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PubMed2004

Chromosomal integrity of freeze-dried mouse spermatozoa after 137Cs gamma-ray irradiation.

This study demonstrated that freeze-dried mouse spermatozoa possess strong resistance to 137Cs gamma-ray irradiation at doses of up to 8 Gy. Freeze-dried mouse spermatozoa were rehydrated and injected into mouse oocytes with an intracytoplasmic sperm injection (ICSI) technique. Most oocytes can be activated after ICSI by using spermatozoa irradiated with gamma-rays before and after freeze-drying. Sperm chromosome complements were analyzed at the first cleavage metaphase. Chromosome aberrations increased in a dose-dependent manner in the spermatozoa irradiated before freeze-drying. However, no increase in oocytes with chromosome aberrations was observed when fertilized by spermatozoa that had been irradiated after freeze-drying, as compared with freeze-dried spermatozoa that had not been irradiated. These results suggest that both the chromosomal integrity of freeze-dried spermatozoa, as well as their ability to activate oocytes, were protected from gamma-ray irradiation at doses at which chromosomal damage is found to be strongly induced in spermatozoa suspended in solution.

Animals↗

PubMed2004

Freeze-drying of mannitol-trehalose-sodium chloride-based formulations: the impact of annealing on dry layer resistance to mass transfer and cake structure.

The objective of this article was to study the mechanism by which annealing increases the primary drying time in mannitol-trehalose-sodium chloride-based formulations. The thermal events occurring during annealing and the glass transition of the frozen solutions were monitored with differential scanning calorimetry (DSC). Manometric temperature measurement was used to evaluate the dry layer resistances during primary drying. The morphologies of the freeze-dried cakes were examined by scanning electron microscopy (SEM). The degrees of crystallinity of mannitol and sodium chloride (NaCl) in freeze-dried cakes were determined by powder X-ray diffraction (XRD). DSC results indicated that annealing during freezing did not increase the glass transition temperature (Tg') significantly, but there was a distinct decrease of deltaCp at Tg' with annealing, suggesting a decrease in amorphous content. SEM revealed that most mannitol crystallized as the delta-form during annealing at -23 degrees C, and further crystallized as the alpha-form, together with NaCl crystallization, during subsequent annealing at -33 degrees C. The powder XRD results demonstrated that annealing caused crystal growth of mannitol and NaCl, and thus prevented the partial collapse observed without annealing. However, the highly crystallized mannitol blocked the pathways for water vapor escape, contributing to the increase in the dry layer resistance and thus the longer times for primary drying. Freeze-dried cakes without annealing had lower dry layer resistances because partial collapse created larger channels for water vapor escape. Therefore, two-step annealing in freezing makes mannitol-trehalose-sodium chloride-based formulations robust in freeze-drying, but annealing increases the dry layer resistances, thereby extending primary drying.

Chemistry, Pharmaceutical↗

PubMed1982

Sensitivity and specificity of immunohistological methods on freeze-dried paraffin sections.

The application of freeze-dried paraffin sections for immunohistology eliminates many of the problems associated with the cryostat technique: the sections are thin sufficiently, the cell morphology is improved, there are no diffusion artifacts and the intensity of immunohistological reactions is superior to that obtained on cryostat sections. The preservation of antogenicity in freeze-dried paraffin sections is sufficient for the demonstration of light chain monotypia in lymphomas of B-cell origin. Indirect immunofluorescence is the method of choice for demonstrating surface Ig in freeze-dried paraffin sections.

Animals↗

PubMed2003

Effect of pH value of freeze-drying solution on the chromosome integrity and developmental ability of mouse spermatozoa.

The nuclei of freeze-dried mouse spermatozoa are able to retain their chromosome integrity and developmental potential. To optimize the conditions of freeze-drying, we examined whether pH values of the freeze-drying solution affect the chromosome integrity and developmental potential of sperm nuclei. The sperm freeze-drying solution we used contained a high concentration (50 mM) of calcium-chelating EGTA. Sperm chromosomes were examined at the metaphase of the first mitosis after injection of freeze-dried spermatozoa into matured oocytes. The developmental potential of sperm nuclei was assessed by examining the development of fetuses in midgestation. The results showed that both sperm chromosomes and sperm developmental potential are maintained better when the freeze-drying solution was slightly alkaline (pH 8.0) rather than near neutral or acidic (pH 7.4-6.0). The data indicated that the chromosome integrity and developmental ability of mouse spermatozoa are affected by the pH value of freeze-drying solution.

Animals↗

PubMed2006

Freeze-drying of nanoparticles: formulation, process and storage considerations.

Freeze-drying has been considered as a good technique to improve the long-term stability of colloidal nanoparticles. The poor stability in an aqueous medium of these systems forms a real barrier against the clinical use of nanoparticles. This article reviews the state of the art of freeze-drying nanoparticles. It discusses the most important parameters that influence the success of freeze-drying of these fragile systems, and provides an overview of nanoparticles freeze-drying process and formulation strategies with a focus on the impact of formulation and process on particle stability.

Chemical Phenomena↗

PubMed1991

The effects of formulation variables on the stability of freeze-dried human growth hormone.

Formulation often has a dramatic effect on degradation of proteins during the freeze-drying process as well as impacting on the "shelf-life" stability of the freeze-dried product. This research presents the results of a formulation optimization study of the "in-process" and shelf-life stability of freeze-dried human growth hormone (hGH). Chemical decomposition via methionine oxidation and deamidation of asparagine residues as well as irreversible aggregation were characterized by HPLC assay methodology. In-process degradation and stability of low moisture freeze-dried solids were studied at 25 and 40 degrees C in a nominal nitrogen headspace (approximately 0.5% O2). Formulation variables included pH, level of salts, and the nature of the lyoprotectant. Studies of the effect of shear on aggregation in solutions indicated that shear comparable to that experienced during filtration does not induce aggregation. Irreversible changes in hGH during the freeze-drying process were minimal, but chemical decomposition via methionine oxidation and asparagine deamidation and aggregation did occur on storage of the freeze-dried solid. Decomposition via methionine oxidation was significant. A combination of mannitol and glycine, where the glycine remains amorphous, provided the greatest protection against decomposition and aggregation. It is postulated that an excipient system that remains at least partially amorphous is necessary for stabilization. However, the observation that dextran 40 formulations showed poor stability toward aggregation demonstrates that an amorphous excipient system is not a sufficient condition for stability. Stability of the solid was optimal when produced from solutions in the pH range, 7-7.5, with severe aggregation being observed at high pH. The level of sodium phosphate buffer affected stability of the solid, although this relationship was complex. Freeze-drying in the presence of NaCl produced severe aggregation and precipitation during the freeze-drying process as well as acceleration of oxidation and/or deamidation.

Buffers↗

PubMed1967

Influence of platen temperatures and relative humidity during storage on the survival of freeze-dried Salmonella typhimurium.

Salmonella typhimurium survived freeze-drying at a platen temperature of 120 F (48.9 C) and also, though to a much lesser degree, at 160 F (82.6 C). The extent of the survival at these temperatures was dependent on the composition of the model system employed. The incidence of damage immediately after freeze-drying was greater for cells dried at the higher platen temperature and was influenced by the composition of the menstruum in which the cells were dried. In model systems having protein-dominant isotherms, survival during subsequent storage depended greatly on relative humidity, with recovery highest at relative humidities below those corresponding to moisture contents at which a monomolecular layer is formed. In menstrua having a higher sugar content, survival was best at low relative humidities corresponding to a very low equilibrium moisture content in the model system used. Damage during storage tended to be a function of the composition of the gels in which the organisms were freeze-dried, and also depended greatly on the presence of air and on the relative humidity. The maximal percentage of damage usually occurred at the low relative humidities as storage time increased.

Freeze Drying↗

PubMed1999

A comparison of the inter- and intragranular drug migration in tray- and freeze-dried granules and compacts.

The influence of the drying process (tray- or freeze-drying), the granulation liquid viscosity, and the drying time during tray-drying on the inter- and intragranular migration of both a water-soluble and a poorly water-soluble compound in a granulation mass and in a compact were quantitatively assessed. Quinidine bisulfate (QBS) and riboflavin (RB) were mixed with a model excipient (glass microspheres) and granulated with an aqueous povidone solution. The drug concentration at different locations inside the granulated mass and in the compacts after drying was determined spectrophotometrically. The RB distribution in the compacts was more homogeneous than the QBS distribution. For both drugs it was clear that the intragranular migration decreased as the granulation liquid viscosity increased. The RB distribution in oven-dried granulation masses showed no migration problem, whereas the QBS samples were characterized by drug-enriched outer layers and drug-depleted inner regions, indicating a strong migration phenomenon. The drug distribution in all freeze-dried samples was homogeneous. A homogeneous distribution of a small-dosed water-soluble and a water-insoluble drug in granules and in compacts was obtained by the use of freeze-drying. The intragranular migration of QBS was prevented by the use of twice the amount of binder compared to that in RB compacts.

Desiccation↗

PubMed1999

Viability and thermal stability of a strain of Saccharomyces cerevisiae freeze-dried in different sugar and polymer matrices.

The viability and thermal stability of a freeze-dried yeast strain were studied in relation to some physical properties of the matrices in which the cells were freeze-dried. Samples of inoculum with solutions of the matrix components [polyvinylpyrrolidone (PVP), maltose, trehalose, maltodextrins, or mixtures of maltodextrin and trehalose] and controls without matrices were freeze-dried and then equilibrated at several relative humidities. Viability was determined before and after freeze-drying and after heat treatment (100 min at 70 degrees C). Freeze-drying with trehalose, PVP, maltose or 1.8-kDa maltodextrin, and mixtures of maltodextrin/trehalose increased viability in comparison with controls. The 3.6-kDa maltodextrin was ineffective at protecting the cells during freeze-drying. The glass transition temperature (Tg), which depends on moisture content, was indicated as a possible factor to determine the stability of labile materials. Protective effects of the excipients during thermal treatment were analysed in relation to the physical changes (collapse or structural shrinkage) which were dependent on the Tg of the systems. The presence of a certain amount of amorphous disaccharides during freeze-drying and heating was found to be a critical factor for ensuring cell viability, which was protected even in rubbery (above Tg) matrices.

Freeze Drying↗

PubMed1976

The physico-chemical basis for the freeze-drying process.

To the extent that the final form and quality of a freeze-dried product depends on the way the freeze-drying is conducted, an understanding of the many factors involved is most important. The numerous effects of the design and mode of operation of the freeze-drying equipment on the course of the process need to be known, as do the properties intrinsic to the material to be freeze-dried. Much can be learned and predicted from the study of the "supplemented phase diagram", a series of experimental plots describing the equilibrium and the non-equilibrium phase behavior of the system in question. Such diagrams map and distinguish eutectic and amorphous phase behavior. Further information is available from gravimetric studies allowing the construction of "desorption isotherms", the plots describing the loss of sorbed water accompanying the sublimation of ice, frequently termed "secondary drying". These plots relate the water retained by the product to the "water activity", or relative humidity at different temperatures. Observations in the freeze-drying microscope contribute additional information, in that they reveal the actual course of the process at the microscopic level. These and other laboratory findings facilitate the analysis and comparison of pilot-plant and commercical scale processing experiences. Where scientific and engineering factors appear to interrelate, the nature and extent of the interdependence can often be determined.

Crystallization↗

PubMed2004

Protein powders for encapsulation: a comparison of spray-freeze drying and spray drying of darbepoetin alfa.

PURPOSE: To evaluate spray-freeze drying and spray drying processes for fabricating micron-sized particles of darbepoetin alfa (NESP, Aranesp) with uniform size distribution and retention of protein integrity, requirements for encapsulation. METHODS: Darbepoetin alfa was spray-freeze dried using ultrasonic atomization at 120 kHz and 25 kHz and spray dried at bench-top and pilot scales. Reconstituted powders were evaluated by size exclusion chromatography and UV/VIS spectroscopy. Powder physical properties were also characterized. RESULTS: Spray-freeze drying resulted in aggregation of darbepoetin alfa. Aggregates (primarily insoluble) formed on drying and/or reconstitution. Particle size distributions were broad (span > or = 3.6) at both nozzle frequencies. Annealing before drying reduced aggregate levels slightly but increased particle size over 5-fold. Spray drying at inlet temperatures up to 135 degrees C (and outlet temperatures up to 95 degrees C) showed little impact on integrity. Integrity at bench-top and pilot scales was identical, with 0.2% dimer and no high molecular weight or insoluble aggregates detected. Particle size was small (< or = 2.3 microm) with uniform distribution (span < or = 1.4) at both process scales. CONCLUSIONS: Under the conditions tested spray drying, conducted at bench-top and pilot scales with commercially available equipment, was superior to spray-freeze drying for the fabrication of darbepoetin alfa particles for encapsulation.

Chemistry, Pharmaceutical↗

PubMed2005

Airborne PCB as a contamination risk on freeze-drying of sediment.

In a study of polychlorinated biphenyls (PCB) in freeze-dried sediments from Swedish lakes, the samples were found to contain unexpectedly high concentrations of PCBs. In one of the lakes (Björken), e.g. the concentration of PCB-28 was found to be 189 ng/g dw after freeze-drying compared to 7 ng/g dw after air-drying of the sediment and therefore our hypothesis was that the contamination had occurred during the freeze-drying procedure. Hence, a dry reference sediment (RS) was used in order to identify potential sources of PCB contamination. The investigation included freeze-drying of the dry RS in five different laboratories, exposure to the air in the freeze-drying room, storage at room temperature and air-drying in a certain fume hood and in a laminar flow clean bench. The pattern and concentrations of PCBs detected in the exposed RS were strongly influenced by low chlorinated PCB congeners under all of these conditions with exception for air-drying in the fume hood. Storage in the laboratory air resulted in a slight elevation in the concentrations of low chlorinated PCB congeners, whereas higher concentrations of all PCB congeners were observed after freeze-drying. In all experiments the contamination appeared to be due to adsorption of PCB from the laboratory air. On the basis of these findings we recommend that reference material be subjected to the entire procedure, including sample preparation, in order to monitor contamination and that a procedure involving solvent extraction of wet sediment samples is used, whenever possible, instead of procedures involving dry samples.

Adsorption↗

PubMed1978

Freeze-dried allogeneic segmental cortical-bone grafts in dogs.

Forty-four adult male mongrel dogs were used to compare segmental cortical freeze-dried allogeneic bone grafts with fresh autogenous, freeze-dried autogenous, and fresh allogeneic segmental cortical grafts. Group I consisted of bilateral fresh autografts as external controls; Group II, a fresh autograft on one side for internal control and a freeze-dried autogenous graft on the other side to evaluate the effect of freeze-drying on repair; Group III, a fresh autograft on one side and a fresh allograft on the other side to measure the differences between autogenous and allogeneic grafts; and Group IV, a fresh autograft on one side and a freeze-dried allogeneic graft on the other to see if freeze-drying altered the repair of allogeneic grafts. The grafts were analyzed qualitatively over a six-month period by the use of interval roentgenograms to determine the times of graft-host union and the incidence of fatigue fractures. Six months after operation, the repair processes in the four groups were compared quantitatively with respect to biological repair and physical strength using torsional stress-testing, tetracycline labeling, and microradiography. The results indicate both qualitatively and quantitatively that: (1) fresh bilateral segmental cortical autografts show reproducible characteristics, so that the canine fibula furnishes a satisfactory model (Group I); (2) freeze-drying does not inhibit the repair process per se (Group II); (3) fresh allografts are rejected in varying degrees of intensity (Group III); and (4) freeze-drying does not protect cortical allogeneic grafts from such rejection (Group IV).

Animals↗

PubMed1980

Conductive coatings studied on inflated lung in the frozen-hydrated and freeze-dried states.

Evaporative coatings of platinum-carbon and gold on freeze-fractured mouse lung, preserved in frozen-hydrated and freeze-dried states, are studied in secondary emission mode by low temperature scanning electron microscopy. Unfixed, inflated frozen lung is freeze-etched in high vacuum, metal coated and observed frozen-hydrated and freeze-dried, to demonstrate sample shrinkage and coating deformation upon drying the sample. Buckling and cracking of the coatings occur as a result of freeze-drying. Correlative TEM thin film analysis of the coatings is used to estimate film thickness and reticularity. Freeze-drying biological samples is a common SEM preparative technique which causes morphological artifacts due to both freezing and drying. Frozen-hydrated low temperature-SEM investigation can selectively study these artifacts morphologically to distinguish between freezing and drying artifacts.

Animals↗

PubMed2004

Design of freeze-drying processes for pharmaceuticals: practical advice.

Design of freeze-drying processes is often approached with a "trial and error" experimental plan or, worse yet, the protocol used in the first laboratory run is adopted without further attempts at optimization. Consequently, commercial freeze-drying processes are often neither robust nor efficient. It is our thesis that design of an "optimized" freeze-drying process is not particularly difficult for most products, as long as some simple rules based on well-accepted scientific principles are followed. It is the purpose of this review to discuss the scientific foundations of the freeze-drying process design and then to consolidate these principles into a set of guidelines for rational process design and optimization. General advice is given concerning common stability issues with proteins, but unusual and difficult stability issues are beyond the scope of this review. Control of ice nucleation and crystallization during the freezing step is discussed, and the impact of freezing on the rest of the process and final product quality is reviewed. Representative freezing protocols are presented. The significance of the collapse temperature and the thermal transition, denoted Tg', are discussed, and procedures for the selection of the "target product temperature" for primary drying are presented. Furthermore, guidelines are given for selection of the optimal shelf temperature and chamber pressure settings required to achieve the target product temperature without thermal and/or mass transfer overload of the freeze dryer. Finally, guidelines and "rules" for optimization of secondary drying and representative secondary drying protocols are presented.

Chemical Phenomena↗

PubMed1989

The effect of cooling rate, freeze-drying suspending fluid and culture age on the preservation of Campylobacter pylori.

The effects of freezing rate, suspending fluid and age of culture on the ability of four strains of Campylobacter pylori to survive and recover from freeze-drying were examined. Freeze-drying by standard procedures generally resulted in an overall loss in viability of between 3 and 7 log units. The exact cause of poor recovery by C. pylori was not established but strain differences were detected, with NCTC 11637 (type strain) surviving better than NCTC 11638 and NCTC 11639. Recovery of the poorest growing strain (NE 26695) was notably more erratic. The largest loss in viability occurred at the primary drying stage. Losses resulting from freezing and secondary drying were less marked and the rate of freezing had only a marginal effect on recovery. Nineteen different freeze-drying suspending fluids were investigated. Overall the best recovery results were obtained with 5% inositol-broth (or horse serum) plus 25% glucose, at pH 7.0, in which loss of viability was typically about 4 log units. Other factors, such as age of culture and number of viable bacteria in the before-dry suspension, did not have a significant effect on survival. We conclude from these results that C. pylori can survive freeze-drying, albeit in small numbers, but the degree of recovery is apparently largely strain dependent.

Campylobacter↗

PubMed2000

Freeze-drying allows double nonradioactive ISH and antigenic labeling.

Because tissue freeze-drying is an excellent way to preserve antigenic conformation, we have tested the feasibility of this technique to reveal nonradioactive in situ hybridization (ISH) of tissue mRNA. We have compared mRNA detection after different methods of tissue preservation, freeze-drying, cryosectioning, and formaldehyde or methanol fixation. Our results show that nonradioactive ISH is more sensitive for tissues preserved by freeze-drying than for other tissue preparations. We have demonstrated that freeze-drying allows combination of ISH and immunohistochemistry for simultaneous detection of mRNA and antigen because with this technique of tissue preservation ISH does not affect the sensitivity or the amount of the detected antigens. This work underscores the fact that tissue freeze-drying is an easy, convenient, and reliable technique for both ISH and immunohistochemistry and achieves excellent structural conditions for nonradioactive detection.

Animals↗

PubMed2001

A Pilot Study Evaluating Sodium Hyaluronate as a Carrier For Freeze-dried Demineralized Bone Powder.

Freeze-dried demineralized bone (FDDB) is widely used in orthopaedic and neurosurgery. However, it is difficult to deliver the FDDB by itself and surgeons have been seeking a convenient and easy-to-use form of FDDB. This study was designed to evaluate sodium hyaluronate (HA) as a carrier or delivery vehicle. HA is a viscous and biocompatible high molecular weight hydrogel. It was tested as a viscous carrier to facilitate the delivery of freeze-dried demineralized bone into a bony defect. The natural bone forming properties of FDDB were evaluated in an athymic mouse model and shown to be as active when dispersed in HA as compared to a saline control. Particle sizes in the range studied (125-850 microm) had no effect on FDDB efficacy. All exhibited active bone formation. Similarly, two different carrier viscosities were formulated to achieve both a malleable putty and a more flowable paste. These, too, both exhibited active bone formation. The use of a pH 7.4 buffer in the HA formulation gave optimum results in the model. Additional studies are underway to further evaluate the surgical efficacy of FDDB suspended in HA.

Journal Article↗