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PubMed1993

The adverse effect of glycation of human serum albumin on its preservative activity in the freeze-drying and accelerated degradation of alkaline phosphatase.

The effects of a glycated protein additive on the stability of freeze-dried biological standards were studied using alkaline phosphatase as a model. Alkaline phosphatase was formulated with artificially glycated albumin, freeze-dried, sealed into glass ampoules and subjected to accelerated degradation studies at temperatures from -20 to 56 degrees C. Alkaline phosphatase, freeze-dried without an additive in neutral buffer, lost over 95% of its activity, but when freeze-dried with human serum albumin it retained approximately 70% of the initial activity. Both deliberately glycated and native albumin protected approximately 70% of the initial activity on freeze-drying and this protection was maintained during storage of the freeze-dried product at temperatures of 20 degrees C or below for up to 16 weeks. At 37 degrees C or above, alkaline phosphatase activity was lost in a time- and temperature-related manner with changes appearing in the SDS-PAGE gels and FPLC chromatograms but, with the artificially glycated albumin formulations, this loss of activity and the changes in the gels and chromatograms happened earlier and at lower temperatures. Formulations with trehalose at 1% w/v and 15% w/v, but without albumin, preserved some 40% of alkaline phosphatase activity following freeze-drying. Further, approximately half of that activity was maintained after 16 weeks' storage at all temperatures up to 56 degrees C by the 15% trehalose without albumin.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1976

A comparison of the ultrastructure of spray-frozen and freeze-etched or freeze-dried bull and boar spermatozoa with that after chemical fixation.

The ultrastructure of bull and boar spermatozoa was investigated following different cryopreparation methods and chemical fixation. Spray-freezing was used for cryofixation in both freeze-etching and freeze-drying studies. Freeze-etching of boar spermatozoa revealed that the arrangement of the postnuclear striations differed from that in the bull. Freeze-drying gave excellent results for structural preservation, which were equal to those of chemical fixation. Some structural details not visible in chemically fixed cells were detected in freeze-dried and vacuum-embedded bull and boar spermatozoa, e.g. the arrangement of the lamellar nuclear contents, known from freeze-fractures, and a fine lamellar structure of the acrosomal contents. Cryofixation by spray-freezing combined with freeze-drying makes any contact of the cells with fixatives, buffer solutions and dehydration media unnecessary, and potentially provides all the advantages of ultrathin sectioning required for histochemical studies.

Acrosome↗
PubMed1985

Effect of drying medium on residual moisture content and viability of freeze-dried lactic Acid bacteria.

The effect of various substances on the relationship between residual moisture content and the viability of freeze-dried lactic acid bacteria has been studied. Compounds such as polymers, which display considerable ability in displacing water, showed no protective action during freeze-drying. Adonitol, on the other hand, produced the smallest change in water content at various times during drying and allowed the highest rate of survival.

Journal Article↗
PubMed1983

Reconstruction of osseous defects with freeze-dried allogeneic and autogenous bone. Clinical and histologic assessment.

Freeze-dried allogeneic bone combined with autogenous particulate cancellous bone and marrow was used for reconstruction in 56 patients. With the exception of 3 patients who had infection postoperatively, the patients had complete acceptance of the composite graft system. Histologic examination at 2, 3, 12, 18, and 24 months demonstrated early incorporation, revascularization, resorption of the freeze-dried component, and formation of a bone ossicle that continued to remodel and form mature lamellar bone. This study suggests that freeze-dried allogeneic bone can serve as a latticework along which osteocompetent cells can proliferate, that composite grafts are readily accepted and will form a satisfactory and predictable bone ossicle over a considerable defect, and that freeze-dried allogeneic bone induces the host mesenchymal cells to differentiate into mature osteoblasts and aids in the formation of a long-lasting functional bone graft.

Biopsy↗
PubMed1999

Surface characterisation of freeze-dried protein/carbohydrate mixtures.

In the present investigation freeze-drying of proteins (BSA or trypsin) together with various carbohydrates, i.e. lactose, sucrose, mannitol, alpha-cyclodextrin and dextrin, has been studied with particular emphasis on the surface composition of the freeze-dried powders. The proteins were found to be over-represented on the powder surface as compared to the bulk concentration of protein. The mechanism behind the surface accumulation is believed to be that proteins adsorb preferentially over carbohydrates to the ice/liquid interface in the frozen sample. The degree of surface accumulation depended on the carbohydrate used, and was increased in annealed samples compared to reference samples. The activity of trypsin was fairly well preserved (58-90%) in the freeze-dried powders, but depended on the carbohydrate excipient, whilst the surface composition had little effect on the activity. The activity preservation was improved when the protein concentration was raised from 1 to 10% in the solids. The surface composition of powders containing mixtures of mannitol and dextrin as excipients depended on the ratio between the two carbohydrates, with the lowest surface coverage of protein obtained in 50/50 mixtures.

Calorimetry, Differential Scanning↗
PubMed2002

Protein spray freeze drying. 2. Effect of formulation variables on particle size and stability.

Spray freeze drying produces protein particles suitable for microencapsulation into polymeric microspheres intended for sustained release. Accessibility of encapsulated protein particles to the microsphere surface increases as the protein particle size is increased. Thus, it is desirable that the encapsulated protein particle size be minimized to limit initial release. We have investigated the effect of formulation on spray freeze-dried bovine serum albumin (BSA) as a model protein. Atomization conditions were fixed such that in the absence of excipient, the particle size of the sonicated powder was submicron, and there was substantial protein degradation (loss of monomer). Addition of low concentrations of surfactants (up to the CMC) or mannitol (up to the point where it tended to crystallize upon dehydration) resulted in partial stabilization without impacting particle size. Trehalose was successful in stabilizing the protein; however, there was a marked increase in particle size at the highest levels tested. Ammonium sulfate provided partial stabilization, but also tended to form crystals and increase particle size. FTIR measurements showed a loss of native secondary structure upon spray freeze drying that was ameliorated by addition of trehalose. Other excipients did not prevent structural perturbations. In general, stabilization of spray freeze-dried BSA was related to lowering of the specific surface area in the powder. A balance must be achieved when spray freeze drying proteins intended for encapsulation in sustained-release systems.

Chemistry, Pharmaceutical↗
PubMed2006

Trehalose and hyaluronic acid coordinately stabilized freeze-dried pancreatic kininogenase.

The ability and mechanisms of stabilization of freeze-dried formulations of pancreatic kininogenase (PKase) by carbohydrates were evaluated. Activity and structure of PKase were examined after freeze-drying and rehydration in presence with or without a carbohydrate. Addition of trehalose, lactose, sucrose, hyaluronic acid (HA) or a combination of trehalose and HA to PKase formulations prior to freeze-drying step increases the stability of PKase during freeze-drying, storage and rehydration as measured by activity preservation. The combination of trehalose and HA is the most effective for the stabilization of PKase. Addition of HA alone to a formulation does not affect protein structure, but it increases glass-transition temperature (Tg) and stability of lyophilized PKase in presence of trehalose during dehydration, storage and rehydration processes. Therefore, trehalose and HA offer complementary properties that improve the stability of PKase during dehydration, storage and rehydration.

Calorimetry, Differential Scanning↗
PubMed2003

Tolerance of the mouse sperm nuclei to freeze-drying depends on their disulfide status.

Mouse spermatozoa from the caudae epididymides could be freeze-dried without losing their ability to support normal development. Immature spermatozoa from the testes, in contrast, were damaged by freeze-drying. However, immature spermatozoa became resistant to freeze-drying after their treatment with diamide, which oxidizes free -SH groups. Conversely, epididymal spermatozoa were damaged by freeze-drying if first treated with dithiothreitol (DTT), which reduces -SS- bonds. The potential for freeze-drying damage seems likely to relate to the -SS- status of sperm proteins, in particular its protamines.

Animals↗
PubMed1977

Variables of the rubella hemagglutination tests employing freeze-dried erythrocytes.

The variables which affect the interaction between freeze-dried one-day-old chick erythrocytes and rubella hemagglutinin prepared from rubella-infected porcine kidney cells were defined and evaluated. The sensitivity of the hemagglutination (HA) reaction is much greater at pH 6.0 to 6.2 than at pH 7.0 to 7.5 HEPES (N-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid) diluent with added Ca+ or Mg2+ ion gave four- to eightfold higher HA titers than one without divalent cations. The development of agglutinated and non-agglutinated erythrocyte patterns depended much upon the concentrations of gelatin and albumin in the HEPES diluent. Gelatin especially was essential to obtain stable and clearly distinguishable patterns. Optimal conditions for the agglutination of freeze-dried erythrocytes by rubella hemagglutinin were provided when a HEPES-buffered saline at pH 6.2, containing 10(-3) M CaCl2, 0.2 per cent bovine serum albumin, and 0.0025 per cent gelatin was employed throughout as a diluent for serum, hemagglutinin, and freeze-dried erythrocyte suspension. This diluent gave maximally sensitive and reproducible results in rubella HA and hemagglutination-inhibition (HI) tests employing freeze-dried erythrocytes.

Animals↗
PubMed2003

Impaction bone grafting with freeze-dried irradiated bone. Part I. Femoral implant stability: cadaver experiments in a hip simulator.

Processed freeze-dried irradiated allografts seem to be used less than instead of fresh-frozen allografts for impaction bone grafting in revision hip arthroplasties. Although biologically acceptable, their use is discouraged because of their questionable mechanical properties following freeze-drying and irradiation procedures. To address this question, we impacted freeze-dried grafts in 6 cadaveric femurs and loaded with a cemented Charnley prosthesis. The routinely used fresh-frozen allografts were used as controls in the contralateral side. These constructs were compared simultaneously in a walking hip simulator for their stability during 900,000 loading cycles. The mechanical parameters were axial inducible displacement and subsidence of the implant. The former parameter was lower in the implant mounted on freeze-dried impacted grafts than that mounted on the fresh-frozen bone. The latter parameter was also lower in the freeze-dried group. At the end of the test, we found no implant loosening in either group and their 'pull out' resulted in cement-prosthesis debonding, which showed the mechanical integrity of the impacted grafts. Freeze-dried grafts provide more stable fixation of the stem than fresh-frozen morselized grafts, when tested in a hip simulator.

Adult↗
PubMed1992

Evaluation of the physical stability of freeze-dried sucrose-containing formulations by differential scanning calorimetry.

Freeze-dried samples of sucrose with buffer salts, amino acids, or dextran have been analyzed with differential scanning calorimetry (DSC) to evaluate the use of DSC thermograms in predicting the physical storage stability. The glass transition temperature, Tg, of the amorphous cake, crystallization, and melting of sucrose are observed with DSC. Tg appeared to be an important characteristic of the physical stability of the amorphous freeze-dried cake. A storage temperature above Tg results in collapse or shrinkage of the cake, which for a sucrose-based formulation, may be accompanied by crystallization of the sucrose. The Tg of the amorphous sucrose is influenced by other components present in the cake. Dextran-40 raised Tg, while the addition of glycine to the formulation lowered Tg. The residual moisture content strongly influences Tg, since water acts as a plasticizer of the system; the higher the moisture content, the lower the Tg and the less physically stable the freeze-dried cake. Crystallization of amorphous sucrose is shown to be inhibited by high molecular weight components or ionic compounds. DSC analysis of freeze-dried cakes proved to be a powerful tool in formulation studies.

Alcohols↗
PubMed1994

Pronuclear formation and cleavage of mammalian eggs after microsurgical injection of freeze-dried sperm nuclei.

A study was conducted to evaluate the ability of mammalian oocytes to develop to the pronuclear stage and beyond if injected with freeze-dried sperm nuclei. The rate of development of hamster eggs to the pronuclear stage after microinjection of freeze-dried hamster sperm nuclei was 90%. The pronuclear formation rate of hamster eggs injected with freeze-dried human sperm nuclei was 85%. On the other hand, the rates for eggs injected with untreated sperm nuclei were 84% and 89% respectively. The survival rate of rabbit eggs microinjected with freeze-dried rabbit sperm nuclei was 64%, the fertilisation rate 56% and the cleavage rate 38%. The survival, fertilisation and cleavage rates of eggs injected with non-freeze-dried sperm nuclei were 78%, 45% and 34%, respectively. There was no difference between eggs injected with freeze-dried sperm nuclei and those injected with sperm nuclei that had not been freeze-dried. Of the viable rabbit eggs injected with freeze-dried rabbit sperm nuclei, 6% developed to the 6- to 8-cell stage 62 h after injection.

Animals↗
PubMed1997

Characterization of the glycation of albumin in freeze-dried and frozen human serum.

Human serum albumin (HSA) in fresh frozen and freeze-dried serum reference materials was examined by mass spectrometry and a variety of affinity chromatography techniques. The relative molecular mass distribution of HSA in fresh frozen serum was found to be identical to that of an HSA standard. However, the HSA in the freeze-dried reference serum exhibited a relative molecular mass distribution that was shifted to higher mass, broader, and substantially more heterogeneous than that of HSA in fresh frozen serum. A proteolytic cyanogen bromide digestion of the HSA from freeze-dried serum contained adducts approximately 162 u higher in mass than digest fragments 124-298 and 447-548, suggesting glycation. The presence of glycation on fragments 124-298 and 447-548 correlates with the known sites of HSA glycation. Glycation was further confirmed by the mass spectral analysis of the retained and unretained fractions from glycoaffinity chromatography of HSA from freeze-dried serum. The relative molecular weight of the HSA in the retained fraction indicated the presence of a doubly glycated species. The chemical heterogeneity of Cys-34, the site of the only free thiol in HSA, was examined and found not to be a substantial source of molecular mass heterogeneity for HSA from either fresh frozen of freeze-dried serum.

Blood Specimen Collection↗
PubMed2006

Effect of ginseng polysaccharide on the stability of lactic acid bacteria during freeze-drying process and storage.

Lactic acid bacteria (LAB) quickly attenuate or are killed during the freeze-drying process and storage. The effect of some natural polysaccharides, which are known as potent antitumor and immunomodulating substances, on the viability of the LAB, Lactobacillus acidophilus and Bifidobacterium breve, on freeze-drying and storage were investigated. Among the polysaccharides tested, red ginseng polysaccharide (RGP) and chitosan significantly inhibited the cell death of the LAB during freeze-drying, and fucoidan and RGP most potently protected the cell death of the LAB during storage. The stabilities of the LAB on the addition of RGP and fucoidan were comparable to that of skimmed milk. However, white ginseng polysaccharide (WGP) did not promote storage stability. When 5% skimmed milk/5% RGP treated LAB were freeze-dried and stored, their viabilities were found to be significantly higher those treated with 5% or 10% RGP. The stabilizing effect of 5% RGP/5% skimmed milk during LAB freeze-drying and storage stability was comparable to that of treatment with 10% skimmed milk. Based on these findings, we believe that RGP beneficially improves the stability of LAB during the freeze-dry process and storage.

Animals↗
PubMed2001

Viability, efficacy, and storage stability of freeze-dried biocontrol agent Candida sake using different protective and rehydration media.

Viability, efficacy against Penicillium expansum on Golden Delicious apples, and storage stability of freeze-dried Candida sake strain CPA-1 were studied. The effect of several protective agents and rehydration media was investigated in the freeze drying of C. sake. Skimmed milk at 10% concentration was a good rehydration medium for all protectants tested. In general, good viability results were obtained when the same solution was used as a protectant and as a rehydration medium. The best survival was obtained when C. sake cells were protected with 10% lactose + 10% skimmed milk and rehydrated with skimmed milk (85% viability). The potential for biocontrol of the best freeze-dried treatments against P. expansum on apples was compared with that of fresh cells. Freeze-dried treatments at 1 x 10(7) CFU/ml reduced the incidence of decay by 45 to 66%. The best biocontrol effect was obtained with cells that had been freeze dried using 10% lactose + 10% skimmed milk as a protectant and 1% peptone as a rehydration medium, with a 66% reduction in rot incidence. However, in all treatments, the efficacy of freeze-dried cells was significantly lower than fresh cells. The stability of freeze-dried samples decreased during storage and was influenced by storage temperature. In the best treatment, storage of C. sake cells for 60 days at 4 degrees C resulte in final concentrations of 2.5 x 10(8) CFU/ml, which was a 10-fold reduction in relation to the initial starting concentration of cells prior to freeze drying.

Candida↗
PubMed1975

Freeze-drying from tertiary butanol in the preparation of endocardium for scanning electron microscopy.

The scanning electron microscope appearances and shrinkage of blocks of canine endocardium prepared by freeze-drying directly, by freeze-drying after replacing tissue water with tertiary butanol (2-methyl propan-2-ol) and by critical point drying were compared. All three methods demonstrated endothelial cells which showed nuclear prominences, microvilli and intercellular boundaries. The microvilli varied in size and number from dog to dog but were generally less well defined in specimens freeze-dried from water. Shrinkage due to t-butanol dehydration was significantly less than that which occurred in ethanol in the critical point drying method. Overall the reduction in surface area was significantly less in specimens freeze-dried directly at -65 C (6.8%) than in those dried from t-butanol at -20 C (15.4%) and those prepared bly critical point drying (22.1%). However the amount of shrinkage observed in t-butanol treated tissue was not significantly different from that which was critical point dried. It was not possible to distinguish between comparable samples prepared by these two methods on the basis of their scanning electron microscopic appearances. Thus the relative simplicity and convenience of the t-butanol method, together with its saving of time, its use of standard freeze-drying equipment and the avoidance of ice-crystal artefact justify its consideration as an alternative method of preparing wet biological tissue for scanning electron microscopy.

Animals↗
PubMed2003

Solute crystallization in mannitol-glycine systems--implications on protein stabilization in freeze-dried formulations.

The use of mannitol in combination with glycine has resulted in stable freeze-dried protein formulations. Our objectives were to (1) study solute crystallization in ternary systems containing mannitol, glycine, and water during all the stages of freeze drying as a function of processing conditions and formulation variables; (2) investigate the effect of sodium phosphate buffer salts on the crystallization of both mannitol and glycine and vice versa; and (3) investigate the effects of these excipients in a freeze-dried lactate dehydrogenase (LDH) formulation. X-ray powder diffractometry (XRD) and differential scanning calorimetry (DSC) were used to study the frozen aqueous solutions. Phase transitions during primary and secondary drying were monitored by simulating the entire freeze-drying process in situ in the sample chamber of the diffractometer. LDH activity after freeze drying was determined spectrophotometrically. In frozen aqueous solutions containing mannitol and glycine, each solute influenced the extent of crystallization of the other. The solutes crystallized as delta-mannitol and beta-glycine during primary drying. Glycine had a stronger tendency to crystallize, while it was easier to influence mannitol crystallization. The buffer salts inhibited the crystallization of mannitol and glycine. However, in some cases, during primary drying, glycine crystallization was followed by that of disodium hydrogen phosphate dodecahydrate. The latter underwent dehydration forming an amorphous anhydrate. It was possible to correlate the extent of crystallization of mannitol and glycine in the lyophile with the retention of protein activity. An increase in buffer concentration decreased the crystallinity of mannitol and glycine. This translated to increased retention of protein activity.

Buffers↗