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PubMed1985

Freeze-dried vaccine against Rinderpest: stability and activity study.

A freeze-dried vaccine against Rinderpest was prepared from modified virus multiplied in calf kidney cell culture. Characteristics of the vaccine are as follows: high titre after freeze-drying (10(4) CCID50/dose), well-adapted freeze-drying stabilizer which ensures maintenance of the infective titre of the vaccinal virus, even under severe conditions (3.5 days at +45 degrees C), use of an appropriate solvent: magnesium sulphate molar solution or more simply physiological saline (for stability after reconstitution even at high temperatures--up to 4 h at +45 degrees C). The activity of the vaccine, tested in cattle by antibody titration and resistance to specific challenge perfectly satisfies requirements set by the WHO and OIE.

Animals↗

PubMed1995

Increased stabilizing effects of amphiphilic excipients on freeze-drying of lactate dehydrogenase (LDH) by dispersion into sugar matrices.

PURPOSE: The stabilizing effect of amphiphilic excipients and sugars against protein inactivation during freeze-drying was studied in relation to their physical states in freeze-dried cakes. METHODS: Physical states of amphiphilic excipients were studied by powder X-ray diffractometry and differential scanning calorimetry (DSC). Stabilizing effects of excipients were studied using lactate dehydrogenase (LDH) as a model protein. RESULTS: Although poly(ethylene glycols) (PEGs) 1000 to 2000 crystallized when freeze-dried alone, the addition of sugars decreased their crystallinity by dispersing PEG into sugar-dominant matrices. Sugars species, molecular weight of PEGs, and buffer concentration also affected the crystallinity of PEGs. Sugars also dispersed some of other amphiphilic excipients, which tended to crystallize or become "microscopically liquid" when freeze-dried without sugar. Only the amphiphilic excipients that remained amorphous solid state protected the enzyme during freeze-drying in the absence of sugars. However, combinations of sucrose and all the amphiphilic excipients studied increased the stabilizing effects markedly. The remaining activities were greater than the sum of their individual ones. CONCLUSIONS: Various amphiphilic excipients are good stabilizers for freeze-drying of proteins when dispersed into sugar-dominant matrices.

Dose-Response Relationship, Drug↗

PubMed1976

An improved freeze-dried bilirubin preparation.

We describe the conditions for successful freeze-drying of bilirubin solutions in the presence of dimethylsulfoxide and human serum albumin. Freeze-dried bilirubin preparations rehydrate readily, rapidly resulting in clear solutions. The preparations exhibit excellent homogeneity and stability at -20 degrees C. Accelerated storage tests indicate that the freeze-dried product would also be stable at higher temperatures but our results do not permit, at the present time, to make accurate predictions regarding the stability at 4 degrees C or 0 degrees C. We believe that our approach will permit manufacturers of bilirubin calibrators for clinical use to improve the quality of their products.

Animals↗

PubMed1997

Enhanced permeability of freeze-dried liposomal bilayers upon rehydration.

Until now, studies on the protection of liposomes against freeze-drying damage have mainly focused on the bilayer integrity during the freezing or drying step of this process. Here, we investigated the bilayer permeability of freeze-dried, lyoprotected liposomes to a nonbilayer interacting compound after rehydration, by monitoring the leak-in kinetics of externally added carboxyfluorescein (CF). The results showed that freeze-drying and rehydration of DPPC:DPPG 10:1 liposomes with sucrose in- and outside the vesicles caused a temporary increase in the bilayer permeability for CF, which leveled off after approximately 20 h. The amount of CF/mol phospholipid which leaked into the vesicles increased with vesicle size (range 0.1-1 micro m) / lamellarity. Reduction of the number of bilayers in 1-1 micro m) vesicles enhanced the permeability to CF after freeze-drying and rehydration. The presence of CHOL decreased CF leak-in rates into 1 micro m MLVs consisting of DPPC:DPPG 10:1, but not into 0.1-micro m unilamellar vesicles. In the absence of sucrose similar leak-in profiles as a function of time were found after rehydration, suggesting that repacking processes of the bilayer were responsible for the enhanced permeability after freeze-drying and dehydration both with and without sucrose. The effect of size and lamellarity on the CF leak-in correlated with the retention of encapsulated CF after freeze-drying and rehydration, but no correlation was found with the effect of lipid composition. Both small (0.1 micro m) lyoprotected liposomes made of DPPC:DPPG 10:1 and DPPC:DPPG:CHOL 10:1:4 were highly permeable during the rehydration step itself. The results indicate that, despite the presence of the lyoprotectant, "repacking" of the bilayer components takes place both during and after rehydration. This eventually leads to regaining of its barrier function.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1992

Scanning electron microscopic and light microscopic observations on morphological changes of freeze-dried bone implantation in rats: comparison with fresh autogenous bone transplantation.

Bone remodelling after the implantation of freeze-dried autogenous bone in rat parietal bone was compared with fresh autogenous bone transplantation, using a scanning electron and light microscope revealed the time intervals after transplantation/implantation. The light microscope revealed the time delay of the bone remodelling in the implantation, compared with the transplantations. The scanning electron microscope showed that the differences between the two groups were in the states of bone union and bone resorption. In the fresh bone group, the newly-formed bone filled the spaces between host and the transplanted bones at 2 to 3 weeks after the transplantation: the newly-formed bone fused and melted into the transplanted bone. New bone formation was more dominant on the bone surface in the dura mater side than in the skin side. The union was almost completed at 5 weeks. In freeze-dried bone implantation, the bone union in the contact space was very poor and the implanted bone was mainly covered by the new bone, which developed from the host bone surface in the dura mater side at 2 to 3 weeks after the implantation. What is noteworthy is that bone resorbed areas showing numerous Howship's lacunae were mainly observed on the host bone surface in the vicinity of newly-formed bone. However in freeze-dried bone implantation, the bone resorption was greater on the host and implanted bone surface than that of fresh bone transplantation: the resorption of host bone was considerably larger at certain periods after freeze-dried bone implantation. The present results show that the healing process of freeze-dried bone implantation, even though autogenous bone was used, differed from that of fresh autogenous bone transplantation, and the differences are concerned not only with time sequences but also with qualitative changes. This suggests that the host would have some different responses to the freeze-dried autogenous bone from fresh materials.

Animals↗

PubMed1990

Electrophoretic analysis of type I collagen from bovine Achilles tendon: comparison between the extracted raw material and the freeze-dried product.

The present study was aimed at verifying if the freeze-drying process has any effect on the polypeptide composition of the collagen extracted from bovine Achilles tendon in an acetic acid gelified form. Data from our laboratories showed that the freeze-drying process renders the collagen gel essentially insoluble; under these conditions the collagen sample can no longer be analyzed by gel electrphoresis. We found that treatment of the sample with pepsin in acid environment, followed by precipitation with ammonium sulphate yields an insoluble fraction that is susceptible of being analyzed by polyacrylamide gel electrophoresis. The The electrophoresis, run under standard conditions, shows that six major subunits, corresponding to alpha, beta and gamma polypeptides, can be revealed in the sample treated in this way. So the freeze-dried collagen exhibits a polypeptidic composition that is basically identical to that shown by the collagen gel, with regard to the fraction precipitated with ammonium sulphate. Otherwise the pattern of the enzymatic hydrolysis was investigated by measuring the hydroxyproline content, and so the collagen content using the 7.46 conversion factor from hydroxyproline to the scleroprotein collagen, in the various steps of the hydrolysis itself: the analytical results showed no differences between the freeze-dried collagen and the gelified form; this confirms that the lyophilization process does not alter the polypeptidic composition of the collagen in any way.

Achilles Tendon↗

PubMed1997

Electron Microscopy of Modified Aluminum Alkoxide Microstructures on Freeze-Drying

Direct imaging of microstructures in chelated aluminum alkoxide systems subjected to freeze-drying and freeze-thaw-drying is reported using cryo-scanning electron microscopy (SEM) and SEM techniques. Aluminum sec-butoxide was chelated with a beta-diketoester in different molar ratios. This system was gelled by hydrolysis under intense sonication. The sonogels formed were subjected to cryo-SEM. Samples for cryo-SEM were made by freezing the chelated alkoxide samples in liquid nitrogen and freeze-fracturing them inside the cryo-SEM and monitoring the apparent morphological changes on exposure to the cryo-SEM environment including intentional sample heating. Microstructures of unchelated, 1:1 chelated and 1:2 chelated (alkoxide:chelate) sonogels after freezing, thawing, and drying were also imaged by SEM techniques. Heterogeneity introduced by chelation of alkoxide is proposed as the cause for the formation of flakes in freeze-thaw-dried chelated alkoxide gels. Microstructures of 1:1 chelated freeze dried alkoxide were compared with the freeze-thaw-dried gels. The absence of flakes in the chelated samples when subjected to in situ freeze-drying indicated reversible nature of the gels.

Journal Article↗

PubMed2002

Sequential dependence of freeze-drying and irradiation on biomechanical properties of rat bone.

Femurs of Sprague-Dawley rats were subjected to in vitro 3-Mrad irradiation and/or freeze-drying to investigate whether these processes have an order-dependent effect on the biomechanical properties of bone. Four experimental groups were designated-irradiated (RAD); freeze-dried (FD); irradiated, then freeze-dried (RAD/FD); and freeze-dried, then irradiated (FD/RAD). After the various treatments, the bones were inspected for microfractures and then torsion-tested. Microfractures were seen in more than 90% of the specimens that had undergone freeze-drying alone or with irradiation. Regarding the normalized relative ratios of torque, there was a statistically significant difference (P < .05) between the RAD group (1.0) and the 3 other groups (FD, .32; RAD/FD, .40; FD/RAD, .14). Differences among the FD, RAD/FD, and FD/RAD groups were not significant. However, a trend was bones were weaker than FD and noted: FD/RAD RAD/FD bones.

Animals↗

PubMed1987

Freeze-dried segmental fibular allografts in azathioprine-treated dogs.

The successful use of a bone allograft may be negated by the host's immune response. This investigation assessed the efficacy of combining freeze-dried cortical allografts in three to six weeks azathioprine-immunosuppressed dogs. Forty-eight of 94 adult mongrel dogs were initiated for this study, and 46 of 94 were previously published and recompiled. The dogs were divided into five groups and followed for six months: Group I consisted of bilateral fresh autografts as an external control; Group II assessed the effect of freeze-drying on autogenous bone; Group III compared fresh autografts with fresh allografts; Group IV assessed the effect of freeze-drying on allografts; and Group V assessed the combined effect of placing freeze-dried allografts in immunosuppressed hosts. Biweekly roentgenograms were made to evaluate the time to union and the incidence of graft fatigue failure. Mechanical graft strength was assessed by rapid torsional loading to failure at the time of sacrifice. Biologic repair was assessed with the use of tetracycline and microradiographic techniques. The incorporation and repair of a fresh cortical autograft is better than that of a freeze-dried autograft because of fractures, nonunion, or delayed union of graft-host junctions; freeze-dried autografts have increased peripheral and internal resorption, yet an increased peripheral bony callus maintains normal graft strength; freeze-dried and fresh allografts are similar in roentgenographic characteristics, mechanical strength, and in the mechanism of graft incorporation; the use of three or six weeks azathioprine therapy did not improve the fate of freeze-dried allografts.

Animals↗

PubMed1981

Freeze-drying shrinkage of glutaraldehyde fixed liver.

Dimensional changes were recorded during the freeze drying (FD) of 1 mm cubes of glutaraldehyde (GA) fixed adult mouse liver. The areas of the front faces of these blocks was measured using a Quantimet 720 image analysing computer system. Interpolating the first straight line portion of the graphs of size versus time backwards to the origin allowed the determination of the original size even if the specimen was covered with some surface-water ice at the beginning of the experiment. GA fixed mouse liver shrinks 7.3% linearly during freeze drying. This gross shrinkage is not increased if the cold stage temperature is raised to approximately 263 K from approximately 223 K, but the rate of drying as monitored by the rate of shrinkage is greatly increased. If morphological specimens are to be prepared for scanning electron microscopy (SEM), freeze drying can be completed rapidly after an initial period at approximately 223 K, since ice recrystallization leading to increased ice crystal artefact will occur in the deep layers which will not be visible in the SEM. Shrinkage of single cells followed during freeze drying in the SEM showed similar gross dimensional changes of about 7.5% linear shrinkage to occur at and below 198 K.

Animals↗

PubMed2003

Preparation of interconnected highly porous polymeric structures by a replication and freeze-drying process.

Three-dimensional degradable porous polymeric structures with high porosities (93-98%) and well-interconnected pore networks have been prepared by freeze-drying polymer solutions in the presence of a leachable template followed by leaching of the template. Templates of the pore network were prepared by fusing sugar or salt particles to form a well-connected structure. The interstices of the template were then filled with a polymer solution (5-15% w/v) in 1,4-dioxane, followed by freeze-drying of the solvent. Subsequent leaching of the sugar template ensures the connectivity of the pore network. The scaffold architecture consists of relatively large interconnected pores modeled after the template and smaller pores resulting from the freeze-drying process. The total porosity of the resultant porous structures is determined by the interstitial space of the leachable template and by the polymer concentration in the freeze-drying solution. The freezing temperature also has an effect on the final morphology of the porous structures. Compared with freeze-drying and combination of freeze-drying /particulate leaching techniques, this method facilitates higher interconnectivity of the scaffolds. Porous structures have been prepared from several relevant polymers in the biomedical and tissue-engineering field: poly(D,L-lactide) (PDLLA), 1000PEOT70PBT30, a segmented poly(ether ester) based on polyethylene oxide and polybutylene terephthalate, and poly(epsilon-caprolactone) (PCL). The mechanical properties of the porous structures prepared by this technique depend on the nature of the polymer, porosity, and the freezing temperature. With porosities in the range of 95-97%, the compression moduli of scaffolds prepared from the different polymers could be varied between 13.0 and 301.5 kPa.

Biocompatible Materials↗

PubMed1987

Stabilization of phosphofructokinase with sugars during freeze-drying: characterization of enhanced protection in the presence of divalent cations.

Phosphofructokinase purified from rabbit skeletal muscle is fully inactivated after freeze-drying and dissolution. The addition of trehalose or maltose to the enzyme solution prior to freeze-drying results in a recovery of up to 80% of the original activity. Slightly less stabilization is imparted by sucrose, whereas glucose and galactose at concentrations up to 500 mM are relatively ineffective at protecting phosphofructokinase. Addition of ionic zinc to enzyme-sugar mixtures prior to freeze-drying greatly enhances the stabilization imparted by the above sugars. This effect is not simply due to the summation of the individual protective capacities of zinc and the sugar. Zinc alone affords no protection, but a high degree of stabilization is achieved when zinc is added to a sugar solution, even when the sugar is at a concentration at which, by itself, it is totally ineffective. In the presence of a constant sugar concentration (100 mM), freeze-dry stabilization of phosphofructokinase is increased as the concentration of zinc is increased. When the zinc concentration is held constant (0.9 mM) and the sugar concentration varied, the maximum stabilization is noted with less than 200 mM sugar. At higher solute concentrations the degree of enhancement decreases such that with 500 mM sugar the addition of zinc results in only a slight increase in protection. Several other organic solutes (proline, 4-hydroxyproline, glycine, trimethylamine N-oxide, glycerol and myo-inositol) that afford cryoprotection to phosphofructokinase, an effect enhanced by the addition of zinc, do not stabilize the enzyme during freeze-drying, even if zinc is present. The addition of ionic copper, cadmium, nickel, cobalt, calcium and manganese to trehalose-phosphofructokinase solutions prior to freeze-drying also increases the percentage of activity recovered after dissolution. Magnesium is ineffective in this respect.

Animals↗

PubMed1970

Characterization of injury incurred by Escherichia coli upon freeze-drying.

When cells of Escherichia coli ML30 were suspended in 2% gelatin and frozen at -40 C, no appreciable metabolic damage or death occurred. After freeze-drying for 8 hr at a platen temperature of 49 C and rehydration with a mineral salts medium, survival of the cells was 0.6%. Metabolic damage of the survivors was found to be 23%. Permeability alterations were detected by several criteria. Freeze-dried cells were susceptible to antibiotics normally ineffective against E. coli and leakage of ribonucleic acid (RNA) occurred. Analysis of ribosomal extracts of rehydrated freeze-dried cells demonstrated the presence of appreciable degradation products. Permeability alterations were shown to be reversible by the observation that antibiotic susceptibility was a time-dependent process and that the gratuitous inducer of beta-galactosidase was not concentrated by freeze-dried cells until the injured cells had been incubated in a nutrient medium for 300 min or more. At approximately the same time, metabolic damage was repaired. RNA synthesis preceded protein synthesis by about 150 min, and deoxyribonucleic acid synthesis occurred with the resumption of normal growth. This was interpreted to be the result of repair of RNA taking place before protein synthesis and growth could resume. A pronounced increase in the lag time of freeze-dried cells was also observed. Peptides and Casamino Acids shortened the lag time for freeze-dried cells but not for the controls. Glycerol and glucose were found to be better carbon sources for growth of freeze-dried cells than sodium lactate or sodium succinate.

Amino Acids↗

PubMed2002

Low-temperature brewing by freeze-dried immobilized cells.

We propose a novel biocatalyst in brewing. A cryotolerant strain of Saccharomyces cerevisiae was immobilized on delignified cellulosic material followed by freeze-drying of the immobilized cells without the use of any cryoprotectant. The freeze-dried immobilized biocatalyst was used in repeated-batch fermentation of wort and showed reduced fermentation time and increased productivities as compared with free freeze-dried cells (FFDCs). It also demonstrated suitability for low-temperature brewing (5 and 0 degrees C). The fermentation time in repeated-batch fermentations at 15 degrees C was 1.5-2 d for a period of 13 mo, showing a high operational stability of the system. At 0 degrees C the freeze-dried immobilized biocatalyst showed a 2- to 3.5-fold decrease in fermentation time in comparison with FFDCs. Polyphenol contents, bitterness, and diacetyl concentration were lower in beers produced by freeze-dried immobilized cells as compared with FFDCs. At 0 degrees C polyphenols were 40% lower than at 15 degrees C. Higher alcohols were reduced and ethyl acetate increased in comparison with FFDCs. Amyl alcohols at 0 degrees C were lower than half of their content at 15 degrees C, while ethyl acetate was 31 mg/L at 0 degrees C and 18 mg/L at 15 degrees C. These data justify the improved aroma and taste of beers produced by freeze-dried immobilized biocatalyst mainly at low temperatures.

Beer↗

PubMed1997

Development of freeze-dried albumin-free formulation of recombinant factor VIII SQ.

PURPOSE: To develop a stable freeze-dried formulation of recombinant factor VIII-SQ (r-VIII SQ) without the addition of albumin. METHODS: Different formulations were evaluated for their protective effect during sterile filtration, freeze-thawing, freeze-drying, reconstitution and long term storage. Factor VIII activity (VIII:C), visual inspection, clarity, solubility, moisture content and soluble aggregates and/ or fragments were assayed. RESULTS: A combination of non-crystallising excipients (L-histidine and sucrose), a non-ionic surfactant (polysorbate 80) and a crystalline bulking agent (sodium chloride) was found to preserve the factor VIII activity during formulation, freeze-drying and storage. Calcium chloride was included to prevent dissociation of the heavy and light chains of r-VIII SQ. Sodium chloride was chosen as the primary bulking agent since the concentration of sodium chloride necessary for dissolution of r-VIII SQ in the buffer will inhibit the crystallization of many potential cake formers. It was found that L-histidine, besides functioning as a buffer, also protected r-VIII SQ during freeze-drying and storage. A pH close to 7 was found to be optimal. Some potential macromolecular stabilisers, PEG 4000, Haes-steril and Haemaccel, were evaluated but they did not improve the recovery of VIII:C. The freeze-dried formulation was stable for at least two years at 7 degrees C and for at least one year at 25 degrees C. The reconstituted solution was stable for at least 100 hours at 25 degrees C. CONCLUSIONS: The albumin-free formulation resulted in consistently high recovery of VIII:C, very low aggregate formation and good storage stability. The stability of the reconstituted solution makes the formulation suitable for continuous administration via infusion pump. The formulation strategy described here may also be useful for other proteins which require a high ionic strength.

Adsorption↗

PubMed2004

Effect of collapse on the stability of freeze-dried recombinant factor VIII and alpha-amylase.

Recombinant Factor VIII (rFVIII) and alpha-amylase were used as model proteins to examine the effect of freeze-drying process conditions on the long-term stability of these proteins as freeze-dried solids. The same sucrose/glycine formulation was used for all treatments. Three freeze-drying protocols were used-an "aggressive" and a "conservative" cycle that both produced pharmaceutically acceptable product, and a protocol that produced a collapsed matrix. For rFVIII, there was no difference in the biological activity versus the time profile for product freeze-dried under the three different conditions when stored at 5 or 25 degrees C. At 40 degrees C, however, the stability of the collapsed product appeared to be better than that of product freeze-dried with no collapse. Also, the level of residual moisture in the collapsed product was higher than that of the product with no collapse. For alpha-amylase, there was no significant difference in the stability profile at any of the temperatures over the time course of the study. The results support the conclusion that collapse is not necessarily detrimental to the long-term stability of freeze-dried proteins.

Animals↗

PubMed1995

The biomechanical properties of deep freezing and freeze drying bones and their biomechanical changes after in-vivo allograft.

This study measured the physical properties in bending of the rat femur and compression of the rat first tail vertebra subjected to deep freezing at -80 degrees C for 2 weeks, 6 weeks, 12 weeks and freeze drying. This study also measured the mechanical changes after in vivo allograft of fresh bone, deep freezing(2, 6, 12 weeks) and freeze drying. Analysis for deep freezing groups showed a mean 7.2% decrease in bending strength and 11.0% decrease in compressive strength when compared with the control group, but there was no statistical difference in the duration of deep freezing. The groups of in vivo graft after deep freezing showed 23.1% and 22.2% decrease in bending and compressive strength. There was no statistical difference in the duration of deep freezing. The freeze drying group showed a 9.7% decrease in bending strength and no significant difference in compressive strength. The group of in vivo graft after freeze drying showed a 30.1% and a 41.3% decrease in bending and compressive strength. The above results suggested that there would be some mechanical limitation in using freeze dried graft for supporting implants.

Animals↗

PubMed2006

Freeze-dried rehydrated human blood platelets regulate intracellular pH.

BACKGROUND: Long-term storage of platelets (PLTs) in the dry state would greatly improve options for PLT storage. Whether trehalose-loaded freeze-dried and rehydrated PLTs could regulate intracellular pH (pHi) was evaluated. STUDY DESIGN AND METHODS: Previously it was shown that human PLTs can be successfully preserved by freeze-drying with trehalose. Trehalose-loaded freeze-dried rehydrated PLTs and fresh control PLTs were labeled with the pH dye BCECF-AM. pHi was measured in resting cells, cells acidified with nigericin, and cells treated with thrombin. The sodium-proton pump was blocked by treatment with 5-(N-methyl-N-isobutyl)amiloride (MIA). RESULTS: The pHi of rehydrated PLTs is the same as that of fresh control PLTs, 7.27+/-0.03 (SD; n=5) and 7.27+/-0.02 (n=5), respectively. Nigericin treatment of cells showed that the recovery in pHi was Na+-dependent and followed Michaelis-Menten kinetics. The Vmax values (DeltapH/9 sec) were 0.21+/-0.039 (n=3) and 0.22+/-0.025 (n=3) for rehydrated and control PLTs, respectively. The exchange constants were 17.7+/-2.3 mmol per L (n=3) and 17.0+/-1.9 mmol per L (n=3) for rehydrated and control PLTs, respectively. Treatment of cells with MIA showed that NHE1 remained sensitive to the inhibitor after freeze-drying and rehydration. CONCLUSION: The results show that the pHi regulation system is largely preserved during freeze-drying and rehydration of PLTs.

Blood Platelets↗