Freeze Drying Research Papers - Academia.edu (original) (raw)

"""Excipients are the integral part of pharmaceutical products development to achieve desired product profile(stability and efcacy). This review deals with understanding of the physicochemical properties of excipients used
in parenteral formulation development for solution, suspension and lyophilized drug products. However, in spite of proper excipients selection, judicious use during formulation manufacturing process based on their critical properties is also important to avoid negative effects such as loss of drug solubility, activity and stability. This paper deals with proper selection of excipients in lyophilized drug products which gives high critical temperature, good bulking properties avoiding melt back and collapse with improved dried product appearance. We have also emphasized on appropriate selection of excipients for solution, suspension injectable dosage forms and linking their physiochemical properties with optimum manufacturing method with suitable case studies. This review will highlight various excipient related issues optimizing product performance with documented references and practical approaches based on scientific justification. The reader will gain better understanding of excipients complexity during stability studies and resolving problems with practical approach."

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- Pharmacy, Pharmaceutical Technology, Proteomics, Pharmaceutics
- by astrid lobo Gajiwala
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- Evaluation, Treatment, Gamma Rays, Cell

We measured apolipoproteins (apo) A-I and B by rate immunonephelometry (rate INA) during Phase 1 of the National Health and Nutrition Examination Survey (NHANES) III. We also made the measurements by radial immunodiffusion (RID) in a 20% subset of the samples. Aliquots of this subset were also analyzed in the Northwest Lipid Research Laboratories by fixed-time INA calibrated to the World Health Organization (WHO)-International Federation of Clinical Chemistry (IFCC) First International Reference Materials for Apolipoproteins A-I and B. The CVs for the rate INA and RID measurements were: apoA-I, 4.5-7.7% and 2.5-7.6%, respectively; apoB, 2.3-5.3% and 2.3-6.4%, respectively. In NHANES III, rate INA values (x) can be transformed to WHO-IFCC Reference Material-based values (y) as follows: for apoA-I, y = 0.87x + 251.8 mg/L (r = 0.93, SEslope = 0.13, SEintercept = 17, n = 708); for apoB (mg/L), y = 1.068x + 112.8 mg/L (r = 0.98, SEslope = 0.08, SEintercept = 7, n = 646).

- by John Albers
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- Quality Control, Clinical Chemistry, Medical Biotechnology, Clinical
- by Jan Roodenburg
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- Biomedical Engineering, Raman Spectroscopy, Connective tissue, Cluster Analysis

Successful use of liposomes as immunological adjuvants in vaccines requires simple, easy to scale up technology capable of high-yield antigen entrapment. Recent work from this laboratory has led to the development of techniques that can generate liposomes of various sizes containing soluble antigens such as proteins or particulate antigens such as whole, live, or attenuated bacteria or viruses. Entrapment of proteins is carried out by the dehydration-rehydration procedure, which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free antigens. Upon rehydration, the large multilamellar vesicles that are formed incorporate up to 80% of the antigen used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped antigen still associated with the vesicles. A similar technique applied to the entrapment of particulate antigens (e.g., Bacillus s...

- by Gregory Gregoriadis
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- Vaccines, Liposomes, Peptides, Proteins
- by Marina Maizato
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- Biomedical Engineering, Artificial Organs, Cytotoxicity, Artificial

Water activity-temperature state diagrams for Lactobacillus acidophilus freeze-dried in a sucrose or a lactose matrix were established based on determination of stabilized glass transition temperatures by differential scanning calorimetry during equilibration with respect to water activity at fixed temperatures. The bacteria in the lactose matrix had higher stabilized glass transition temperatures for all a w investigated. The survival of Lactobacillus acidophilus determined as colony forming units for up to 10 weeks of storage at 20 C for (i) a w ¼ 0.11 with both freeze-dried matrices in the glassy state, (ii) a w ¼ 0.23 with the bacteria in the lactose matrix in a glassy state but with the bacteria in sucrose matrix in the nonglassy state, and (iii) a w ¼ 0.43 with both freeze-dried matrices in a nonglassy state showed that the nature of the sugar was more important for storage stability than the physical state of the matrix with the nonreducing sucrose providing better stability than the reducing lactose. V

- by Jens Risbo
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- Engineering, Technology, Water, Food Microbiology

It was investigated the effect of particle size, use of infrared radiation, and type of freeze-drying (vacuum or atmospheric) on some nutritional properties of blueberries. Particle size and type of freeze-drying had negative effects on ascorbic acid and total polyphenols content, while the use of infrared radiation had positive effects on them. Particle size negatively affected antioxidant activity. Ascorbic acid was significantly decreased in freeze-dried blueberries compared to fresh fruit, in all the conditions studied. The total polyphenols content was significantly lower in atmospheric freezedried blueberries compared to the fresh ones, unlike in vacuum freeze-drying, where this nutritional property was increased. Antioxidant activity of freeze-dried blueberries did not differ significantly from that found in fresh fruits. In order to minimize the impairment of the nutritional properties of freeze-dried blueberries, it is suggested to use infrared radiation, a relatively small particle size and vacuum freeze-drying.

- by Ruben Darío Martínez Bustos
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- Nutrition and Dietetics, Food, Flavonoids, Food Preservation

Cyclosporine (CsA), commercially available as iv or oral Sandimrnune, is a potent immunosuppressant which can induce doserelated nephrotoxocity. In addition, the iv product contains a solubilizing agent, Cremophore EL, which in itself is reported to be nephrotoxic and can induce, in sensitized patients, anaphylactic reactions. Solubilization of CsA with liposomes or lipid emulsions could provide a suitable alternative dosage form for iv administration. With this in mind, male New Zealand white rabbits were given iv CsA (10 mglkg) in three different dosage forms: (7) CsA:liposornes; (2) CsA:lntralipid (soybean oil and phospholipids); and (3) the commercially available Sandimmune (cyclosporine). The CsA concentration in whole blood samples was analyzed by HPLC. The terminal disposition half-life of CsA (t,,, p) ranged from 400 to 475 min and was not statistically different among the three groups. However, the distribution characteristics of CsA changed dramatically depending on the dosage form. The volume of distribution of CsA at steady state (Vd,,) in Sandimmune was 2.7 ? 0.2 Ukg and was significantly lower than that of either lntralipid (10.6-+ 2.7 Ukg) or liposomes (7.4-+ 2.3 Ukg). A significantly lower total body clearance (TBC) of CsA also was seen for Sandimmune (1 2.7 f 0.3 rnuminlkg) as compared with that of either lntralipid (24.4 f 8.2 mUminlkg) or liposomes (18.9 5 3.9 mUmin1kg). Since CsA is extensively bound to lipoproteins, it is surprising that both test vehicles showed a different distribution pattern. It is speculated that in addition to the benefit of removing Cremophore EL from the product, the different distribution of CsA:liposomes or CsA:lntralipid may prove useful in altering the nephrotoxocity and immunosuppression of CsA during iv therapy.

- by Suresh Venkataram
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- Pharmacokinetics, Pharmaceutical Chemistry, Dosage Form, Lipids

The stability of live-attenuated viral vaccines is important for immunization efficacy. Here, the thermostabilities of lyophilized live-attenuated mumps vaccine formulations in two different stabilizers, a trehalose dihydrate-based stabilizer and a stabilizer containing sucrose, human serum albumin and sorbitol were investigated using accelerated stability tests at 4 • C, 25 • C and 37 • C at time points between 4 h (every 4 h for the first 24 h) and 1 week. Even under the harshest storage conditions of 37 • C for 1 week, the 50% cell culture infective dose (CCID 50 ) determined from titrations in Vero cells dropped by less than 10-fold using each stabilizer formulation and thus complied with the World Health Organization's requirements for the potency of live-attenuated mumps vaccines. However, as the half-life of the RS-12 strain mumps virus infectivity was lengthened substantially at elevated temperatures using the trehalose dihydrate (TD)-based stabilizer, this stabilizer is recommended for vaccine use.

- by Mohammad Taqavian and +1
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- Microbiology, Medical Microbiology, Cercopithecus aethiops, Temperature

Background: Poly-L-lactic acid gained U.S. Food and Drug Administration approval for use in human immunodeficiency virus-related facial lipoatrophy in August of 2004. Since that time, it has become available for use in the United States for human immunodeficiency virus facial lipoatrophy patients and for off-label uses in other areas for soft-tissue contouring. This article is intended to enumerate reconstitution, injection techniques, management, and avoidance of complications. Methods: The authors have pooled their experiences to arrive at a consensus opinion for recommendations on treatment protocols for injectable poly-L-lactic acid use. Results: This article prescribes techniques to achieve safer, consistent results while minimizing risks of complications with injectable poly-L-lactic acid. Although the product has been used widely in Europe since 1999, physicians in the United States have only recently begun to explore the uses of Sculptra as a volumizing agent in the face and the body. U.S. physicians have benefited from the European experience with this product, including early problems secondary to overaggressive use, low-volume reconstitution, higher volume injection of product at one session, and inadequate time between injection sessions. Conclusions: The authors therefore have opted for a more conservative approach in their treatment recommendations. Higher volume dilution (8 to 12 cc), fewer vials used at each session, injections placed in the subcutaneous plane without any product being placed in the dermis, adequate time between injection sessions (at least 6 weeks), and postinjection patient massage should decrease the risks and avoid the potential complications associated with poly-L-lactic acid soft-tissue augmentation.

- by Miles Graivier
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- Face, Medicine, Cellulose, Massage

Aims: To compare the technological robustness of two antifungal Lactobacillus plantarum isolates and to assess their ability to inhibit growth of the spoilage yeast Rhodotorula mucilaginosa in two different refrigerated foods. Methods and Results: The effects of freeze-drying, thermal treatments and varying salt concentrations on the viability of two antifungal lactic acid bacteria (LAB) were examined. Antifungal compound(s) contained in the supernatant of both isolates were compared to commercially available food preservatives. Both isolates were used as dairy starter adjuncts in yoghurt and inoculants in orange juice to determine the antiyeast activity towards R. mucilaginosa. Yeast growth was retarded by the tested isolates in both food settings with one of the isolates, Lact. plantarum 16, being the most potent inhibitor. Conclusions: Both lactobacilli exhibited considerable robustness to withstand processing treatments commonly encountered in a food industrial setting. The isolates were shown to possess potent antifungal activity in both in vivo and in vitro food models. Significance and Impact of the Study: The studied antifungal lactobacilli may represent safer and consumer-friendly alternatives to the use of chemical preservatives. This is the first report of antifungal Lact. plantarum exerting protective potential in yoghurt and orange juice.

- by Douwe Van Sinderen
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- Applied microbiology, Multidisciplinary, Food Microbiology, Food Preservation

Vial ''Fogging'' is a phenomenon observed after lyophilization due to drug product creeping upwards along the inner vial surface. After the freeze-drying process, a haze of dried powder is visible inside the drug product vial, making it barely acceptable for commercial distribution from a cosmetic point of view. Development studies were performed to identify the root cause for fogging during manufacturing of a lyophilized monoclonal antibody drug product. The results of the studies indicate that drug product creeping occurs during the filling process, leading to vial fogging after lyophilization. Glass quality/ inner surface, glass conversion/vial processing (vial ''history'') and formulation excipients, e.g., surfactants (three different surfactants were tested), all affect glass fogging to a certain degree. Results showed that the main factor to control fogging is primarily the inner vial surface hydrophilicity/hydrophobicity. While Duran vials were not capable of reliably improving the level of fogging, hydrophobic containers provided reliable means to improve the cosmetic appearance due to reduction in fogging. Varying vial depyrogenation treatment conditions did not lead to satisfying results in removal of the fogging effect. Processing conditions of the vial after filling with drug product had a strong impact on reducing but not eliminating fogging.

- by Dinu Eric
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- Thrombin, Clinical Sciences, Storage, Solutions

Glass transition temperatures were determined for dehydrated lactose/salt mixtures with various water contents and water activities, and state diagrams were established. Crystallization behaviour was studied for pure amorphous lactose stored at various relative water vapour pressures (RVP). Furthermore, glass transitions temperatures and time-dependent lactose crystallization of freeze-dried lactose and lactose/CaCl 2 , lactose/NaCl, lactose/MgCl 2 and lactose/KCl mixtures in molar ratios of 9:1 were determined. Glass transition temperatures (T g) of lactose powder as determined by differential scanning calorimetry (DSC) was lower than that of lactose/CaCl 2 (9:1) , and lactose/MgCl 2 (9:1), but it was slightly higher than the T g of lactose/NaCl (9:1), and lactose/KCl (9:1). Lactose/KCl had the lowest glass transition temperature, but it had about the same crystallization temperature as lactose/NaCl, and lactose/MgCl 2. The glass transition temperatures decreased as water contents increased. The critical water contents and water activities at 23 1C were predicted using data on glass transition temperature and water sorption. Pure lactose had a different critical water activity and water content from lactose/salt mixtures. The critical values of lactose/CaCl 2 (9:1) were the highest. Loss of sorbed water, indicating lactose crystallization, was observed in lactose and lactose/salt mixtures stored above the critical RVP.

- by Ashok Shrestha
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- Animal Production, Differential scanning calorimetry, Salt, Water Activity

We describe the preparation of a lyophilized material containing purified human pancreatic a-amylase and the certification of its catalytic concentration. The enzyme was purified from human pancreas by ammonium sulphate precipitation and chromatography successively on DEAE-Sephacel, CM-Sepharose and Sephadex G-75. The purified enzyme Nonstandard abbreviations: IFCC, International Federation had a specific activity of 52.9 kU/g protein and was >99% pure on polyacrylamide gel electrophoresis. Only trace amounts of lipase and lactate dehydrogenase were detected in the purified fraction. The purified pancreatic a-amylase had a molar mass of 57 500 g/mol and an isoelectric point at 7.1. The material was prepared by diluting the purified or-amylase in a matrix containing PIPES buffer 25 mmol/1, pH 7.0, sodium chloride 50 mmol/l, calcium chloride 1.5 mmol/1, EDTA 0.5 mmol/1 and human serum albumin 30 g/l, dispensing in ampoules and freeze-drying. The ampoules were homogeneous and the yearly loss of activity on the basis of accelerated degradation studies was less than 0.01% at -20°C. The certified value for or-amylase catalytic concentration in the reconstituted reference material is 555 U/1 _+ 11 U/I when measured by the specified method at 37°C. The material can be used to verify the comparability of results from different laboratories, for intra-laboratory quality control or for calibration of a-amylase catalytic concentration measurements.

- by Ferruccio Ceriotti
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- Catalysis, Kinetics, Quality Control, Clinical Chemistry

Background: Because of the wide variation in the ability of human demineralized freeze-dried bone allograft (DFDBA) to reproducibly induce new bone formation, there is a need for a reliable measure of bone induction activity. In this study we examined an immature osteoprogenitor cell line for its potential utility in measuring the activity of DFDBA in vitro.

- by David Cochran
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- Dentistry, Stem Cells, Bone Morphogenetic Proteins, Cell Division

The aim of this work was to characterize lipid nanoparticles from a rheological point of view, intended for drug delivery after parenteral administration. The conditions to obtain a re-dispersible powder using freeze-drying and spray-drying have also been investigated. Lipid nanoparticles (179.9 ± 6.2 nm) were prepared with the high pressure homogenization technique, using previously established optimal conditions (lipid volume fraction of 0.121), though particle size increased (285.9 ± 4.3 nm) in suspensions produced with higher lipid volume fractions (0.255). Rheology evidenced an expected increase of viscosity with the volume fraction and Newtonian behaviour was observed for volume fractions up to 0.161, while higher volume fractions showed shear thinning and shear thickening. In the suspension with a volume fraction of 0.255, a change of the complex modulus was observed at low shear stress. Freeze-drying and nano spray-drying were suitable only when trehalose was employed as an additive. In the former case, particle size was increased by 18% (198.7 ± 1.1 nm) using 20 fold water dilution. With spray-drying, the use of 20 fold dilution in water:ethanol (8:2) led to particle dimensions of 207.7 ±10.0 ( size 20%). In conclusion, cetylpalmitate nanoparticles seem to be suitable for parenteral application, up to volume fractions of 0.16, and pharmaceutical operations, which submit suspensions to shear stress, should not be a critical issue.

- by Alessandro Di Michele
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- Chemical Engineering, Biomedical Engineering, Rheology, Nanoparticles

Microtubules represent cytoplasmic structures that are indispensable for the maintenance of cell morphology and motility generation. Due to their regular structural organization, microtubules have become of great interest for preparation of in vitro nanotransport systems. However, tubulin, the major building protein of microtubules, is a thermolabile protein and is usually stored at À80°C to preserve its conformation and polymerization properties. Here we describe a novel method for freeze-drying of assemblycompetent tubulin in the presence of a nonreducing sugar trehalose. Even after prolonged storage at ambient temperature, rehydrated tubulin is capable of binding antimitotic drugs and assembling to microtubules that bind microtubule-associated proteins in the usual way. Electron microscopy confirmed that rehydrated tubulin assembles into normal microtubules that are able to generate motility by interaction with the motor protein kinesin in a cell-free environment. Freeze-drying also preserved preformed microtubules. Rehydrated tubulin and microtubules can be used for preparation of diverse in vitro and in vivo assays as well as for preparation of bionanodevices.

- by Vadym Sulimenko
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- Analytical Chemistry, Electron Microscopy, Protein Stability, Cell morphology

The moderate heat treatment of amphotericin B (AmB) in its micellar form (M-AmB) results in superaggregates (H-AmB) that present a substantially lower toxicity and similar activity. The aim of this work was to evaluate the H-AmB behavior after a freeze-drying process. H-AmB and M-AmB micelles were evaluated before and after freeze-drying concerning their physicochemical and biological properties by spectrophotometry and activity/toxicity assay, respectively. Four concentrations of M-AmB and H-AmB were studied aiming to correlate their aggregation state and the respective biological behavior: 50 mg L −1 , 5 mg L −1 , 0.5 mg L −1 , and 0.05 mg L −1. Then, potassium leakage and hemoglobin leakage from red blood cells were used to evaluate the acute and chronic toxicity, respectively. The efficacy of M-AmB and H-AmB formulations was assessed by potassium leakage from Candida albicans and by the broth microdilution method. After heating, in addition to an evident turbidity, a slight blueshift from 327 to 323 nm was also observed at the concentrations of 50 and 5 mg L −1 for H-AmB. Additionally, an increase in the absorbance at 323 nm at the concentration of 0.5 mg L −1 was detected. Concerning the toxicity, H-AmB caused significantly lower hemoglobin leakage than M-AmB. These results were observed for H-AmB before and after freeze-drying. However, there was no difference between H-AmB and M-AmB concerning their activity. Accordingly, the freeze-drying cycle did not show any influence on the behavior of heated formulations, highlighting the suitability of such a method to produce a new AmB product with a long shelf life and with both greater efficiency and less toxicity.

- by Christian Silva
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- Chemistry, Pharmaceutical Technology, Pharmaceutical Chemistry, Medicine

Background: Embryonic enamel matrix proteins are involved in the formation of acellular cementum during development of the periodontal attachment apparatus, suggesting that these proteins might be used clinically to promote periodontal regeneration. At present, it is unknown if these proteins are osteoinductive, osteoconductive, or osteopromotive. To address this question, we examined the ability of a commercially prepared embryonic porcine enamel matrix derivative to induce new bone formation in nude mouse calf muscle, or to enhance the bone induction ability of a demineralized freeze-dried bone allograft (DFDBA). Methods: Porcine fetal enamel matrix derivative (EMD) was implanted bilaterally in the calf muscle of 4 male Nu/Nu mice per treatment group (N = 8 implants): 2 mg EMD alone; 4 mg EMD alone; inactive human DFDBA alone; inactive DFDBA + 2 mg EMD; inactive DFDBA + 4 mg EMD; active DFDBA alone; active DFDBA + 2 mg EMD; and active DFDBA + 4 mg EMD. Implants were harvested after 56 days and examined histologically for bone induction using a semi-quantitative score and histomorphometrically for area of new bone, cortical bone, bone marrow, and residual DFDBA. Results: Implants containing inactive DFDBA, 2 mg EMD, 4 mg EMD, and inactive DFDBA + 2 or 4 mg EMD did not induce new bone. Active DFDBA and active DFDBA + 2 mg EMD induced new bone to a similar extent. In contrast, active DFDBA + 4 mg EMD resulted in enhanced bone induction, area of new bone, and cortical bone. Residual DFDBA was also increased in this group. Conclusions: EMD is not osteoinductive. However, it is osteopromotive, due in part to its osteoconductive properties, but a threshold concentration is required.

- by Barbara Boyan
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- Dentistry, Skeletal muscle biology, Soft Tissue Preservation, Bone Regeneration

Upon the removal of water, proteins undergo a major, reversible rearrangement of their secondary structure, as revealed by FTIR spectroscopy. We have found herein that for recombinant human albumin (rHA) the extent of this structural change does not depend significantly either on the composition of the aqueous solution prior to lyophilization (protein concentration, pH, and the presence of excipients such as dextran or NaCI) or on the mode of dehydration (lyophilization, spray drying, or rotary evaporation), even though these factors profoundly affect rHA's solid-state stability against moisture-induced aggregation. In all cases, the a-helix content of rHA drops from 58% in solution to 25-35% in the dehydrated state, the /3-sheet content rises from 0 to 10-20%, and unordered structures increase from 40% to 50-60%. We have also investigated another model protein, hen egg-white lysozyme, and confirmed that it too undergoes a significant alteration of the secondary structure upon lyophilization. The extent of this structural reorganization has been found to be insensitive to the pH of the aqueous solution prior to lyophilization from pH 1.9 to 5.1, even though the thermal transition temperature (T m) in aqueous solution over this range varies by 30°C.

- by Prashant Mishra
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- Water, Protein Folding, FTIR spectroscopy, Protein Stability

Progesterone rapidly dissolves in an aqueous solution containing hydroxypropyl-b-cyclodextrin (HPBCD) at 1:2 molar ratio, forming a soluble inclusion complex. After filtration and freeze-drying of the final solution, the final powder was examined by SEM, DSC, TGA, XRD, Raman, and FTIR spectroscopy. Experimental results confirm that an inclusion complex exists in the solid state and possible structure of the complex is briefly discussed. ß

T . H . A L -S AM A RR AI A ND J. S CH MI D . 2000. We have developed a new, simple and effective method for extraction of fungal genomic DNA. The initial steps involved suspension of freeze-dried mycelium in buffer containing sodium dodecyl sulphate, detachment of DNA from polysaccharides by mild shearing, NaCl precipitation of polysaccharides and protein, chloroform extraction and ethanol precipitation. The ethanol precipitate was then subjected to a second round of mild shearing, NaCl precipitation, chloroform extraction and ethanol precipitation. The procedure required approximately 1 h to perform. The method yielded 8-32 mg of high molecular weight DNA per 30 mg of freeze-dried mycelium when tested on six fungal species: Aspergillus niger, A. flavus, Fusarium graminarum, Neotyphodium lolii, Penicillium citrinum and Rhizopus nigricanes. The DNA was digestible with EcoRI, HindIII, SalI and BamHI. For the slow-growing N. lolii, a modification of the method was developed that removed the agar residue from colonies grown on agar plates by centrifugation at 13 000 rev min −1 in the presence of CsCl. The modified method yielded 1·5-2 mg of high molecular weight DNA per colony.

- by Jan Schmid
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- Fungi, Multidisciplinary, Fusarium, Ethanol

Fruit and vegetable consumption has consistently been associated with decreased risk of a number of aerodigestive tract cancers, including esophageal cancer. We have taken a "food-based" chemopreventive approach to evaluate the inhibitory potential of lyophilized black raspberries (LBRs) against N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in the F344 rat, during initiation and postinitiation phases of carcinogenesis. Anti-initiation studies included a 30-week tumorigenicity bioassay, quantification of DNA adducts, and NMBA metabolism study. Feeding 5 and 10% LBRs, for 2 weeks prior to NMBA treatment (0.25 mg/kg, weekly for 15 weeks) and throughout a 30-week bioassay, significantly reduced tumor multiplicity (39 and 49%, respectively). In a short-term bioassay, 5 and 10% LBRs inhibited formation of the promutagenic adduct O(6)-methylguanine (O(6)-meGua) by 73 and 80%, respectively, after a single dose of NMBA at 0.25 mg/kg. Feeding 5% LBRs also signif...

- by Gary D Stoner
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- Cancer, Biology, Diet, Medicine

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- by Andrew Lamb
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- Microbiology, Medical Microbiology, Pharmaceutical Chemistry, Escherichia coli

A sensitive method for the determination of the bismuth concentration in human serum is described. Analyses were carried out by inductively coupled plasma-mass spectromctry (ICP-MS), after a simple dilution of the samples with nitric acid. The detection limit of the applied method is O&o7 &I whereas relaitive standard deviations varied from 57 to 13,6% Determination of reference values in human serum of healthy adults gave a range from <0.007 to I (19 persons), Bismuth concentrations in serum were also measu and after the intake of therapeutic doses of colloidal bismuth subcitrate (CBS). About I S-2 h afux the intake of one tablet of CBS, the bismuth concentration in serum was found to have increased 5bi483 times (two apparentiy healthy volunteers and six hos;jitalized patients), showing that the serum bismuth concentrations can increase by several or&~ of magnitude during the intake of CBS. During the intake of four tablets of CBS per day by two apparently healthy volunteers, bismuth concentrations in serum after an overnight fast were I'ound to be, respectively, 5.56 and 8.1 &l on day 15 and 4.28 and 13,6 &I on day 29. After stopping the therapy, the concentration of bismuth in serum slowly returned towards normal over a period of months. Ke)v WON&: Bismuth; Human strum; Colloidal bismuth subcitrate; Inductively coupled plasma-mass spectrometry (ICP-MS) l Corresponding author. 0009-898U93/$06.00 0 1993 Elsevier Science Publishers B.V. All rights reserved. SSDf 0009-898 I (93)05601 -A

- by Hans Vanhoe
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- Mass Spectrometry, Colloids, Clinical Sciences, Bismuth

Preparation of drug-loaded freeze-dried (FD) liposomes, designed for delivery to lungs after rehydration/nebulization was investigated. Rifampicin (RIF) incorporating multilamelar (MLV) and dried rehydrated vesicles (DRV); composed of phosphatidylcholine (PC), dipalmitoyloglycero-PC (DPPC) or distearoyloglycero-PC (DSPC), containing or not Cholesterol (Chol), were prepared. Vesicles were characterized for encapsulation efficiency (EE%), size distribution, zeta-potential, stability during freeze drying (FD) and nebulization (nebulization efficiency (NE%) and retention of RIF after nebulization (NER%)). Mucoadhesion and toxicity in A549 cells was measured. RIF EE% was not affected by liposome type but lipid composition was important; Synthetic lipid vesicles (DPPC and DSPC) had higher EE% compared to PC. As Chol increased EE% decreased. Freeze drying (FD) had no effect on EE%, however trehalose decreased EE% possibly due to RIF displacement. NER% was highly affected by lipid composition. Results of NE% and NER% for RIF-loaded liposomes show that DSPC/Chol (2:1) is the best composition for RIF delivery in vesicular form to lungs, by nebulization. Mucoadhesion and A549 cell toxicity studies were in line with this conclusion, however if mucoadhesion is required, improvement may be needed.

- by Pavlos Klepetsanis
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- Thermodynamics, Drug delivery, Pharmaceutical Chemistry, Aerosols

Purpose (1) To determine the effect of solution pH before lyophilization, over the range of 1.5 to 10, on the salt and polymorphic forms of glycine crystallizing in frozen solutions and in lyophiles. (2) To quantify glycine crystallization during freezing and annealing as a function of solution pH before lyophilization. (3) To study the effect of phosphate buffer concentration on the extent of glycine crystallization before and after annealing. Materials and Methods Glycine solutions (10% w/v), with initial pH ranging from 1.5 to 10, were cooled to −50°C, and the crystallized glycine phases were identified using a laboratory X-ray source. Over the same pH range, glycine phases in lyophiles obtained from annealed solutions (0.25, 2 and 10% w/v glycine), were characterized by synchrotron X-ray diffractometry (SXRD). In the pH range of 3.0 to 5.9, the extent of glycine crystallization during annealing was monitored by SXRD. Additionally, the effect of phosphate buffer concentration (50 to 200 mM) on the extent of glycine crystallization during freezing, followed by annealing, was determined. Results In frozen solutions, β-glycine was detected when the initial solution pH was ≥ 4. In the lyophiles, in addition to β- and γ-glycine, glycine HCl, diglycine HCl, and sodium glycinate were also identified. In the pH range of 3.0 to 5.9, decreasing the pH reduced the extent of glycine crystallization in the frozen solution. When the initial pH was fixed at 7.4, and the buffer concentration was increased from 50 to 200 mM, the extent of glycine crystallization in frozen solutions decreased with an increase in buffer concentration. Conclusion Both solution pH and solute concentration before lyophilization influenced the salt and polymorphic forms of glycine crystallizing in frozen solutions and in lyophiles. The extent of glycine crystallization in frozen solutions was affected by the initial pH and buffer concentration of solutions. The high sensitivity of SXRD allowed simultaneous detection and quantification of multiple crystalline phases.

- by Larry Gatlin
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- Polymorphism, Pharmaceutical Technology, Crystallization, Pharmaceutical

In this study, structure and biomechanical properties of freeze-dried decellularized porcine pulmonary heart valves were investigated. Heart valves were dissected from porcine hearts. The tissues were decellularized and separated in three groups: (1) without lyoprotectant, (2) with 5% sucrose, and (3) with a mixture of 2.5% sucrose and 2.5% hydroxyl ethylene starch (HES), and then underwent freeze-drying. Freeze-drying in the absence of lyoprotectants caused an overall more disintegrated appearance of the histological architecture of the porcine valves, especially between the fibrosa and the ventricularis layers. Freeze-dried tissues with lyoprotectants have a looser network of collagen and elastic fibers with bigger pore sizes. Tissue freeze-dried in the absence of lyoprotecants had the largest pore sizes, whereas the tissue freeze-dried in the presence of protectants showed pores of intermediate sizes between the decellularized tissue and the unprotected freeze-dried samples. Tissue freeze-dried with sucrose alone displayed less porosity than tissue freeze-dried with the sucrose/HES mixture, whereas no significant differences in biomechanical properties were observed. Decellularization decreased the elastic modulus of artery tissue. The elastic modulus of freeze-dried tissue without protectants resembled that of decellularized tissue. The elastic modulus values of freeze-dried tissue stabilized by lyoprotectants were greater than those of decellularized tissue, but similar to those of native tissue.

- by Andres Hilfiker
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- Biomedical Engineering, Tissue Engineering, Porosity, Tissue

Nanospheres containing ketoprofen (Keto) and polymer eudragit RS were prepared using an emulsion solvent evaporation method. The ultrasonic probe (VCX500, vibracell) was used as a tool to disperse oil phase into aqueous phase leading to water/oil emulsion. Nanoparticles were successfully prepared and their morphologies and diameters were confirmed by transmission electron microscope (TEM) and dynamic light scattering (DLS), respectively. The result showed that particles were spherical with submicron size. The particle size was dependent on the RS concentration, emulsification tools and the types of organic solvents. For the encapsulation ability, Keto-loaded RS nanoparticle showed 9.8% of Keto in nanoparticle, which was evaluated by high-performance liquid chromatography (HPLC). Moreover, the drug release behavior of Keto-loaded eudragit RS nanoparticle was also investigated in vitro at pH 7.4 and compared to referential profenid.

- by Osaf Laghari
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- Materials Engineering, Technology, Biomedical Engineering, Water

Photodynamic therapy (PDT) is a promising option in the treatment of cancer. Efficient photosensitizers are available but many of them have insufficient physico-chemical properties for parenteral application. We have established nanoparticles consisting of human serum albumin (HSA) as a drug carrier system for 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrine (mTHPP) and 5,10,15,20tertrakis(m-hydroxyphenyl)chlorin (mTHPC), two well-known photosensitizers.

- by Matthias Wacker
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- Water, Nanoparticle, Pharmaceutical Technology, Scanning Electron Microscopy

Résumé Le mitotane (o,p -dichlorodiméthyl dichloroéthane [o,p -DDD]) est utilisé dans le traitement du cancer de la corticosurrénale et parfois chez des patients atteints du syndrome de Cushing. Ce principe actif est très peu soluble dans l'eau et, après administration orale, environ 60 % de la dose initiale est retrouvée dans les selles. La préparation d'une forme soluble pourrait permettre d'améliorer la biodisponibilité. La caractérisation d'un complexe soluble à base de cyclodextrines constitue l'objectif de ce travail. L'inclusion de mitotane dans la méthyl-ß-cyclodextrine a été étudiée en réalisant des diagrammes de solubilité de phase et des expériences de RMN. Pour déterminer le mécanisme d'inclusion, l'o,p -DDD a été comparé à son régioisomère, le p,p -DDD. Nous avons démontré que deux diméthyl-ß-cyclodextrines (DMßCD) peuvent complexer les cycles aromatiques. À partir des diagrammes de solubilité de phase, on observe également que les deux complexations sont très différentes. K 1:1 est compris entre 37 000 et 85 000 mol.l −1 alors que K 1:2 est compris entre 5,3 et 32 mol.l −1 . Les expériences de RMN ont confirmé l'inclusion et ont permis d'obtenir des indications sur la cinétique de dissociation : la partie ortho-chloro subit un équilibre lent comparativement à l'échelle de temps de la RMN, tandis que la partie para-chloro subit un échange rapide.

- by Stéphane Gibaud
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- Algorithms, Kinetics, Magnetic Resonance Spectroscopy, Solubility

The occurrence of cyanobacterial blooms in aquatic environments is increasing in many regions of the world due to progressive eutrophication of water bodies. Because of the production of toxins such as Cylindrospermopsin (CYN), contamination of water with cyanobacteria is a serious health problem around the world. Therefore it is necessary to develop and validate analytical methods that allow us to quantify CYN in real samples in order to alert the public of this toxin. In this work, an analytical method has been developed an optimized for the determination of CYN from Aphanizomenon ovalisporum cultures. The analytical procedure is based on solvent extraction followed by a purification step with graphitized cartridges and CYN quantification by LC-MS/MS. The extraction and purification steps were optimized using a two-level full factorial design with replications. A suitable and practical procedure for assessing the trueness and precision of the proposed method has been applied by using validation standards. The method has been suitably validated: the regression equation was calculated from standards prepared in extracts from lyophilized M. aeruginosa PCC7820 (r 2 Z 0.9999) and the linear range covered is from 5 to 500 mg CYN/L, equivalent to 0.18-18.00 mg CYN/g dry weight lyophilized cells. Limits of detection and quantification were 0.04 and 0.15 mg CYN/g, respectively, the recovery range (%) oscillated between 83 and 94% and intermediate precision (RSD %) values from 5.6 to 11.0%. Moreover, the present method showed to be robust for the three factors considered: the batch of the graphitized carbon cartridges, the flow rate of the sample through the cartridge, and the final redissolved water volume after SPE treatment, which permits its validation. The validated method has been applied to different lyophilized cultures of A. ovalisporum (LEGE X-001) to evaluate CYN content. This procedure can be used for determining CYN in lyophilized natural blooms samples in environmental studies.

- by Tiina M Nieminen
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- Analytical Chemistry, Graphite, Calibration, Plant tissue Culture Techniques

A series of experiments was performed on lyophylized melanosomes in order to analyze the melanin in the natural state as polymerized into these organelles and to verify in such biological amorphous material the possibility of obtaining intensity scattering curves from which Bragg distances can be calculated. The results confirm the feasibility of the method and show that melanins in melanosomes maintain many structural features of the purified form and that the biochemical composition of the organelles can be responsible for the observed differences in the diffractograms. The presence in melanosomes of supramolecular paracrystalline aggregates was also clearly demonstrated.

- by Franco Ugozzoli and +2
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- Biological Sciences, Horses, X ray diffraction, Freeze Drying

The impact of probiotic supplementation of canine-derived strain Lactobacillus fermentum AD1-CCM7421 in freeze-dried form on quantitative composition of microbiota and short-chain fatty acid profile in feces of dogs was demonstrated by two independent studies (straightforward repeated-measures model; study I: a dose of 2 g per dog for 2 weeks, 10 8 CFU/g, n=12; study II: 1 g per dog for 1 week, 10 7 CFU/g, n=11. The results revealed a significant increase of lactic acid bacteria population persisting also after the cessation of probiotic application in both studies. A reduction of clostridia (study I, p sum <0.01) and tested Gram-negative bacterial genera (coliforms, Aeromonas sp., Pseudomonas sp., study II, p<0.05) was also detected. The strain AD1-CCM7421 colonized the canine digestive tract in sufficient numbers (10 5-10 6 CFU/g) and it persisted in the majority of dogs after cessation of probiotic application. An increase of short-chain fatty acid concentrations (study I: butyric, succinic, valeric, formic acid) especially in the early post-treatment phase (p<0.05) most likely led to a decrease of fecal pH value (p<0.05) without negative influence on fecal consistency throughout the studies.

- by A. Lauková
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- Microbiology, Probiotics, Medical Microbiology, Dogs

An efficient freeze-drying cycle for recombinant human interleukin-1 receptor antagonist (rhIL-1ra) formulations, which contained glycine and sucrose as excipients, was developed. Development was based on characterizing the frozen formulations by thermal analysis and by examining the effect of various lyophilization process parameters on the sublimation rate of ice. Thermal analysis showed that the metastable glass of glycine in frozen formulation could be devitrified by slowly warming the frozen product to -15 degrees C. During drying, the sublimation rate of ice was increased as a linear function of the difference between the vapor pressure of ice at the product temperature (PO) and the chamber pressure (PC). Therefore, the product temperature (Tp) was maintained as high as possible at temperatures below Tg' of the formulation, in order to maximize the PO without allowing the collapse of cake. Although various combinations of shelf temperatures and chamber pressures could be u...

- by Byeong Chang
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- Mathematics, Pharmaceutical, Temperature, Proteins

Moisture content is an important parameter for lyophilized vaccines. Currently, Karl Fischer titration is widely used for moisture determination in routine analysis. However, this method is time-consuming, sample destructive and requires environment polluting reagents, as well as the results rely on the random samplings. In this study, near infrared spectroscopy was used as a fast, non-invasive and non-destructive method to determine the moisture content in lyophilized allergy vaccines. Five different vaccine products were investigated, which contained water in the range of 0.17-1.51% (w/w, KF). Different data pre-treatments, wavelength selection and partial least squares regression were applied to construct calibration models. Multi-products model and product-specific models were obtained, which show the possibility of NIR as a rapid method to discriminate whether moisture content fit into the specifications of a pharmaceutical company.

- by Susanne Wrang Bruun
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- Analytical Chemistry, Water, Vaccines, Near Infrared Spectroscopy

Freeze-drying and irradiation are common process used by tissue banks to preserve and sterilize bone allografts. Freeze dried irradiated bone is known to be more brittle. Whether bone brittleness is due to irradiation alone, temperature during irradiation or to a synergetic effect of the freeze-drying-irradiation process was not yet assessed. Using a left-right femoral head symmetry model, 822 compression tests were performed to assess the influence of sequences of a 25 kGy irradiation with and without freeze-drying compared to the unprocessed counterpart. Irradiation of frozen bone did not cause any significant reduction in ultimate strength, stiffness and work to failure. The addition of the freeze-drying process before or after irradiation resulted in a mean drop of 35 and 31% in ultimate strength, 14 and 37% in stiffness and 46 and 37% in work to failure. Unlike irradiation at room temperature, irradiation under dry ice of solventdetergent treated bone seemed to have no detrimental effect on mechanical properties of cancellous bone.

- by Pierre-louis Docquier
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- Gamma Rays, Cancellous bone, Compressive Strength, Sterilization

Effects of wetting and drying conditions on micromeritic, mechanical and disintegration properties of microcrystalline cellulose (MCC) pellets were evaluated. Extrusion/spheronization and three drying methods (fluidized bed, microwaves, and freeze drying) were applied using two wetting liquids (water or water-isopropanol 60:40 w/w) and three MCC types: (standard, silicified, and modified). Additionally, the effects of drying method were compared on highly porous pellets prepared by the incorporation and extraction of pore former (NaCl). It was found that the drying method has the greatest effect on the pellet size and porosity followed by the wetting liquid. The modification of MCC resulted in reduced water retention ability, implying hornification, increased porosity, reduced resistance to deformation and tensile strength of pellets. The disintegration time also decreased markedly due to the modification but only in the low porosity range &amp;amp;amp;amp;amp;amp;amp;lt;37%. Silicification increased greatly the disintegration time of the low porosity pellets (&amp;amp;amp;amp;amp;amp;amp;lt;14%). Combination of water-isopropanol, freeze drying and modified MCC gave the greatest increase in pellet size and porosity. The increase in pellet porosity caused exponential reduction in the resistance to deformation, tensile strength and disintegration time, as expected. Compared to fluidized bed, the freeze drying resulted in 20-30% higher porosity for pellets prepared without pore former and 6% for those with pore former, indicating the possibility of preparing highly porous pellets by employing freeze drying.

- by Kyriakos Kachrimanis and +1
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- Water, Pharmaceutical Technology, Pharmaceutical Chemistry, Cellulose

A major factor in the direct determination of lutein in spinach extracts proved to be obtaining reproducible and stable chromatography of lutein. This was achieved on a C30 column with the mobile phase acetone-0.1 M triethylammonium acetate (TEAA) buffer (pH 7) 9:1 (v/v). Extraction of 10 mg of lyophilized spinach with 10 mL of extraction solvent (ethanol, acetone, ethanol-ethyl acetate 1:1 (v/v), methanol-THF 1:1 (v/v)) for 15 min with magnetic stirring under nitrogen resulted in equal yields of lutein. The yields were enhanced by addition of 15% of 1 M TEAA buffer pH 7 to all four extraction solvents. As confirmed by recovery experiments, no loss of lutein occurred during the extraction. The relative standard deviation from triplicate extractions was less than 5%. The addition of 15% TEAA pH 7 to acetone enhanced the extraction yield of lutein also from unlyophilized spinach. The content of lutein in different spinach samples ranged from 5 to 15 mg/100 g of fresh weight. The first separation is reported of all the carotenoids and chlorophylls on a C18 core-shell column and the addition of 15% of 1 M TEAA buffer pH 7 to acetone also enhanced the extraction yield of ␤-carotene compared to the yield produced by pure acetone.

- by Irena Vovk
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- Engineering, Technology, High Pressure Liquid Chromatography, CHEMICAL SCIENCES
- by Dr Vivek Shukla
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- Fungi, Blood, Bacteria, Anti-Bacterial Agents

The objective of the study was to compare the effectiveness of trehalose with that of melibiose in protecting a monoclonal antibody (rituximab) from aggregation, fragmentation, and secondary structure alterations during processing and subsequent storage. Because reducing disaccharides such as melibiose participate in Maillard reaction with proteins, especially in the presence of water, the lyophilizates were stored under different relative humidity (RH 5%, 11%, and 23%) atmospheres. Freeze drying was shown to cause clear alterations in rituximab secondary structure, an increase in noncovalent protein aggregation, and in some cases fragmentation. However, these changes were less pronounced in the formulation containing melibiose. Storing the lyophilizates under low RH (5%) proved to be most harmful to the stability of rituximab, intensifying secondary structure alterations and increasing protein aggregate content. Again, these changes were less aggravated in the formulation containing melibiose. Surprisingly, the concentration of aggregates larger than 1 :m decreased in some cases during storage at RH 11% and 23%. There was no indication that storage even under the highest RH (23%) would have caused significant amounts of Maillard reaction end products to be formed during 3 months of storage.

- by Anne Juppo
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- Pharmaceutical Chemistry, Atmosphere, Pharmaceutical Sciences, Trehalose

The long-term preservation of valuable fungal cultures can be achieved in several ways and the choice of methodology can be problematical. Firstly, there is the decision whether to use a public service culture collection or in-house' facilities. Secondly, the wide variety of preservation methods available often leads to confusion about which protocol(s) are best suited for speci®c fungi. No method can be universally applied to all fungi. Some species are notoriously dicult to preserve, whilst other fungi can be preserved by almost any method. A decision-based key has been devised, which uses questions related to fungal characters and user facilities and economics to determine the most appropriate method for long-term preservation of cultures. This key should facilitate the decisions of microbiologists when considering preservation of important fungal cultures.

- by Peter Jeffries
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- Technology, Conservation, Biology, Cryopreservation

Freeze-drying of bacterial cells with retained viability and activity after storage requires appropriate formulation, i.e. mixing of physiologically adapted cell populations with suitable protective agents, and control of the freeze-drying process. Product manufacturing may alter the clinical effects of probiotics and it is essential to identify and understand possible factor co-dependencies during manufacturing. The physical solid-state behavior of the formulation and the freeze-drying parameters are critical for bacterial survival and thus process optimization is important, independent of strain. However, the maximum yield achievable is also strain-specific and strain survival is governed by e.g. medium, cell type, physiological state, excipients used, and process. The use of preferred compatible solutes for cross-protection of Lactobacilli during industrial manufacturing may be a natural step to introduce robustness, but knowledge is lacking on how compatible solutes, such as betaine, influence formulation properties and cell survival. This study characterized betaine formulations, with and without sucrose, and tested these with the model lactic acid bacteria Lactobacillus coryniformis Si3. Betaine alone did not act as a lyo-protectant and thus betaine import prior to freeze-drying should be avoided. Differences in protective agents were analyzed by calorimetry, which proved to be a suitable tool for evaluating the characteristics of the freeze-dried end products.

- by Sebastian Hakansson
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- Cryobiology, Probiotics, Lactobacillus, Sucrose

Freeze drying is a complex, time consuming and thus expensive process, hence creating a need for understanding the material behaviour in the process environment and for process optimization. Near-infrared (NIR) spectroscopy offers the opportunity to monitor physicochemical changes of the formulation during freeze-drying. The aim of this work was to examine whether NIR spectroscopy allows in-line monitoring of all components during the entire freeze-drying process of a multi-component pharmaceutical formulation (a solution of fenofibrate and mannitol in a mixture of tertiary-butyl alcohol, and water). To extract useful information of all components in the formulation from the large multivariate data-sets obtained during in-line spectroscopic monitoring, several spectral pre-processing techniques and spectral data analysis techniques such as the mean of selected wavenumbers (Mws), the correlation coefficient (Cor-rCoef) and principal component analysis (PCA) have been evaluated and compared. To find out whether these chemometric techniques are also able to differentiate between changes in the process settings influencing the freeze-drying process of the formulation, freeze-drying processes were performed at four different conditions. Results demonstrated that in-line measurements using NIR spectroscopy were possible in an icy environment and that a further process understanding could be obtained. Data-analysis revealed the crystallization behaviour of each of the four components. In addition, using the three preprocessing techniques allowed observe the sublimation of the solvents. Mws and CorrCoef have proven to be adequate methods for monitoring the main physicochemical changes of product during the processes; this affirmation was confirmed by observing the outputs of PCA for entire processes.

- by W. Hinrichs
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- Analytical Chemistry, Water, Principal Component Analysis, Crystallization

Use of trehalose as a novel drying aid to produce freeze-dried strawberry puree was studied and compared with conventional drying aids for fruit purees, namely sucrose and maltodextrin (MD). Glass transition temperature (Tg), colour, sensory profile and consumer preference were determined. Purees dried with MD and/or trehalose presented higher Tg values as compared to sucrose. Sensory evaluation indicated that samples dried with trehalose retained fresh strawberry aroma and flavour, keeping a good sweetness/sourness balance, while puree dried with sucrose was mostly sweet. Addition of MD caused an increase in products visual viscosity and sourness, attributes which were negatively correlated to preference. Trehalose presented several advantages for increasing the quality of freeze dried fruit purees. It raised Tg, gave an adequate sweetness/sourness balance, and allowed a better perception of fresh strawberry flavour.

- by Mara Galmarini
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- Food Science and Technology, Taste, Strawberry, Sensory Evaluation

An efficient cryopreservation protocol for the safe storage of Fraxinus excelsior L. embryogenic callus cultures is reported. The cryopreservation methods tested included one-step freezing by means of (i) encapsulation-vitrification; or (ii) encapsulation-dehydration; and (iii) slow cooling using the Nalgene Freezing container, Mr Frosty, which produces a temperature decrease of about 1 masculineC min-1 when placed in a -70 degree C freezer. None of the one-step freezing techniques was effective for cryopreservation of encapsulated callus masses, irrespective of the cryoprotective treatment applied, i.e., treatment with the PVS2 vitrification solution or physical dehydration with silica gel before direct immersion in liquid nitrogen. On the contrary, when a slow cooling protocol was applied to embryogenic callus which had been pretreated for 60 min with a 210 g per liter (0.61 M) sucrose-7.5 percent DMSO cryoprotective solution, up to about 1.3 g per Petri dish of proliferating call...

- by M. Lambardi
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- Cryopreservation, Cell Division, Sucrose, Desiccation