Additives to biological substances. III. The moisture content and moisture uptake of commonly used carrier agents undergoing processing conditions similar to those used in the preparation of international biological standards

Aqueous solutions of the carbohydrates alpha alpha'-trehalose, mannitol and lactose, human serum and human albumin were freeze-dried in ampoules and part of each batch was further desiccated over phosphorus pentoxide in a manner similar to that used to prepare international biological standards and related materials. The residual moisture present in the preparations and their uptake of moisture was then measured by Karl Fischer titration. In ampoules open to atmosphere [22 degrees C, 34% relative humidity (RH)] freeze-dried serum, albumin, lactose and alpha alpha'-trehalose all exhibited hygroscopic properties, their moisture contents rising over a period of 24 h, to 10%, 8%, 8% and 7%, respectively. Mannitol when dried to low moisture levels did not exhibit hygroscopic properties, but the residual moisture in different freeze-dried batches was very variable, possibly as a result of that solid's sponge-like and/or crystalline microstructure. When exposed to the same conditions, dried materials in ampoules fitted with polythene capillary leak plugs (used routinely at this Institute during the preparation of Biological Standards) exhibited a considerably reduced rate of moisture uptake. The effect of secondary vacuum drying over phosphorus pentoxide on the moisture content of these preparations is also reported.     

Effect of Arginine on the Aggregation of Protein in Freeze-Dried Formulations Containing Sugars and Polyol: II. BSA Reconstitution and Aggregation

The current paper continues our study on the ability of l-arginine to prevent/reduce the aggregation of proteins that results from the various stresses during the lyophilisation and/or storage of lyophilized protein-based products. The first part of our study, i.e. formulation development, was devoted to the rational design and optimization of an l-arginine containing lyophilized formulation which can resist the natural tendency of l-arginine to absorb atmosphere moisture. Mannitol and trehalose were chosen among other excipients to be included in the protein-based formulation, as mannitol in a combination with l-arginine has been shown to reduce moisture sorption while trehalose provides a degree of lyoprotection. In the present study, a number of formulations, which comprised bovine serum albumin (BSA) with and without l-arginine, and with five different ratios of trehalose-to-mannitol (from 30:70 to 80:20) were lyophilised and assessed. The internal structures and the moisture sorption/retention of the lyophilized formulations were characterised. To study the effect of l-arginine on BSA solid-phase stability, the lyophilized powder was exposed to accelerated storage conditions (high moisture (75% RH) and temperature (22 or 45 °C)) for up to 24 h. The lyophilized BSA formulations were then reconstituted and solution-state protein aggregation assessed by turbidimetry at 360 nm and fluorescence spectroscopy using the thioflavin T assay. It was demonstrated that l-arginine can be used in protein-based freeze-dried formulations to significantly reduce the aggregation of protein during the manufacturing, storage and subsequent reconstitution. The results also revealed the importance of a sufficient amount of mannitol in the arginine-containing formulations.     

Degradation of functional integrity during long-term storage of a freeze-dried biological membrane

Trehalose, and to some extent a few other carbohydrates, is capable of stabilizing the structure and function of isolated biological membranes during lyophilization. In this paper the results of investigations into the long-term stability of the lyophilized membrane-carbohydrate mixtures were reported. The effects of varying water content, oxygen level, and light on the rates of oxidation, browning, and degradation of biological activity were reported. The efficiency with which three carbohydrates stabilized membrane structure was also reported, with glucose shown to be less efficient than maltose or trehalose. Increased water content accelerated loss of biological activity, possibly because, under the same conditions, nonenzymatic browning and photooxidation were accelerated also. Glucose-containing samples were especially unstable at elevated humidities. Efficiency of preservation could be maximized by storage under conditions of low oxygen, low humidity, and dark, and by the inclusion of high levels of trehalose.     

Thermal stability of freeze-dried mammalian interferons. Analysis of freeze-drying conditions and accelerated storage tests for murine interferon.

Conditions of Interferon processing were analyzed to select those that promote stability after freeze-drying. The effects of various preparative methods and treatment conditions were assessed by measuring the retention of biological activity by lyophilized interferon samples in two kinds of accelerated storage tests: the linear nonisothermal stability (LNS) test, a rapid method used for direct comparison of two or more preparations of interferon, and the multiple isothermal storage (MIS) test, a slow method requiring weeks to months to obtain data for the prediction of stability of a given preparation stored under various conditions.The most stable preparations of Newcastle-disease-virus-induced mouse L cell interferon were obtained using the following conditions: 1) perchlorate treatment to inactivate residual inducing virus, 2) nonspecific adsorption using zeolite for partial purification, 3) suspending medium of 0.5% bovine serum albumin in 0.1 m sodium phosphate buffer at pH 7, and 4) sublimation of ice in vacuo with a starting temperature of ?30 °C to a final residual moisture of about 3%. The final product, reference reagent G002-904-511, was stable throughout the course of the LNS test. From an extensive MIS test, this reference interferon was predicted to lose 1000 units of activity in 110 years at 4 °C and 1000 units in 100 days at 37 °C. After 6 years of storage at 37 °C when the predicted residual activity would be about 20% of the original potency, 35% of initial interferon activity remained, confirming the usefulness of the short-term predictive test.     

Viability of lyophilized microorganisms after 50-year storage

The viability of lyophilized microorganisms belonging to different physiological groups was determined after 50-year storage at 2–4°C. During this period, the number of viable cells gradually decreased, in some cases, by 2–3 orders of magnitude. However, after 50-year storage, the ampoules contained considerable amounts of viable cells (in many cultures, 10⁶–10⁹ cells) that were quite sufficient for the culture maintenance. All the studied lyophilized microorganisms (pro- and eukaryotes) retained their viability after 50-year storage, a longer period than those known in literature.     

Experience with studying suspension media in actinomycete lyophilization]

Viability, cultural features and antibiotic-production properties of the organisms producing tetracycline, chlortetracycline, erythromycin, neomycin, oxytetracycline and polymyxin were studied after their storage for 2 years in ampoules at a temperature of 4--10 degrees in lyophilized state with the use of sodium glutamate, polyvinylpyrrolidone, their combination and horse serum. The highest growth rate was observed in most of the cultures lyophilized in sodium glutamate. The growth of the cultures lyophilized in the solution of polyvinylpyrrolidone alone was mainly scanty or moderate. The antibiotic production level in some strains lyophilized in sodium glutamate or its combination with polyvinylpyrrolidone was after storage for 2 years somewhat higher than that in the control. The cultural features, i.e. the colour of the aerial and substrate mycelium and pigment secretion did not significantly differ in the lyophilized cultures and the cultures maintained on agarized media.     

冻干神经生长因子活性稳定性试验研究

用不同的物质作保护剂冻干纯化鼠神经生长因子,经检定以人血白蛋白作为赋形剂和保护剂冻干神经生长因子制品外观洁白细腻,结构强度良好,生物活性符合要求,残存水分低于３％。采用留样观察法检测其稳定性,将制品放置４℃～８℃,在０、０．５、１、２、３、６、９、１２、１８、２４、２５个月分别测定其各自的活性。结果表明神经生长因子冻干制品在４℃～８℃保存,２５个月仍然保持原有生物活性     

Factors influencing changes in moisture content during storage of freeze-dried vaccines in vials

Previous studies have demonstrated that the water content of freeze-dried vaccines increases during storage. The reasons for these variations in water content are discussed in this paper. Different possible mechanisms have been considered: microleakages at the closure sealing point, water vapour transfer through the closure (permeation, loss and uptake of water by the stopper). Several types of vials and closures were studied, under different conditions of storage. Regardless of the vial form selected, the probability of microleakages was low. The stopper would seem to play an important role in the water transfer mechanisms. The moisture content of the stopper determines its equilibrium relative humidity (ERH) and whether there will be moisture transfer between the stopper and the vial contents and between the stopper and the storage atmosphere.     

Comparison of reactors for oxygen-sensitive reactions: reductive dechlorination of chlorophenols by vitamin b(12s)

Serum bottles are frequently used for studies of reductive dechlorination by vitamin B(12), but reducing conditions can be maintained only for several days. This time period is inadequate for evaluating the reductive dechlorination of some slow-reacting aromatic compounds. Sealed glass ampoules maintain reducing conditions for many months, but this method has the disadvantage of disallowing subsampling of the reaction mixture. A glass serum tube was modified for these experiments which not only maintained anoxic conditions for several days but also allowed subsamples to be removed during experiments. The modification was a restriction placed in the middle of the tube by heating in a flame, creating two chambers separated by a narrow neck. The lower chamber contained the oxygen-sensitive reaction mixture. The upper chamber, sealed with a septum and screw cap, was purged with purified nitrogen or argon introduced and vented through fused silica capillaries. Reductive dechlorination of chlorophenols by vitamin B(12) reduced with Ti(III) citrate was monitored in all three reactor types. Sealed ampoules maintained reducing conditions for up to 12 months. The two-chambered reactor maintained reducing conditions longer than the serum vials when frequent samples were taken.     

On the stability of salivary progesterone under various conditions of storage

The concentrations of progesterone in saliva of women exhibited significant decreases when the fluid was stored in plastic vials for 3 days at room temperature or 37 C. The addition of antibiotics or a variety of metabolic poisons to the saliva prior to storage did not prevent the progesterone decrement. However, the addition of albumin (2 g/dl) was protective, suggesting that the protein impeded adsorption of salivary progesterone by the plastic container. Saliva could be maintained at 37 C for 3 days in glass vials or at -20 C in plastic containers for indefinite periods without loss of progesterone titers. These data indicate that a patient under luteal function assessment may collect saliva samples in glass vials at regular intervals during the latter half of her cycle and store them in the freezer compartment of the refrigerator until shipment by mail to the laboratory for progesterone assay. With special care, plastic vials charged with albumin may also be used.     

Moisture-induced aggregation of lyophilized proteins in the solid state

A critical problem in the storage and delivery of pharmaceutical proteins is their aggregation induced by moisture. A model system has been elaborated and investigated to elucidate the mechanism of this phenomenon. When 10 mg of bovine serum albumin lyophilized from an aqueous solution of pH 7.3 are wetted with just 3 muL of a buffered physiological saline solution and incubated in the solid state at 37 degrees C, the protein progressively loses its solubility in water; e.g., after a 24 h incubation 97% of the protein becomes insoluble. This moisture-induced aggregation of albumin has been discovered to be due to an intermolecular S-S bond formation via the thiol-disulfide interchange reaction. The dependence of the extent of the solid-state aggregation on the amount and mode of addition of moisture and the atmosphere, additives, temperature, and history of the protein powder have been investigated. The moisture-induced solid-state aggregation has also been established and studied for three other lyophilized proteins: ovalbumin, glucose oxidase, and beta-lactoglobulin. In all cases, the loss of solubility is caused by thiol-disulfide interchange either alone or in combination with a conformational (noncovalent) process. The aggregation can be minimized by lyophilizing the proteins from acidic aqueous solutions, by adding inorganic salts, by co-lyophilizing the proteins with water-soluble polymers, or by controlling the moisture content at optimal levels.     

Influence of moisture and aeration on oxygen uptakes in Warburg respiratory experiments with litter and soil

Summary Oxygen uptakes by litter and soil from a beech forest were determined in a Warburg apparatus, using material at field moisture content, at 60 per cent water-holding capacity (WHC), and at saturation (>100 per cent WHC), in unshaken and shaken flasks. In unshaken flasks, oxygen uptakes by materials from some horizons were markedly dependent upon their moisture content. Oxygen uptakes were similar with unshaken materials at 60 per cent WHC and with shaken water-saturated materials; this latter method of determination is preferred for routinely measuring respiratory activity under aerobic conditions. Control of moisture content of the systems appeared necessary to estimate respiratory responses to added solutions of glucose. Significant responses to added glucose were found with materials from all horizons under conditions of adequate moisture and aeration but responses were much less with some unshaken water-saturated materials.     

PRODUCTION,PURIFICATION, AND CHEMICAL STABILITY OF RECOMBINANT HUMAN INTERFERON-γ IN LOW OXYGEN TENSION CONDITION: A FORMULATION APPROACH

The low stability of recombinant human interferon-γ (rhIFN-γ) therapeutic protein imposes some restrictions in its medical applications. In the current study, the effect of oxygen tension on the stability of purified rhIFN-γ was investigated. The rhIFN-γ was purified (>99%) by a two-step chromatographic process. Storage vials were filled by purified formulated product under normal atmospheric oxygen and low oxygen tension conditions. At different time intervals, the amounts of rhIFN-γ covalent dimers and deamidated forms were analyzed using analytical high-performance liquid chromatography (HPLC; size exclusion and cation exchange) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods. To determine the biological activity of purified rhIFN-γ, an antiviral activity assay against vesicular stomatitis virus (VSV) was performed. Upon rhIFN-γ long-term storage in a low oxygen tension condition, the amounts of rhIFN-γ covalent dimers and deamidated forms and also the biological activity of rhIFN-γ changed a little. In contrast, by 9 months of storage of rhIFN-γ preparations under normal atmospheric condition, the amount of covalent dimers and deamidated forms increased with time and reached to approximately 3.5% and 11.5% of the initial amount, respectively. The antiviral specific activity of 9-month-old rhIFN-γ preparations decreased to 41% of the initial amount at normal storage condition, while no significant reduction was seen at the low oxygen tension condition. In conclusion, oxygen tension during storage could have a significant impact on rhIFN-γ stability and finally on the quality of pharmaceutical rhIFN-γ product.     

International collaborative study by in vitro bioassays of the first international standard for porcine inhibin.

A lyophilized preparation of inhibin from porcine ovarian follicular fluid, ampoule code 86/690, was made internationally available as a research standard for in vitro bioassays in 1987. A study involving ten participants in eight countries assessed the stability and suitability of this research standard to serve as an international standard. Each of the participants used in vitro assays, the majority of which depended upon the inhibition of release of follicle-stimulating hormone from dispersed rat anterior pituitary cells. The research standard 86/690 was compared with coded ampoules of 86/690 stored under conditions of accelerated thermal degradation and with inhibins from different species. Intra- and interlaboratory variation for estimates of potency of a coded duplicate ampoule of the research standard provided the basis for comparisons of non-identical inhibins, but the fourfold variability of potency estimates for identical ampoules was such that no conclusions about the differences seen for non-identical inhibins could be made. Predictions of stability from consensus estimates of potency of ampoules that have undergone accelerated thermal degradation indicated that the research standard had satisfactory stability. On the basis of this study, the research standard 86/690 was deemed sufficiently stable and suitable to serve as a standard for in vitro bioassays and was established by the World Health Organization Expert Committee on Biological Standardization as the First International Standard for Porcine Inhibin. The possible presence, in biological extracts (standard or sample), of other bioactive proteins, such as activin and follistatin, complicates the quantitative interpretation of bioassay data. A standard of highly purified human inhibin is now required as a standard for immunoassays used for clinical research purposes; sufficient quantities of recombinant human inhibins have recently been donated for ampouling and evaluation by bio- and immunoassay in the subsequent phase of the standardization of inhibins.     

Long-term preservation of some Rhodospirillaceae by freeze-drying

Lyophilization of phototropically grown cultures is difficult as it is essential to maintain strict anaerobic conditions or to avoid the exposure of the cultures to light. During a systematic investigation it was observed that several species of Rhodospirillaceae are able to grow heterotrophically in darkness on organic media and are not damaged by air oxygen under such conditions. Based on this character several oxygenic species of Rhodospirillaceae were grown under heterotrophic conditions and were lyophilized using raffinose (5% w/v) along with skim milk (20% w/v) as a protective agent. More than 30 strains from nine species of Rhodospirillaceae were successfully freeze-dried with this method. All tested cultures proved viable and showed 10–100% survival after lyophilization. During 2–3 years of storage at 9°C no further loss in viability was observed. In such lyophilized cultures no loss in photoautotrophy, diazotrophy or other desirable characters like hydrogen production, pigmentation, etc. was detected. The method is not suited to such anoxygenic Rhodospirillaceae which are not able to grown aerobically in darkness.     

Comparison of Reactors for Oxygen-Sensitive Reactions: Reductive Dechlorination of Chlorophenols by Vitamin B12s

Serum bottles are frequently used for studies of reductive dechlorination by vitamin B₁₂, but reducing conditions can be maintained only for several days. This time period is inadequate for evaluating the reductive dechlorination of some slow-reacting aromatic compounds. Sealed glass ampoules maintain reducing conditions for many months, but this method has the disadvantage of disallowing subsampling of the reaction mixture. A glass serum tube was modified for these experiments which not only maintained anoxic conditions for several days but also allowed subsamples to be removed during experiments. The modification was a restriction placed in the middle of the tube by heating in a flame, creating two chambers separated by a narrow neck. The lower chamber contained the oxygen-sensitive reaction mixture. The upper chamber, sealed with a septum and screw cap, was purged with purified nitrogen or argon introduced and vented through fused silica capillaries. Reductive dechlorination of chlorophenols by vitamin B₁₂ reduced with Ti(III) citrate was monitored in all three reactor types. Sealed ampoules maintained reducing conditions for up to 12 months. The two-chambered reactor maintained reducing conditions longer than the serum vials when frequent samples were taken.     

Viability of Bacillus popilliae after Lyophilization of Liquid Nitrogen Frozen Cells

The per cent viability of Bacillus popilliae after lyophilization of liquid nitrogen frozen cells was determined. Lyophilization of 9- to 12-hr cells which had been suspended in 5% sodium glutamate plus 0.5% gum tragacanth, frozen in liquid nitrogen vapor, and dried 4 to 5 hr with the ampoules exposed to room temperature resulted in survival of 64.6% of the original cells. After storage of these lyophilized preparations for 6 months at room temperature, 10.5% of the original cells were still viable.     

Do-It-Yourself device for recovery of cryopreserved samples accidentally dropped into cryogenic storage tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 °C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues (1,2). The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn' (2), which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces (2). In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks (1). These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials (3). However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use. In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.     

The Influence of Temperature, Moisture, and Oxygen on Period of Seed Viability in Barley, Broad Beans, and Peas

The viability of seeds of barley, broad beans, and peas hasbeen examined in hermetic storage over a range of temperaturesfrom 25? to 45? C and over a range of moisture contents fromabout 12 to 18 per cent. It was found that the survival curvesunder nearly all conditions can be described as negative cumulativenormal distributions. Under very severe storage conditions,however, when the mean viability period is of the order of aweek or less, the survival curves may become slightly skew.The spread of the distribution is linearly proportional to themean viability period. There is a negative linear relationshipbetween log mean viability period and both temperature and moisturecontent. Because of these relationships it is possible to predictthe percentage germination of these species after any givenperiod under a wide range of storage conditions. This patternof loss of viability in barley, broad beans, and peas is consistentwith that previously shown for wheat and rice. Oxygen has been shown to have a deleterious effect on the viabilityof barley, beans, and peas. Most of the deleterious effect isproduced by increasing the oxygen level from 0 per cent (nominal)to 21 per cent at atmospheric pressure; a further increase to100 per cent has comparatively little effect. The deleteriouseffect of oxygen is more pronounced at high moisture contents.Experiments at a low moisture content (12 per cent) have demonstratedthat the effect of oxygen is independent of the activity ofmicro-organisms. There is also some indication that the effectof oxygen is independent of the rate of seed respiration. The gas-exchange of pea seeds has been investigated in hermeticstorage at 25? C and 18.4 per cent moisture content. The seedsshowed a constant rate of gas-exchange and a constant R.Q. (0.63)over a period of time during which the concentration of oxygendecreased from 21 to I.4 per cent and the carbon dioxide concentrationrose from 0.03 to 12 per cent.     

A hydrolytic procedure giving reproducible analytical conditions

A hydrolytic procedure which can be routinely performed in a readily reproducible environment is described. The procedure is useful in that it can be applied to batches of samples, giving a high degree of confidence that similar conditions exist within and among batches. The method makes use of a special polythene capillary leak plug which is fitted in the mouth of ampoules (which were back-filled with nitrogen) prior to their being sealed in the open laboratory. This plug acts as an efficient barrier, for several minutes, to gaseous diffusion, allowing only small amounts of oxygen to enter the ampoule. The method is demonstrated by results from amino acid analyses of acid-hydrolyzed samples of bovine serum albumin.