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PubMed2001

Lipid extracted freeze-dried bank bone sterilized with low temperature plasma.

Ethylene oxide gas (EOG) is widely used for sterilization of freeze-dried bone. Recently EOG is regulated for clinical use because of cytotoxicity and environmental pollution concerns. Low Temperature Plasma Sterilization (LTPS) has no toxicity and mutagenicity working with a hydrogen peroxide gas plasma at 45 degrees centigrade. We have now used this method experimentally and clinically to sterilize freeze-dried bone. Cortical bone with a thickness of 5 mm and cancellous bone with a 10 mm cubic size were prepared, washed, defatted, dehydrated by freeze drying and finally sterilized by LTPS. As a Microbiological test, defatted freeze-dried bone was contaminated with B. subtilis, and after then the contaminated bone was treated with LTPS and the bacteria was proven inactive. Mechanical testing on rabbit cortical bone was investigated by compression testing using a mechanical testing machine. No differences between EOG treated and LTPS treated bone were found. From December 1996, 52 patients who had small and middle bone defects were treated with defatted, freeze-dried and LTPS sterilized bone. Clinical result was good. No deep infections, no transmission of virus infections or severe graft absorption were noted. The LTPS bank bone is safe and useful to reconstruction to small bone defects.

Bone Banks↗

PubMed1989

Enzyme histochemistry on freeze-dried, resin-embedded tissue.

We have developed a method for histochemical demonstration of a wide range of enzymes in freeze-dried, resin-embedded tissue. Freeze-dried tissue specimens were embedded without fixation at low temperature (4 degrees C or -20 degrees C) in glycol methacrylate resin or LR Gold resin. Enzyme activity was optimally preserved by embedding the freeze-dried tissue in glycol methacrylate resin. All enzymes studied (oxidoreductases, esterases, peptidases, and phosphatases), except for glucose-6-phosphatase, were readily demonstrated. The enzymes displayed high activity and were accurately localized without diffusion when tissue sections were incubated in aqueous media, addition of colloid stabilizers to the incubating media not being required. Freeze-drying combined with low-temperature resin embedding permits the demonstration of a wide range of enzymes with accurate enzyme localization, high enzyme activity, and excellent tissue morphology.

Animals↗

PubMed2026

Single-cell transcriptomics reveals heterogeneous stress responses and Mg2+-mediated survival mechanisms in Lactobacillus delbrueckii subsp. bulgaricus during freeze-drying and storage.

Maintaining the viability of lactic acid bacteria during dehydration and subsequent storage remains a significant challenge. Here, we employed single-cell RNA sequencing to reveal the heterogeneous stress responses of Lactobacillus delbrueckii subsp. bulgaricus, identifying seven distinct transcriptional clusters across the liquid culture, freeze-drying, and storage phases. The dominant clusters in the freeze-drying and storage were not completely consistent, showing significant functional differentiation. Genomic stability may be important for survival during freeze-drying and storage, while intracellular energy homeostasis appears important for viability during storage. The magnesium transporter mgtB was highly expressed in clusters tolerant to freeze-drying and storage, suggesting a critical role for Mg2+ homeostasis. Further experimental validation confirmed that Mg2+ treatment significantly bolstered stress resistance, increasing immediate post-freeze-drying survival by over 2-fold (up to 92.90%) and post-storage survival by over 5-fold (up to 5.98%). Proteomic data indicated that Mg2+ supplementation correlated with the maintenance of several biological functions potentially relevant to bacterial survival during freeze-drying and storage, including DNA repair, translation, and central carbon metabolism. These findings provide a map of microbial stress resistance through population heterogeneity and offer a potential strategy that may be adapted for enhancing the stability of other industrial lactic acid bacteria products.

Freeze Drying↗

PubMed2004

The bioequivalence of liquid and freeze-dried formulations of recombinant human chorionic gonadotrophin.

OBJECTIVE: To compare the bioavailability and tolerability of liquid and freeze-dried formulations of recombinant human chorionic gonadotrophin (r-hCG). SUBJECTS AND METHODS: In an open-label, randomised, single-centre, Phase I study, healthy adult volunteers (18-50 years of age) received single injections of r-hCG 250 microg from reconstituted freeze-dried (1.0 mL of 250 microg/mL) and liquid (0.5 mL of 250 microg/0.5 mL) formulations in random order, separated by a 10-day wash-out period. Pharmacokinetics (C(max), AUC, AUC(last), t(max)) and local and systemic tolerability were assessed. RESULTS: Pharmacokinetic properties of the two formulations were very similar, with mean C(max) 125 mIU/mL (liquid formulation) vs 129 mIU/mL (freeze-dried formulation), mean AUC 10,350 mIU.h/mL vs 10,480 mIU(.)h/mL, mean AUC(last) 10,050 mIU.h/mL vs 10,210 mIU.h/mL, and median t(max) 20 vs 24h. The 90% confidence intervals of the ratios of the treatment means for C(max), AUC and AUC(last) all fell within the pre-defined FDA acceptance range of 0.8-1.25, demonstrating the bioequivalence of the two formulations. Both formulations were equally well tolerated; the most frequent adverse events were headache and nausea. CONCLUSION: The liquid formulation of r-hCG was shown to be bioequivalent to the freeze-dried formulation, with no clinically significant differences in tolerability. The liquid formulation of r-hCG can be expected to provide the same efficacy and tolerability as the freeze-dried formulation when used to trigger final follicular maturation in women undergoing therapies for assisted reproduction, together with a greater convenience of use.

Adult↗

PubMed1973

Stability of freeze-dried plasma prepared from patients on oral anticoagulants.

The suitability of freeze-dried plasmas from patients on oral anticoagulants to serve as reference material in the calibration of thromboplastins used in the control of oral anticoagulant treatment was assessed in two centres. One pooled normal plasma and four pooled plasmas from patients at different levels of anticoagulation were collected into HEPES-citrate containing Trasylol. The plasmas, in 1 ml volumes, were freeze dried in siliconized ampoules under the optimal conditions used for biological standards. Small aliquots of the same plasmas were stored at -70 degrees C. Five different thromboplastins were used to carry out the tests on these plasmas. There was little difference in the clotting times obtained with five reference thromboplastins (two human, two rabbit, and one bovine reagent) on frozen and freeze-dried plasma, if the latter was tested immediately after complete reconstitution or within six hours of reconstitution if kept at +4 degrees C. Reconstituted plasmas showed a marked shortening of clotting times when stored at 22 and 37 degrees C if thromboplastins sensitive to activation of factor VII were used. In contrast, when thromboplastins sensitive to factor V were used, prothrombin times in plasmas stored at 22 and 37 degrees C became prolonged due to the loss of factor V.Freeze-dried plasmas from patients on oral anticoagulants can be used to calibrate thromboplastins, provided they are used immediately after complete reconstitution or kept at +4 degrees C for use within four to six hours of reconstitution.

Animals↗

PubMedDate not supplied

[Retention of volatile organic compounds in a freeze-dried model food gel (author's transl)].

In this work the results obtained during freeze-drying of a food model gel are reported. The model system was constituted by 12,5% sucrose, 2,5% pectin and five organic volatile substances: acetone, ethyl acetate, methanol, ethanol and n-propanol. In particular, it was studied the effect of granulation and the effect of frozen material thickness on retention of volatiles. Other experiments were made to study volatiles distribution inside the freeze-dried materials; the stripping action of warm air and of water vapor flux through the freeze-dried layers, and eventually the holding capacity of dried layers to "capture" the volatiles. All the data suggested a theory about the different mechanisms which influence the retention and the remotion of volatiles during freeze-drying of foods.

Food Preservation↗

PubMed1998

Preparation and preservation of freeze-dried cells of acetic acid bacteria with aldehyde oxidase activity.

Freeze-dried cells of acetic acid bacteria were prepared to use as an additive for manufacturing and processing foods. When the freeze-dried cells were stored for 1 week at 5 degrees C, however, more than 50% of the original activity of aldehyde oxidase (AOX) was lost. It was found that this decrease in AOX was caused by damage to both the membrane-bound aldehyde dehydrogenase and terminal oxidase activities involved in the aldehyde oxidase electron transport system of acetic acid bacteria. The addition of 30% sucrose to the cell suspension prepared in a McIlvaine buffer (pH 6) before lyophilization was found to be effective for preventing the decrease of AOX activity. Cells freeze-dried in this way lost no AOX activity at all during first 3 weeks of storage at 5 degrees C and, even after 9 weeks, 80% of the original activity remained.

Acetobacter↗

PubMed1976

Optimization in freeze-drying.

The author assumes that success in freeze-drying must be judged in economic terms. Following a summary of the reasons given for adopting freeze-drying, and an historical outline to explain the changing emphasis in the performance requirements of equipment, desirable features in design and operation are reviewed as the basis for a charter for plant manufacturers and an aid to comparison between different types of plant. Ways of appraising value for money are examined and a novel method is proposed for maximizing throughput by optimising the conditions in the freeze-dryer.

Freeze Drying↗

PubMed1971

Stability of prothrombin and factor VII in freeze-dried plasma.

The stability of prothrombin and factor VII was studied using accelerated degradation tests in three preparations of freeze-dried pooled normal plasmas. In a previous report (Brozović, Gurd, Robertson, and Bangham, 1971) factor X was shown to be relatively unstable in these preparations of freeze-dried plasma: it was calculated that up to 8% of the original factor X activity would be lost after 10 years at -20 degrees C, up to 54% at 4 degrees C, and up to 90% at room temperature. The losses of factor VII activity were estimated to be negligible at -20 degrees C, between 2 and 18% at 4 degrees C, and between 20 and 70% of the original activity at 20 degrees C, after 10 years of storage. Prothrombin was found to be less stable than factor VII: the expected loss in 10 years at -20 degrees C may be up to 4%, at 4 degrees C up to 30%, and at 20 degrees C up to 83% of the initial activity. These findings indicate that in freeze-dried plasma prothrombin as well as factor X may be insufficiently stable for plasma to serve as long-term reference material for the standardization of the one-stage prothrombin time. Moreover, the loss of prothrombin and factor X in freeze-dried plasma stored at 4 degrees C may be so high that when it is required to preserve these factors it may be necessary to store freeze-dried plasma at lower temperatures.

Cold Temperature↗

PubMed1989

[Study on the formation of fluorapatite with freeze-drying silicahydro gel method].

Resultant products and gel substance, formed with silicahydro gel method as well as with freeze-drying silicahydro gel method by use of tray, were compared and examined to materialize clinical application of gel method easily and the following results were obtained: 1. Evaluation of gel substance in control group gel (silicahydro gel) and in experimental group gel (gel prepared by the addition of deionized water to freeze-drying silicahydro gel) As the results of measuring the estimation of F- by ion chromatography and pH value, no clear-cut distinction between control group gel and experimental group gel was recognized, however, it was implied by naked-eye observations and through scanning electron microscope with the passage of time that experimental group gel had been inferior to control group gel. 2. Evaluation of resultant products in control group gel (resultant products formed with silicahydro gel method) and in experimental group gel (resultant products formed with freeze-drying silicahydro gel method) Naked-eye observations of reacting progress, the pH measurements, the calculation of Ca/P ratio, qualitative analyses by X-ray diffraction (reference; the measurements of crystallite size, lattice imperfection and lattice constant) and composition analyses by infrared absorption spectrum were carried out and as the results, resultant products in control group gel proved to be more favorable than those in experimental group gel. From the foregoing outcomes, it was suggested that fluorapatite of favorable crystallinity had been possible to be produced more with silicahydro gel method than with freeze-drying silicahydro gel method, and that further investigation inclusive of gel substance would be required in the event of clinically applying freeze-drying silicahydro gel method effectively.

Apatites↗

PubMed1988

X-ray microanalysis of freeze-dried and frozen-hydrated cryosections.

The elemental composition and the ultrastructure of biological cells were studied by scanning transmission electron microscopy (STEM) combined with energy dispersive X-ray microanalysis. The preparation technique involves cryofixation, cryoultramicrotomy, cryotransfer, and freeze-drying of samples. Freeze-dried cryosections 100-nm thick appeared to be appropriate for measuring the distribution of diffusible elements and water in different compartments of the cells. The lateral analytical resolution was less than 50 nm, depending on ice crystal damage and section thickness. The detection limit was in the range of 10 mmol/kg dry weight for all elements with an atomic number higher than 12; for sodium and magnesium the detection limits were about 30 and 20 mmol/kg dry weight, respectively. The darkfield intensity in STEM is linearly related to the mass thickness. Thus, it becomes possible to measure the water content in intracellular compartments by using the darkfield signal of the dry mass remaining after freeze-drying. By combining the X-ray microanalytical data expressed as dry weight concentrations with the measurements of the water content, physiologically more meaningful wet weight concentrations of elements were determined. In comparison to freeze-dried cryosections frozen-hydrated sections showed poor contrast and were very sensitive against radiation damage, resulting in mass loss. The high electron exposure required for recording X-ray spectra made reproducible microanalysis of ultrathin (about 100-nm thick) frozen-hydrated sections impossible. The mass loss could be reduced by carbon coating; however, the improvement achieved thus far is still insufficient for applications in X-ray microanalysis. Therefore, at present only bulk specimens or at least 1-micron thick sections can be used for X-ray microanalysis of frozen-hydrated biological samples.

Animals↗

PubMedDate not supplied

Moisture measurement: a new method for monitoring freeze-drying cycles.

Quality of the final product largely depends on the freeze-drying process. In turn this largely depends on an adequate control of the amount of residual moisture after freeze-drying. Measuring this amount in the chamber of the freeze-dryer to determine the end point of sublimation and the end point of secondary drying provides a reliable control with regard to the methods traditionally used (for example rapid increase in product temperature). The purpose of this study is to evaluate the benefits and disadvantages of the different methods recommended for the monitoring of a freeze-drying cycle. Two systems for the measurement of the moisture in the freeze dryer are evaluated here: the Pirani vacuum gauge, and the moisture sensor. The moisture sensor appears to be the most sensitive and reliable way of determining both the end of sublimation and the end of secondary drying of the full load batch when placed on a freeze-dryer. The immediate benefit for the industry is to allow to scale-up without the risks of under or over estimating the freeze-drying cycle.

Equipment Design↗

PubMed1998

Effect of process conditions on recovery of protein activity after freezing and freeze-drying.

The objective of this research was to gain a better understanding of the degree to which recovery of activity of model proteins after freeze-drying can be maximized by manipulation of freeze-dry process conditions in the absence of protective solutes. Catalase, beta-galactosidase and lactate dehydrogenase (LDH) were used as model proteins. All of the three proteins exhibited a concentration-dependent loss of activity after freezing, with significantly higher recovery at higher concentration. The freezing method and the type of buffer were also important, with sodium phosphate buffer and freezing by immersion of vials in liquid nitrogen associated with the lowest recovery of activity. Differential scanning calorimetry was predictive of the onset of collapse during freeze-drying only for beta-galactosidase. For the other proteins, either no Tg' transition was observed, or the apparent glass transition did not correlate with the microscopically-observed collapse temperature. The time course of activity loss for beta-galactosidase and LDH was compared during freeze-drying under conditions which produced collapse of the dried matrix and conditions which produced retention of microstructure in the dried solid. Recovery of activity decreased continuously during primary drying, with no sharp drop in recovery of activity associated with the onset of collapse. The most important drying process variable affecting recovery of activity was residual moisture level, with a dramatic drop in activity recovery associated with residual moisture levels less than about 10%.

Animals↗

PubMed1995

Stability of freeze dried horseradish peroxidase conjugated monoclonal antibodies used in diagnostic serology.

Two murine monoclonal antibodies, an IgG1 isotype specific for the heavy chain of bovine IgG1, the second an IgG3 isotype and specific for an epitope of the O-polysaccharide of Brucella abortus were conjugated with horseradish peroxidase. The conjugated antibodies were freeze dried in the presence of a number of additives to preserve activity and tested for stability over an 18 month period. Addition of 0.3M trehalose or 0.8% lactalbumin and 3.2% sucrose resulted in the lowest loss of activity if the conjugated antibodies were freeze dried in glass vials. Freeze drying in polypropylene vials resulted in a more rapid rate of deterioration with some additives or an accelerated rate of decline after an initial plateau of lesser loss of activity. The use of polypropylene vials and lactalbumin and sucrose were included in this study because of their low cost compared to glass vials and trehalose. While each antibody behaved differently, addition of trehalose or lactalbumin and sucrose to the conjugated antibodies aided in the preservation of enzyme activity. Both additives provide a suitable method for long term storage of freeze dried or freeze dried and reconstituted monoclonal antibody-enzyme conjugates.

Animals↗

PubMed1965

FREEZE-DRYING OF FOOT-AND-MOUTH DISEASE VIRUS AND STORAGE STABILITY OF THE INFECTIVITY OF DRIED VIRUS AT 4 C.

Foot-and-mouth disease virus, type A, strain 119, propagated in cultures of calf kidney cells and in the tongue epithelium of cattle was used. The process of freeze-drying was conducted in two cycles on unit volumes of 4 ml in Pyrex ampoules, averaging 150 ampoules per run, and was studied separately from the problems of storage. Ampoules containing freeze-dried virus were flame-sealed for either immediate study or storage at 4 C for later reference. Tissue-culture virus dried with various additives had a mean processing loss of 0.8 log LD(50) per ml for six different preparations. Virus freeze-dried in tissue suspension had a mean loss of 0.8 log LD(50) per ml for three different preparations. A second set of preparations was processed and specifically studied for storage quality at 4 C. The virus in 14 freeze-dried tissue-culture preparations had a mean loss of 0.75 log LD(50) per ml while stored at 4 C for 1 year. Virus in four freeze-dried tissue suspensions had a mean loss of 0.05 log LD(50) per ml held at 4 C for 1 year. None of the specific additives used for conservation of the virus during the freeze-drying process or during storage at 4 C contributed significantly to the stability of the virus preparations over and above that observed with the normal growth medium of the tissue culture or the ordinary diluents used in making suspensions of tissue virus.

Animals↗

PubMed2005

Freeze-dried formulations for in vivo gene delivery of PEGylated polyplex micelles with disulfide crosslinked cores to the liver.

A stable, freeze-dried formulation consisting of a core-shell-type polyplex with a poly(ethylene glycol) (PEG) shell (polyplex micelles) was prepared from a polyion complex of plasmid DNA (pDNA) and thiolated PEG-poly(L-lysine) block copolymers. The use of lyoprotectants was avoided by crosslinking the core with disulfide bonds. The crosslinked polyplex micelles (CPMs) showed excellent stability during freeze-drying and reconstitution processes, which is in sharp contrast with the formation of visible agglomerates from the non-crosslinked polyplex micelles (NCPMs) after a similar process. A thiolation degree higher than 13% of the lysine residues was required to achieve sufficient tolerability of the CPMs during the freeze-drying/reconstitution cycle. Dynamic light scattering measurements and atomic force microscopy observations demonstrated that the original size and shape of the CPMs with a thiolation degree of higher than 13% were maintained even after the freeze-drying. Furthermore, the CPMs reconstituted from the freeze-dried state achieved a transfection efficiency as high as that of the original samples. The intravenous injection of the CPM with a thiolation degree of 37% into mice via the orbital vein led to an appreciably uniform gene expression of a yellow fluorescence protein variant (Venus) in the liver, while no gene expression was observed in the case of the free pDNA injection. The procedure of disulfide crosslinking of the polyplex micell core allows the preparation of non-viral gene vectors as a powder formulation without the use of any lyoprotectants. This achievement is certainly useful for pharmaceutical applications and exhibits many advantages, including easy concentration adjustments of dosing samples, long-term storage stability, and large-scale production reproducibility.

Animals↗

PubMed2002

Spray-freeze-drying for protein powder preparation: particle characterization and a case study with trypsinogen stability.

This work investigates the use of spray freeze-drying (SFD) to produce protein loaded particles suitable for epidermal delivery. In the first part of the study, the effects of formulation and process conditions on particle properties are examined. Aqueous solutions of trehalose produce SFD particles in the size range 20-80 microm, with a smooth, textured surface, but having high internal porosity. The latter was visualized using SEM and a novel particle embedding and sectioning technique. Use of an annealing step during the freeze-drying cycle caused the particles to shrink, reducing hereby porosity and also the measured rate of moisture uptake into these amorphous particles. SFD pure mannitol was approximately 40% amorphous, but not hygroscopic. Incorporation of dextran 37,500 into a combined amorphous trehalose/mannitol formulation led to increased particle shrinkage and lower particle porosity on annealing. The model protein trypsinogen lost approximately 15% activity during SFD of solutions containing 50 mg/mL protein, but was only marginally aggregated (1.4%). It is suggested that trypsinogen forms an irreversible partially unfolded state or molten globule on SFD/rehydration. The pure protein was also partially inactivated without aggregation during atomization into air. Surprisingly, neither activity loss nor aggregation were detected on atomization of the protein solution into liquid nitrogen. Quench-freezing of small droplets may reverse the partial unfolding of trypsinogen occurring on atomization into air. The origin of the trypsinogen inactivation during SFD must therefore be the subsequent freeze-drying step of this multistep process. Isolated freeze drying of trypsinogen produces strong aggregation and equivalent inactivation. This result suggests that trypsinogen behaves differently during freeze drying from frozen droplets and from bulk solution in a vial. In the former case the protein forms an irreversible partially unfolded state, whereas in the latter case aggregates are formed. Trypsinogen inactivation during SFD could be completely prevented by the presence of trehalose in the formulation. Electron Spectroscopy for Chemical Analysis (ESCA) showed a high surface excess of the protein in the SFD particles, which was reduced on inclusion of Polysorbate 80, but not trehalose. Taken together, these results help to elucidate the complex destabilization behavior of trypsinogen during SFD.

Chromatography, Gel↗

PubMed2002

Characterization of physical state of mannitol after freeze-drying: effect of acetylsalicylic acid as a second crystalline cosolute.

Freeze-drying of mixed solutes is a preparative technique widely used in the pharmaceutical industry. The presence of an amorphous form or changes in the crystalline form can affect solid state stability. In this work, acetylsalicylic acid (AAS) was chosen as a model drug, and was mixed with mannitol, a commonly used bulking agent in formulation of tablets. Variations in the final freeze-dried crystalline forms were found after changing the ratios of the two co-solutes. Samples were analysed by powder X-ray diffractometry and differential scanning calorimetry. A major amorphous form and a minor crystalline delta-mannitol form were produced during the mannitol freeze-drying process. The crystal form of mannitol in the two-component system depended on the AAS:mannitol ratio. The AAS was mostly crystalline, regardless of the amount of mannitol present. A major delta-mannitol and a minor amorphous form were obtained when AAS was present in a high percentage (75% w/w). When AAS percentages of 50 and 25% (w/w) were present during the drying process, the mannitol was found in a highly crystalline form.

Amides↗