme, processed by freeze-drying, is stabilised primarily by the water substitution mechanism.     

In Vitro Simulation of Solid-Solid Dehydration, Rehydration, and Solidification of Trehalose Dihydrate Using Thermal and Vibrational Spectroscopic Techniques

PURPOSE: The processes of dehydration, rehydration, and solidification of trehalose dihydrate were examined to simulate it in the drying and wetting states. METHODS: Techniques included differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and Fourier transform infrared (FT-IR) microspectroscopy combined with thermal analysis. Trehalose dihydrate was pressed on one KBr pellet (IKBr method) or sealed within two KBr pellets (2KBr method) for FT-IR measurement. RESULTS: On the DSC thermogram, the shoulder between 60 degrees C and 80 degrees C represented a transitional change because no weight loss occurred in this area of the TGA curve. The endothermic peak at 100 degrees C represented dehydration of trehalose dihydrate to anhydrous trehalose; a 9.5% weight loss in the TGA curve occurred from 81 degrees C. The thermal-dependent FT-IR spectra for trehalose dihydrate prepared by the IKBr method changed markedly with temperature in the 1800-1500 cm(-1) region during dehydration. IR peak intensity at 1687 cm(-1) assigned to the bending vibrational mode of solid-like water declined with temperature and decreased sharply at 67 degrees C. Another IR peak at 1640 cm(-1) associated with the bending of liquid water increased at 67 degrees C but disappeared at 79 degrees C as a result of water evaporation. Both peaks for the sample prepared by the 2KBr method changed dramatically at 64 degrees C; peak intensity at 1640 cm(-1) remained constant above 64 degrees C. CONCLUSIONS: A new polymorphic transition of trehalose dihydrate was first evidenced at 64-67 degrees C from both DSC curves and thermal-related FT-IR spectra. This transitional temperature reflected the thermal-dependent transformation from solid-like water to liquid water in the trehalose dihydrate structure during dehydration. During rehydration, trehalose anhydrate was rehydrated to the dihydrate, and liquid water in the dihydrate structure was again transformed to solid-like water. The polymorphic transition within this temperature range seems to correlate with the bioprotective effect of trehalose dihydrate in preserving protein stability.     

Effect of Sucrose/Raffinose Mass Ratios on the Stability of Co-Lyophilized Protein During Storage Above the Tg

Purpose. To examine the potential of raffinose as an excipient in stabilizing protein and to study the effect of sucrose/raffinose mass ratios on the stability of co-lyophilized protein and amorphous solids during storage at an elevated temperature. Methods. Glucose-6-phosphate dehydrogenase (G6PDH) was co-lyophilized with sucrose and raffinose mixed at different mass ratios. The activity of dried G6PDH was monitored during storage at 44°C. Thermal properties of sucrose/raffinose matrices were determined by differential scanning calorimetry (DSC). Results. Mass ratios of sucrose to raffinose did not affect the recovery of G6PDH activity after freeze-drying, but significantly affected the stability of freeze-dried G6PDH during storage. The sucrose-alone formulation offered the best enzyme stabilization during storage. With increasing fraction of raffinose, the G6PDH stability decreased, sugar crystallization inhibited, and crystal-melting temperature increased. Conclusions. Despite the higher T_g of the formulations with higher fraction of raffinose, they provided less protection for G6PDH than did sucrose alone during storage. Our data do not support the prediction from recent thermophysical studies that raffinose should be superior to sucrose and trehalose as a potential excipient or stabilizer.     

Distinct effects of sucrose and trehalose on protein stability during supercritical fluid drying and freeze-drying.

Supercritical fluid (SCF) drying has been proposed as an alternative for freeze-drying to stabilize proteins. Here we studied the influence of sucrose and trehalose during SCF drying on the protein stability and the physical powder characteristics of lysozyme and myoglobin formulations. The results obtained with SCF drying were compared with the results after freeze-drying of the same solutions. Aqueous protein solutions, with or without sugar, were sprayed into a SCF mixture of carbon dioxide and ethanol. The dried products were analyzed by residual water measurements, scanning electron microscopy, X-ray powder diffraction and differential scanning calorimetry. After reconstitution the protein structure was studied by UV/VIS, circular dichroism and fluorescence spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and bioactivity assay (lysozyme). The SCF dried and freeze-dried formulations showed comparable water contents, but their physical properties were substantially different. All freeze-dried cakes were amorphous with fully preserved protein structure. SCF dried sucrose-containing formulations showed agglomerated crystalline particles, whereas SCF dried trehalose-containing formulations appeared to consist of amorphous spherical particles. Particle morphology of excipients-free proteins was protein specific. Nearly all SCF dried lysozyme could be readily reconstituted, but for myoglobin significant fractions of SCF protein did not dissolve, especially in the absence of sugars. Covalent aggregation was not observed for the two proteins. For the recovered soluble fractions, the secondary protein structure was preserved. The tertiary structure was preserved for lysozyme, but not entirely for myoglobin. Surprisingly, during SCF drying trehalose was less protective than sucrose for myoglobin.     

A New Strategy to Stabilize Oxytocin in Aqueous Solutions: I. The Effects of Divalent Metal Ions and Citrate Buffer

In the current study, the effect of metal ions in combination with buffers (citrate, acetate, pH 4.5) on the stability of aqueous solutions of oxytocin was investigated. Both monovalent metal ions (Na(+) and K(+)) and divalent metal ions (Ca(2+), Mg(2+), and Zn(2+)) were tested all as chloride salts. The effect of combinations of buffers and metal ions on the stability of aqueous oxytocin solutions was determined by RP-HPLC and HP-SEC after 4 weeks of storage at either 4°C or 55°C. Addition of sodium or potassium ions to acetate- or citrate-buffered solutions did not increase stability, nor did the addition of divalent metal ions to acetate buffer. However, the stability of aqueous oxytocin in aqueous formulations was improved in the presence of 5 and 10 mM citrate buffer in combination with at least 2 mM CaCl(2), MgCl(2), or ZnCl(2) and depended on the divalent metal ion concentration. Isothermal titration calorimetric measurements were predictive for the stabilization effects observed during the stability study. Formulations in citrate buffer that had an improved stability displayed a strong interaction between oxytocin and Ca(2+), Mg(2+), or Zn(2+), while formulations in acetate buffer did not. In conclusion, our study shows that divalent metal ions in combination with citrate buffer strongly improved the stability of oxytocin in aqueous solutions.     

The effects of freeze-drying on the stability of liposomes to jet nebulization

Egg phosphatidylcholine liposomes were freeze-dried in the presence and absence of trehalose. The lyophilized liposomes were rehydrated and aerosolized using a Pari LC jet nebulizer. The size of the aerosols generated was determined by laser diffraction, which was also used to determine the size distribution of the liposomes before lyophilization, post-rehydration, in the nebulizer post-aerosolization and those deposited in the two stages of a twin impinger. In the absence of trehalose, large liposomes and vesicle aggregates were produced on rehydration, which were rapidly reduced in size on nebulization. Liposomes with a mean size of 1 or 2.5 microm, freeze-dried with trehalose, had a mean size less than 3 microm following rehydration and exhibited enhanced stability to nebulization. Liposomes of 1 microm before freeze-drying were evenly distributed within aerosols generated by the nebulizer, whilst aerosols generated from 2.5 microm liposomes were fractionated in the twin impinger with the largest liposomes collected in the upper stage.     

Spray-drying of proteins: effects of sorbitol and trehalose on aggregation and FT-IR amide I spectrum of an immunoglobulin G.

An immunoglobulin G (IgG) was spray-dried on a Buchi 190 laboratory spray-dryer at inlet and outlet air temperatures of 130 and 190 degrees C, respectively. The IgG solution contains initially 115 mg/ml IgG plus 50 mg/ml sorbitol. After dialysis, at least 80% of low molecular weight component was removed. After spray-drying the dialyzed IgG and immediate redissolution of the powder, an increase in aggregates from 1 to 17% occurred. A major shift towards increase beta-sheet structure was detected in the spray-dried solid, which, however, reverted to native structure on redissolution of the powder. A correlation between aggregation determined by size exclusion chromatography and alterations in secondary structure determined by Fourier transformation infra-red spectroscopy could not therefore be established. On spray-drying a non-dialyzed, sorbitol-containing IgG only some 0.7% aggregates were formed. The sorbitol is therefore evidently able to stabilize partially the IgG during the process of spray-drying. Addition of trehalose to the liquid feed produced quantitatively the same stabilizing action on the IgG during spray-drying as did the sorbitol. This finding again points towards a water replacement stabilization mechanism. The IgG spray-dried powder prepared from the dialyzed liquid feed showed continued substantial aggregation on dry storage at 25 degrees C. This was substantially less in the non-dialyzed, sorbitol-containing spray-dried powder. Addition of trehalose to both dialyzed and non-dialyzed system produced substantial improvement in storage stability and reduction in aggregate formation in storage. The quantitative stabilizing effect of the trehalose was only slightly higher than that of the sorbitol. Taken together, these results indicate that both the sorbitol and trehalose stabilize the IgG primarily by a water replacement mechanism rather than by glassy immobilization. The relevance of this work is its questioning of the importance of the usually considered dominance of glassy stabilization of protein in dried systems of high glass transition temperature, such as trehalose. The low glass transition temperature sorbitol produces almost equal process and storage stability in this case.     

Freeze-drying of squalenoylated nucleoside analogue nanoparticles

Nucleoside analogues are potent anticancer or antiviral agents that however display some limitations (rapid metabolism, induction of resistance). In order to overcome these drawbacks, we recently proposed new prodrugs, in which nucleoside analogues were covalently coupled to squalene (SQ). The resulting amphiphilic compounds spontaneously formed nanoparticles (NPs) and displayed a promising efficacy both in vitro and in vivo. Since long-term stability is essential for further clinical development we needed to develop a laboratory-scale freeze-drying protocol in order to improve the colloidal stability of those NPs. Squalenoylated gemcitabine (SQdFdC) has been successfully freeze-dried with trehalose (10%, w/w) as a cryoprotectant. Concentrations of SQdFdC up to 4 mg/mL after freeze-drying and rehydration have been obtained, which is necessary for in vivo studies. Stability measurements by dynamic light scattering showed that trehalose had a stabilizing effect on SQdFdC NPs, and that freeze-dried SQdFdC NPs could be stored up to four months at room temperature before rehydration, without loss of stability. In vitro cytotoxicity studies on three murine cell lines showed that SQdFdC NPs retained their cytotoxic activity after freeze-drying. We showed that this freeze-drying protocol could also be applied to squalenoylated didanosine (SQddI) and zalcitabine (SQddC). Overall, these results allow for the use of freeze-dried NPs in upcoming preclinical trials of the different squalenoylated compounds developed in our laboratory.