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PubMed2005

Fos-like immunoreactivity in the brain associated with freezing or escape induced by inhibition of either glutamic acid decarboxylase or GABAA receptors in the dorsal periaqueductal gray.

GABAergic neurons exert tonic control over the neural substrates of aversion in the dorsal periaqueductal gray (dPAG). It has been shown that electrical stimulation of this region at freezing or escape thresholds activates different neural circuits in the brain. Since electrical stimulation activates cell bodies and fibers of passage, it is necessary to use chemical stimulation that activates only post-synaptic receptors. To investigate this issue further, reduction of GABA transmission was performed with local injections of either the GABA-A receptor antagonist bicuculline or the glutamic acid decarboxylase (GAD) inhibitor semicarbazide into the dorsolateral periaqueductal gray (dlPAG). Local infusions of semicarbazide (5.0 microg/0.2 microl) or bicuculline (40 ng/0.2 microl) into this region caused freezing and escape, respectively. The results obtained showed that freezing behavior induced by semicarbazide was associated with an increase in Fos expression in the laterodorsal nucleus of the thalamus (LD) and ventrolateral periaqueductal gray (vlPAG), while bicuculline-induced escape was related to widespread increase in Fos labeling, notably in the columns of the periaqueductal gray, hypothalamus nuclei, the central amygdaloid nucleus (Ce), the LD, the cuneiform nucleus (CnF) and the locus coeruleus (LC). Thus, the present data support the notion that freezing and escape behaviors induced by GABA blockade in the dlPAG are neurally segregated: freezing activates only structures that are mainly involved in sensory processing, and bicuculline-induced escape activates structures involved in both sensory processing and motor output of defensive behavior. Therefore, the freezing elicited by activation of dlPAG appears to be related to the acquisition of aversive information, whereas most brain structures involved in the defense reaction are recruited during escape.

Analysis of Variance↗
PubMed2006

Relation of several antioxidant enzymes to rapid freezing resistance in suspension cultured cells from alpine Chorispora bungeana.

Chorispora bungeana Fisch. & C.A. Mey (Crucifer) is a rare alpine subnival plant surviving sudden snowstorms. In this paper, we have attempted to explore possible roles of autoxidation rate (AR) and the antioxidant enzymes associated with cryoprotective mechanisms in the plant cells. The results showed that when the suspension cultures growing at 25 degrees C were suddenly exposed to -8 degrees C for 15 days, 2,3,5-triphenyltetrazolium chloride reduction was not affected within 9 days and AR remained at a low level in comparison with controls. This indicated that the cells maintained considerable amounts of soluble protein and the integrity of the cell membranes was intact during the whole freezing test. Furthermore, on average, the activity of antioxidant enzymes such as superoxide dismutase, dehydroascorbate reductase, ascorbate peroxidase and glutathione reductase were prominently enhanced in the freezing-stressed cells. Peroxidase activity significantly increased soon after freezing, possibly to make up for the early decrease of catalase activity in the cells. Statistical analysis showed negative correlations between resistance to rapid freezing and antioxidant enzyme activity in the cultured cells after exposure from 25 to -8 degrees C, indicating that the reduction of cell viability with freezing activates a combination of antioxidant enzymes that results in intact cells. All of these findings suggest a synergy between these antioxidant enzymes, leading to a low autoxidation rate that contributes to the protection of the cell membranes and plays an important role in the resistance of suspension cultured cells of C. bungeana to sudden freezing.

Alpinia↗
PubMed2005

Physical characterisation of formulations for the development of two stable freeze-dried proteins during both dried and liquid storage.

The development of stable freeze-dried proteins requires maintaining the physical and biological integrity of the protein as well as increasing the efficiency of the manufacturing process. Our objective was to study the effects of various excipients on both the physical characterisation and the dried and liquid stability of two proteins. Thermo-physical properties of 13 formulations were determined using both differential scanning calorimetry and freeze-drying microscopy. The antigenic activity was evaluated immediately after freeze-drying and after subsequent storage in both dried and liquid state. From the comparison between glass transition (T'g) and collapse (T coll) temperatures, we concluded that the collapse temperature was a more relevant parameter than T'g for freeze-drying cycle development and optimisation. One crystalline formulation composed of 4% mannitol and 1% of sucrose protected efficiently both proteins during subsequent storage in dried state (6 months at 25 degrees C) and in liquid state (3 months at 4 degrees C after rehydration). However, the freeze-drying behaviour of this crystalline formulation remained difficult to predict and control. On the other hand, two amorphous formulations composed of 4% of maltodextrin and 0.02% of Tween 80, or 5% of BSA preserved antigenic activity during storage in dried state. The glassy character of these formulations as well as their high collapse temperature values (-9 and -12 degrees C, respectively) should allow simplification and shortening of freeze-drying process.

Calorimetry, Differential Scanning↗
PubMed2005

The influence of freezing and duration of storage on Campylobacter and indicator bacteria in broiler carcasses.

In total, 215 commercially processed broiler carcasses were examined to determine optimum cultural enumeration, the effects of freezing, method of thawing, and duration of frozen storage on levels of Campylobacter spp. and fecal coliforms. Enumeration studies compared MPN procedures to direct plating onto selective mCCDA agar and indicated equivalency for quantitation of Campylobacter spp. Levels of Campylobacter and fecal coliforms were subsequently estimated by direct plating of carcass rinses. Freezing of naturally contaminated carcasses followed by storage at -20 degrees C for 31, 73, 122 and 220 days showed statistically significant (P< or =0.05) reductions in Campylobacter counts initially as compared with counts found on fresh product. Among 5 lots of broilers, levels of Campylobacter on carcasses were reduced by log mean values ranging from 0.65 to 2.87 after freezing and 31 days of storage. Similar reductions due to freezing were not observed for fecal coliforms counts. The level of Campylobacter was reduced by approximately one log immediately after freezing, and remained relatively constant during the 31-220 days of frozen storage. The levels were constant during 7 days of refrigerated storage. After 31 days of frozen storage there was a reduced rate in reduction of counts among broilers thawed at 7 degrees C as compared to thawing at 22 degrees C with either cultural method (MPN and mCCDA). These findings warrant consideration of the public health benefits related to freezing contaminated poultry prior to commercial distribution to reduce Campylobacter exposure levels associated with contaminated carcasses.

Animals↗
PubMed2006

Freeze-drying of nanocapsules: impact of annealing on the drying process.

Freeze-drying process was recently applied to improve the long-term storage stability of nanocapsules. Thermal treatment by annealing is an interesting process to optimize a freeze-drying cycle of these colloidal vectors. The objective of this paper is to investigate the impact of annealing on primary and secondary drying characteristics and on nanocapsules (NC) properties. Nanocapsules were prepared from poly-epsilon-caprolactone (PCL) biodegradable polymer and stabilized by polyvinyl alcohol (PVA), and then freeze-dried with two cryoprotectants: sucrose and poly vinyl pyrrolidone (PVP). Freeze-dried nanocapsules were characterized by size measurement and transmission electron microscopy after reconstitution. The effect of annealing on the kinetics of sublimation, on the mass transfer resistance and on the porosity of the freeze-dried product has been studied in the case of PVP. Finally, the effect of annealing on the kinetic of secondary drying was studied and the results were coupled with the isotherm of sorption. Results showed that PCL nanocapsules could be freeze-dried without any modification of their properties in presence of the two cryoprotectants used. Annealing of nanocapsules suspensions could accelerate the sublimation rate without any modification of nanocapsules size in the case of the two studied cryoprotectants. Such improvement could be explained by the increase of ice crystals size after annealing and by the diminution of mass transfer resistance by the dried layer. The acceleration of sublimation rate seems to depend on the temperature of annealing. The annealing of sucrose solution slows down the secondary drying kinetic whereas no effect is observed in the case of PVP.

Capsules↗
PubMed2005

Rapid degradation of starch in chloroplasts and concomitant accumulation of soluble sugars associated with ABA-induced freezing tolerance in the moss Physcomitrella patens.

Abscisic acid (ABA) has been postulated to play a role in the development of freezing tolerance during the cold acclimation process in higher plants, but its role in cold tolerance in tower land plants has not been elucidated. The moss Physcomitrella patens rapidly developed freezing tolerance when its protonemata were grown in a medium containing ABA, with dramatic changes in the LT50 value from -2 degrees C to over -10 degrees C. We examined physiological and morphological alterations in protonema cells caused by ABA treatment to elucidate early cellular events responsible for rapid enhancement of freezing tolerance. Microscopic observations revealed that ABA treatment for 1 day resulted in a dramatic alteration in the appearance of intracellular organelles. ABA-treated cells had slender chloroplasts, with a reduced amount of starch grains, in comparison with those of non-treated cells. The ABA-treated cells also had several segmented vacuoles while many of non-treated cells had one central vacuole. When frozen to -4 degrees C, freezing injury-associated ultrastructural changes such as formation of aparticulate domains and fracture-jump lesions were frequently observed in the plasma membrane of non-treated protonema cells but not in that of ABA-treated cells. The ABA treatment increased the osmotic concentration of the protonema cells, in correlation with accumulation of free soluble sugars. These results suggest that ABA-induced accumulation of soluble sugars, associated with morphological changes in organelles, mitigated freezing-induced structural damage in the plasma membrane, eventually leading to enhancement of freezing tolerance in the protonema cells.

Abscisic Acid↗
PubMed2006

Pharmacological assessment of the freezing, antinociception, and exploratory behavior organized in the ventrolateral periaqueductal gray.

Opioid and serotonergic mechanisms of the ventrolateral periaqueductal gray (vlPAG) are recruited by conditioned freezing and antinociception. However, it is unclear whether freezing and antinociception induced by stimulation of the vlPAG are interrelated. To address this issue we looked at the effects of the opioid antagonist naltrexone, the 5-HT2 antagonist ketanserin, and the benzodiazepine agonist midazolam injected into the vlPAG on the freezing and antinociception induced by electrical stimulation of this region. This antinociception was evaluated by the tail-flick and formalin tests. To further characterize the involvement of the vlPAG in unconditioned fear, the effects of intra-vlPAG injections of midazolam on the exploratory behavior were also assessed in independent groups of rats submitted to the elevated plus-maze test (EPM). The data obtained showed that: (i) electrical stimulation of the vlPAG causes freezing blocked by midazolam but not by naltrexone and ketanserin; (ii) antinociception generated at the level of the vlPAG is inhibited by naltrexone, ketanserin, and midazolam; (iii) activation of benzodiazepine-mediated mechanisms in the vlPAG increased the exploratory behavior of rats in the closed arms but not the avoidance behavior of open arms of the EPM. Thus, freezing and antinociception generated in the vlPAG are dissociated pharmacologically. Whereas antinociception is a multimediated process sensitive to naltrexone, ketanserin, and midazolam, the freezing induced by vlPAG stimulation was reversed only by the benzodiazepine compound. As injections of midazolam into the vlPAG do not cause anxiolytic effects in the EPM, the aversive stimuli inherent of this test seem to bypass the vlPAG.

Analysis of Variance↗
PubMed2006

Pharmacological and histopathological characterization of a hyperalgesia model induced by freeze lesion.

Induction of a freeze lesion in human skin is an experimental model of hyperalgesia that allows assessing the antihyperalgesic effects of traditional non-steroidal anti-inflammatory drugs (NSAIDs). We have investigated whether this model is also sensitive to selective cyclooxygenase (COX)-2 inhibitors and have characterized morphological substrates of the generated hyperalgesia in the skin. In eight healthy subjects, a freeze lesion was induced and mechanical pain thresholds (MPT) were tested for 5h following administration of the non-selective COX inhibitor diclofenac (75mg), the COX-2-selective inhibitor parecoxib (40mg) or placebo in a randomized, double-blind cross-over study. In five additional healthy subjects, biopsies were taken from normal skin and the area of freezing injury. Induction of the freeze lesion resulted in hyperalgesia expressed by a decrease of MPT after 24h. Diclofenac and parecoxib, but not placebo, statistically significantly elevated MPT. Histochemical and Western blot analyses of skin biopsies revealed a strong upregulation of COX-2, a slight decrease of COX-1 and activation of nuclear factor kappa B (NF-kappaB) in the area of the freezing injury. These findings indicate that the freeze lesion model is sensitive to NSAIDs including selective COX-2 inhibitors, and that NF-kappaB-dependent COX-2 upregulation contributes to the hyperalgesia in this model.

Adult↗
PubMed2004

Transgenic tobacco plants accumulating osmolytes show reduced oxidative damage under freezing stress.

We studied the reaction to the oxidative component of freezing in several tobacco lines, transformed with genes coding for enzymes involved in the synthesis of osmoprotectants (proline, fructan or glycine betaine) along with their wild type. The levels of some oxidative stress markers (leakage of electrolytes, hydrogen peroxide and malondialdehyde) as well as the activity of antioxidative enzymes catalase (EC 1.11.1.6.) and guaiacol peroxidase (EC 1.11.1.7.) have been followed at acclimation, 12 and 24 h freezing and at recovery. Freezing for 24 h resulted in severe damages for the wild type. A corresponding increase of electrolyte leakage, hydrogen peroxide and malondialdehyde contents, a rise of peroxidase activity and inhibition of catalase activity occurred in the non-transformants. Similar, but significantly lower trend of the same parameters has been found for the transgenic lines. Moreover, the oxidative markers returned to their normal levels when the transformants were able to recover from freezing. It could be speculated that transfer of genes, coding for accumulation of osmoprotectants, is related to reduced intensity of freezing-induced oxidative processes. Our lines and model system could serve as a good prerequisite for additional studies to gain further insights into the complex role of osmoprotectants in freezing tolerance.

Acclimatization↗
PubMed2006

A vaccine cold chain freezing study in PNG highlights technology needs for hot climate countries.

Fourteen data loggers were packed with vaccine vials at the national vaccine store, Port Moresby, Papua New Guinea (PNG), and sent to peripheral locations in the health system. The temperatures that the data loggers recorded during their passage along the cold chain indicated that heat damage was unlikely, but that all vials were exposed to freezing temperatures at some time. The commonest place where freezing conditions existed was during transport. The freezing conditions were likely induced by packing the vials too close to the ice packs that were themselves too cold, and with insufficient insulation between them. This situation was rectified and a repeat dispatch of data loggers demonstrated that the system had indeed been rectified. Avoiding freeze damage becomes even more important as the price of freeze-sensitive vaccines increases with the introduction of more multiple-antigen vaccines. This low-cost high-tech method of evaluating the cold chain function is highly recommended for developing and industrialized nations and should be used on a regular basis to check the integrity of the vaccine cold chain. The study highlights the need for technological solutions to avoid vaccine freezing, particularly in hot climate countries.

Drug Stability↗
PubMed1984

Antigenicity of sperm cells after freezing and thawing.

Freezing and thawing is thought to result in removal of spermatozoal membrane antigens. We investigated the presence of sperm antigens before and after freezing and thawing by means of the immunoperoxidase assay (IPAMA), sperm immobilization test (SIT), and separation of proteins by gel electrophoresis. The results of the IPAMA and SIT assays showed no difference in the membrane antigens before and after freezing and thawing. Analysis of surface proteins by gel electrophoresis demonstrated that freezing and thawing did not remove any particular group of proteins from the surface membrane of spermatozoa. According to the evidence of the three tests performed, there is no meaningful removal of antigens from the sperm cell surface membrane by the process of freezing, preservation, and thawing when carried out by the specific methods used. This work does not support the suggestion that in cases of immunologic incompatibility between spermatozoa and cervical mucus it would be possible to overcome the couple's infertility by employing the process of freezing, preservation, and thawing.

Antigens, Surface↗
PubMed2001

Effect of freeze drying and protectants on viability of the biocontrol yeast Candida sake.

The effects of freezing method, freeze drying process, and the use of protective agents on the viability of the biocontrol yeast Candida sake were studied. Freezing at -20 degrees C was the best method to preserve the viability of C. sake cells after freeze drying using 10% skim milk as a protectant (28.9% survival). Liquid nitrogen freezing caused the highest level of damage to the cells with viability < 10%. Different concentrations of exogenous substances including sugars, polyols, polymers and nitrogen compounds were tested either alone or in combination with skim milk. There was little or no effect when additives were used at 1% concentration. Galactose, raffinose and sodium glutamate at 10% were the best protective agents tested alone but the viability of freeze-dried C. sake cells was always < 20%. Survival of yeast cells was increased from 0.2% to 30-40% by using appropriate protective media containing combinations of skim milk and other protectants such as 5% or 10% lactose or glucose, and 10% fructose or sucrose.

Animals↗
PubMed2000

Effect of freezing and thawing rates on the post-thaw viability of boar spermatozoa frozen in FlatPacks and Maxi-straws.

The effects of different freezing and thawing rates on the post-thaw motility and membrane integrity of boar spermatozoa, processed as split samples in Maxi-straws or flat PET-plastic packages (FlatPack) were studied. A programmable freezing device was used to obtain freezing rates of either 20, 50 or 80 degrees C/min. Thawing of the samples was performed in a bath of circulating water; for 40s at 50 degrees C or 27s at 70 degrees C for Maxi-straws and 23s at 35 degrees C, 13s at 50 degrees C or 8s at 70 degrees C for the FlatPacks. Sperm motility was assessed both visually and with a computer assisted semen analysis (CASA) apparatus, while plasma membrane integrity was assessed using the fluorescent probes Calcein AM and ethidium homodimer-1. Temperature changes during freezing and thawing were monitored in both forms of packaging. Values for motile spermatozoa, sperm velocity and lateral head displacement variables were significantly (p<0.05) higher for samples frozen in FlatPacks than in Maxi-straws, with superior results at higher thawing rates. Freezing at 50 degrees C/min yielded better motility than 20 or 80 degrees C/min, although the effect was rather small. Neither freezing rate nor thawing rate had any effect on membrane integrity (p>0.05). A significant boar effect was seen for several parameters. The most striking difference in temperature courses between containers was a 4-5-fold lowering of the thawing rate, between -20 and 0 degrees C, in the center of the Maxi-straw, compared with the FlatPack. This is apparently due to the insulating effect of the thawed water in the periphery of the Maxi-straw. The improvement in sperm motility seen when using the FlatPack appears to be related to the rapid thawing throughout the sample, which decreases the risk of cell damage due to recrystallization during thawing. Since sperm motility patterns have been reported to be correlated with fertility both in vitro and in vivo it is speculated that the use of the FlatPack might improve the results when using frozen-thawed boar spermatozoa for artificial insemination.

Animals↗
PubMed2005

Bioactive compounds of grapefruit (Citrus paradisi Cv. Rio Red) respond differently to postharvest irradiation, storage, and freeze drying.

In the present study, the effect of irradiation, storage, and freeze drying on grapefruit bioactive compounds was investigated. Grapefruits were exposed to one of two irradiation doses: 0 (control) or 300 Gy (137Cs, a proposed treatment against fruit flies) and then stored for up to 6 days. At the last storage time point (6 days after harvest), grapefruit pulp from control and irradiated fruits was freeze-dried. Bioactive compounds were extracted from Rio Red grapefruit pulp and analyzed with reverse phase liquid chromatography while volatile compounds were analyzed using gas chromatography. Freeze-dried pulp from irradiated fruits had a higher (P < or = 0.05) flavonoid content (naringin and narirutin) as compared to the freeze-dried pulp from the control fruits. Freeze-drying treatment reduced (P < or = 0.05) the lycopene content, but the reduction (P < or = 0.05) in beta-carotene content occurred only in the control fruit. Reduction in d-limonene and myrcene was observed in the irradiated fruits at 6 days after harvest and in the freeze-dried samples. These results warrant testing of the effect of postharvest treatments and processing on bioactive compounds in functional systems as they have varied effects on different bioactive compounds of grapefruit.

Ascorbic Acid↗
PubMed2000

Activation of mitogen-activated protein kinases during natural freezing and thawing in the wood frog.

The responses of mitogen-activated protein kinase (MAPK) family members, including ERK (extracellular signal-regulated kinase), JNK (c-Jun NH2-terminal kinase), and p38, in the metabolic responses to whole animal freezing (up to 24 h frozen at -2.5 degrees C) and thawing (up to 4 h at 5 degrees C after a 12 h freeze) were examined in four organs (liver, kidney, heart, brain) of the freeze-tolerant wood frog Rana sylvatica. Levels of the active phosphorylated form of p38 increased within 20 min as an early response to freezing in liver and kidney but rose later (after 12 h) in heart. Both JNK and p38 were activated during thawing in liver, kidney and heart with temporally-distinct patterns in each organ. The only MAPK response to freeze/thaw in frog brain was a transient elevation of p38 after 90 min thawing. ERK activity did not respond to freeze/thaw in any organ. The levels of c-Fos increased during freezing in kidney and brain whereas c-Jun was unaffected by freeze/thaw. Organ-specific responses by MAPKs, particularly p38, suggest that these may have roles in regulating metabolic or gene expression responses that may be adaptive in dealing with freezing stress or metabolic recovery during thawing.

Acclimatization↗
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↗
PubMed1995

Glycine crystallization during freezing: the effects of salt form, pH, and ionic strength.

PURPOSE: The purpose of the study is to characterize glycine crystallization during freezing of aqueous solutions as a function of the glycine salt form (i.e., neutral glycine, glycine hydrochloride, and sodium glycinate), pH, and ionic strength. METHODS: Crystallization was studied by thermal analysis, microscopy, x-ray diffraction, and pulsed Fourier transform nmr spectroscopy. RESULTS: A solution of neutral glycine with no additives undergoes rapid secondary crystallization during freezing, forming the beta polymorph, with a eutectic melting temperature of -3.4 degrees C. Glycine hydrochloride solutions undergo secondary crystallization relatively slowly, and the eutectic melting temperature is -28 degrees C. Sodium glycinate crystallizes from frozen solution at an intermediate rate, forming a eutectic mixture with a melting temperature of -17.8 degrees C. Where secondary crystallization does not occur rapidly, a complex glass transition is observed in the -70 degrees to -85 degrees C temperature range in the DSC thermograms of all systems studied. Rates of secondary crystallization and the type of crystal formed are influenced by solution pH relative the the pKs of glycine, and also by the change in ionic strength caused by adjustment of pH. Increased ionic strength significantly slows the crystallization of neutral glycine and promotes formation of the gamma polymorph. Thermal treatment or extended holding times during the freezing process may be necessary in order to promote secondary crystallization and prevent collapse during freeze drying. CONCLUSIONS: The results underscore the importance of recognizing that seemingly minor changes in formulation conditions can have profound effects on the physical chemistry of freezing and freeze drying.

Calorimetry, Differential Scanning↗
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↗