The gravimetric method for the determination of residual moisture in freeze-dried biological products

The gravimetric test for the determination of residual moisture in freeze-dried biological products performed in a humidity- and temperature-controlled room with the use of scrupulous gravimetric analytical technique can be used to accurately determine residual moisture in freeze-dried biological products such as antihemophilic factor (human) or honey bee venom allergenic extract. This method determines the first water of hydration of sodium tartrate dihydrate (7.93%) to within 1.3% of the calculated value with a relative standard deviation of 0.3% for 10 replicates. For this gravimetric procedure, freeze-dried samples containing from 1.12 to 4.4% residual moisture had relative standard deviations ranging from 3.6 to 9.1%. Samples containing less than 1.0% residual moisture by the gravimetric method such as intravenous immune globulin and antihemophilic factor (human) had relative standard deviations ranging from 16.7 to 47.0%. Relative standard deviations for residual moisture tests performed on comparable samples by the Karl Fischer and thermogravimetric methods showed similar variability.     

A comparison of vials with ampoules for the storage of biological reference materials.

Lyophilization is a key strategy in the stabilization of biological materials. Protection of the lyophilized material from an oxidizing atmosphere is essential if stability is to be maintained under long term storage. Vials of lyophilized albumin closed by two methods and ampoules of albumin have been examined for moisture and oxygen ingress after storage both under conditions of stress for two months and under thermally accelerated conditions for one year. The results show that the gas and moisture contents of ampoules do not detectably change even under conditions of stress, in contrast to vials for which this study shows clearly detectable moisture ingress and suggests some oxygen ingress. This is consistent with the results of other studies. Thus, although vials may be satisfactory under constrained conditions of temperature and storage for limited periods of time, and are presently used satisfactorily for some working standards, it would be prudent to continue to use ampoules for storage of international reference materials which are intended for indefinite storage and for which stability is an essential requirement.     

影响生物制品冻干粉针剂水分的探讨

探讨生物制品冻干粉针剂样品放置一时间后残余水分增高的原因。进行了水分测定,真空度检测,二甲硅油和丁基橡胶药用瓶塞干燥失重的检测。冻干后每只丁基橡胶药用瓶塞平均含水分0.00224g。结果表明丁基橡胶药用瓶塞灭苗,干燥和冻干过程中去除水分不彻底是引起样品中水分升高的直接原因。     

Freeze-Drying of Foot-and-Mouth Disease Virus and Storage Stability of the Infectivity of Dried Virus at 4 C

Foot-and-mouth disease virus, type A, strain 119, propagated in cultures of calf kidney cells and in the tongue epithelium of cattle was used. The process of freeze-drying was conducted in two cycles on unit volumes of 4 ml in Pyrex ampoules, averaging 150 ampoules per run, and was studied separately from the problems of storage. Ampoules containing freeze-dried virus were flame-sealed for either immediate study or storage at 4 C for later reference. Tissue-culture virus dried with various additives had a mean processing loss of 0.8 log LD₅₀ per ml for six different preparations. Virus freeze-dried in tissue suspension had a mean loss of 0.8 log LD₅₀ per ml for three different preparations. A second set of preparations was processed and specifically studied for storage quality at 4 C. The virus in 14 freeze-dried tissue-culture preparations had a mean loss of 0.75 log LD₅₀ per ml while stored at 4 C for 1 year. Virus in four freeze-dried tissue suspensions had a mean loss of 0.05 log LD₅₀ per ml held at 4 C for 1 year. None of the specific additives used for conservation of the virus during the freeze-drying process or during storage at 4 C contributed significantly to the stability of the virus preparations over and above that observed with the normal growth medium of the tissue culture or the ordinary diluents used in making suspensions of tissue virus.     

冻干麻疹减毒活疫苗残余水分影响因素的探讨

影响冻干制品残余水分的因素很多,但西林瓶胶塞的含水量是其关键因素。使用经干烤工艺处理和未干烤处理的药用溴丁基胶塞进行麻疹减毒活疫苗冻干试验,结果显示,用未经干烤工艺处理及处理的药用溴丁基胶塞冻干的麻疹减毒活疫苗,存储18个月后,水分上升的平均值分别为2.03%和0.08%,经单一因素的方差分析,两者有统计学意义(P0.01)。证明经处理的药用溴丁基胶塞,对保持麻疹减毒活疫苗在存储期内的残余水分稳定作用效果明显。     

Stabilities of Suspensions of Influenza Virus Dried by Sublimation of Ice In Vacuo to Different Contents of Residual Moisture and Sealed Under Different Gases

Suspensions of influenza virus were dried by sublimation of ice in vacuo to contents of residual moisture of 2.8, 1.6, or 0.6%. After drying, the preparations were sealed under helium, argon, nitrogen, or a mixture of oxygen and nitrogen (30% O(2), 70% N(2)). Stabilities of the several preparations were determined by an accelerated storage test. Based on the times predicted for the dried preparations stored at preselected temperatures to lose 1 log of infectivity titer, the order of stabilities in relation to residual moisture, gas was as follows: 1.6%, He > 0.6%, He > 2.8%, He > 1.6%, Ar > 2.8%, N(2) > 2.8%, Ar > 1.6%, N(2) > 2.8%, O(2) > 0.6%, Ar > 0.6%, N(2) > 1.6%, O(2) > 0.6%, O(2). The stability of the preparation sealed under helium and dried to the content of residual moisture found best for this gas (1.6%) resulted in an increased stability of the order of years as compared to the other preparations tested.     

99mTc-neolactosylated human serum albumin for imaging the hepatic asialoglycoprotein receptor

99mTc-labeled diethylenetriaminepentaacetic acid (DTPA)-coupled neogalactosyl human serum albumin (GSA) is used as an imaging agent for asialoglycoprotein receptor of the liver. However, its labeling is inconvenient because it should be incubated for 30 min at 50 degrees C. In addition, the conjugated DTPAs can cause decrease of pI and denaturation of protein. Therefore, we developed an improved agent 99mTc-neolactosyl human serum albumin (LSA) which contains a terminal galactose. LSA was synthesized by conjugating lactose to human serum albumin by the formation of a Schiff's base and successive reduction with sodium cyanoborohydride. The number of conjugated lactose molecules per LSA was 40.7 +/- 12.3. To simplify the labeling procedure, we used a direct labeling method that adopts a high affinity 99mTc binding site concept in antibody labeling. The produced LSA was reduced by beta-mercaptoethanol to generate sulfhydryl groups and purified by PD-10 size-exclusion column. The number of generated sulfhydryl groups per LSA was 21.9 +/- 3.0. Medronate and stannous chloride were added to the reduced LSA and freeze-dried. Finally, 99mTc-pertechnetate (37 MBq, 1 mL) was added to the vial and incubated for 10 min at room temperature. The labeling efficiency of 99mTc-LSA was higher than 98%, and the stability in human serum at 37 degrees C for 24 h was over 90%. Biodistribution study using balb/c mice and imaging study using SD rats showed high initial liver uptake and slow increase in the intestine due to hepatobiliary excretion after metabolism in the hepatocytes. Negligible spleen uptake was found while 99mTc-tin colloid showed significant amount of spleen uptake due to reticuloendothelial uptake. In conclusion, an improved agent, 99mTc-LSA, for imaging asialoglycoprotein receptor of the liver was successfully developed which showed a simple labeling procedure, high labeling efficiency, high stability, and high initial liver uptake.     

Proceedings: Stability and residual moisture content of dried vaccinia virus

Dried products of unpurified vaccinia virus containing 5% sodium glutamate or 5% peptone as a protectant, and the product without any protectant were prepared from calf dermal pulp.In these products the relation between the stabilities and the residual moisture content were examined.The stability of all dried products remained unaffected when the residual moisture content was kept below about 4%.     

Near infrared reflectance spectroscopy (NIRS) to predict biological parameters of maize silage: effects of particle comminution, oven drying temperature and the presence of residual moisture

Maize silage nutritive quality is routinely determined by near infrared reflectance spectroscopy (NIRS). However, little is known about the impact of sample preparation on the accuracy of the calibration to predict biological traits. A sample population of 48 maize silages representing a wide range of physiological maturities was used in a study to determine the impact of different sample preparation procedures (i.e., drying regimes; the presence or absence of residual moisture; the degree of particle comminution) on resultant NIR prediction statistics. All silages were scanned using a total of 12 combinations of sample pre-treatments. Each sample preparation combination was subjected to three multivariate regression techniques to give a total of 36 predictions per biological trait. Increased sample preparations procedure, relative to scanning the unprocessed whole plant (WP) material, always resulted in a numerical minimisation of model statistics. However, the ability of each of the treatments to significantly minimise the model statistics differed. Particle comminution was the most important factor, oven-drying regime was intermediate, and residual moisture presence was the least important. Models to predict various biological parameters of maize silage will be improved if material is subjected to a high degree of particle comminution (i.e., having been passed through a 1 mm screen) and developed on plant material previously dried at 60 °C. The extra effort in terms of time and cost required to remove sample residual moisture cannot be justified.     

Stability of Suspensions of Influenza Virus Dried to Different Contents of Residual Moisture by Sublimation In Vacuo

After freezing, suspensions of influenza virus were dried by sublimation of water in vacuo to contents of residual moisture of 3.2, 2.1, 1.7, 1, or 0.4%. The stability of the several suspensions was determined by an accelerated storage test. Based on the times predicted for the dried preparations stored at different temperatures to lose 1 log of infectivity titer, the order of stability in relation to residual moistures was as follows: 1.7% > 2.1% > 1% > 3.2% > 0.4%.     

水分和磷素对木荷不同种源苗木生长和磷效率的影响

以用木荷中心产区的浙江龙泉、福建建瓯、尤溪和江西吉安4个代表性优良种源为试验材料,以广西产红荷为对照,设置不同水分处理和磷素水平的盆栽试验,研究水分和磷素对木荷种源苗木生长和生理指标的影响.结果表明：在不同的水分和磷素处理下,4个参试种源的苗木生长、根系形态参数和磷素吸收效率等均存在显著的遗传差异,福建建瓯和浙江龙泉种源苗木生长量大、根系发达、磷素吸收效率高,生长表现明显优于福建尤溪和江西吉安种源,而广西产红荷则保持其原产地速生、抗旱、耐瘠的特性.土壤水分和磷素对木荷种源苗木生长影响显著.适宜水分条件下,种源苗木径生长、干物质量、根系参数和磷素吸收效率较干旱胁迫条件下高18.5%～105.6%,高磷水平下种源苗木上述性状较低磷处理高37.5%～286.2%.但在干旱和低磷胁迫下,木荷将光合产物更多地分配至地下根系,磷素利用效率也较高,这可能是木荷适应干旱和低磷胁迫的重要生理机制.相对于显著的种源、水分和磷素主效应,其间的交互作用则可以忽略.     

Physical properties and compact analysis of commonly used direct compression binders

This study investigated the basic physico-chemical property and binding functionality of commonly used commercial direct compression binders/fillers. The compressibility of these materials was also analyzed using compression parameters derived from the Heckel, Kawakita, and Cooper-Eaton equations. Five classes of excipients were evaluated, including microcrystalline cellulose (MCC), starch, lactose, dicalcium phosphate (DCP), and sugar. In general, the starch category exhibited the highest moisture content followed by MCC, DCP, lactose, and finally sugars; DCP displayed the highest density, followed by sugar, lactose, starch, and MCC; the material particle size is highly processing dependent. The data also demonstrated that MCC had moderate flowability, excellent compressibility, and extremely good compact hardness; with some exceptions, starch, lactose, and sugar generally exhibited moderate flowability, compressibility, and hardness; DCP had excellent flowability, but poor compressibility and hardness. This research additionally confirmed the binding mechanism that had been well documented: MCC performs as binder because of its plastic deformation under pressure; fragmentation is the predominant mechanism in the case of lactose and DCP; starch and sugar perform by both mechanism.     

Freeze drying of histocompatibility typing sera.

This report describes a unit employed for the freeze drying of histocompatibility typing serum using a 50-hr cycle. This unit will process approximately 3200 3-ml vials with a final residual moisture content of less than 2%. The system employs dry ice-alcohol cooled circulating baths to maintain the condensers below ?60 °C, and two shelf cooling baths to maintain the product at required temperatures during the freeze drying process.The results of a 5-yr study of the effect of residual moisture as a function of time and storage temperature is also included. Studies conducted to date indicate that with residual moistures below 2%, freeze dried histocompatibility sera can be stored at +4 °C without the loss of significant tissue typing factors. Solubility of all serum was lost when stored at +37 °C or higher during this same 5-year period.     

Additives to biological substances. V--The stability of lactose as a carrier for Biological Standards

The stability of freeze-dried lactose has been studied, by accelerated degradation, after being ampouled under the conditions employed for the preparation of International Standards and Reference Preparations and also under less stringent conditions which might facilitate degradation. The possible formation of the reactive product, 5-hydroxymethyl-furfural, has been monitored over a period of 10 years at temperatures up to 56 degrees C. No evidence has been obtained to suggest that the formation of this compound would present a hazard to the stability of standards prepared by the procedures customarily employed.     

Stabilities of Dried Suspensions of Influenza Virus Sealed in a Vacuum or Under Different Gases

Suspensions of purified influenza virus, dried to a 1.4% content of residual moisture by sublimation of ice in vacuo, were sealed in a vacuum or under different gases of high purity. The stabilities of the several preparations were determined by an accelerated storage test. Based on the times predicted for the dried preparations stored at different temperatures to lose 1 log of infectivity titer, the order of stabilities in relation to sealing in vacuum or under different gases was as follows: helium > hydrogen > vacuum > argon > nitrogen > oxygen > carbon dioxide.     

Freeze-drying of solutions of serum albumin containing dimethylsulfoxide.

Although several publications have emphasized the inadvisability of drying biologic materials containing dimethylsulfoxide (DMSO) by sublimation of ice in vacuo, our studies showed a relationship to exist between the relative concentrations of serum albumin, human, and dimethylsulfoxide for successful or unsuccessful freeze-drying of albumin-DMSO solutions. The cycle of freeze-drying for the successful drying of an albumin-DMSO solution was a modification of the cycle used for the successful drying of suspensions of measles virus by sublimation of ice in vacuo. Using nuclear magnetic resonance spectroscopy, a strong, sharp signal for DMSO was obtained in preparations of freeze-dried albumin-DMSO solutions rehydrated with deuterium oxide, D₂O.     

Oxygen delivery and myocardial function in rabbit hearts perfused with cell-free hemoglobin.

Isolated rabbit hearts were perfused with Krebs-Henseleit buffer that contained 1.5 g/dl hemoglobin Ao [HbAo; PO2 at which half-saturation of hemoglobin occurs = 12 Torr], human hemoglobin cross-linked between alpha-chains with bis(3,5-dibromosalicyl)fumarate (alpha alpha-Hb; PO2 at which half-saturation of hemoglobin occurs = 30 Torr), or fatty acid-free bovine serum albumin (BSA). Myocardial performance and oxygen uptake were determined at different aortic PO2's [arterial PO2 (PaO2)] by use of an isovolumic Langendorff preparation. Function and oxygen uptake were comparable among the three different groups of hearts at an average mean PaO2 of 557 Torr. As PaO2 decreased, myocardial function was preserved better in hearts perfused with hemoglobin than in hearts perfused with Krebs-Henseleit buffer alone or with BSA. Hearts perfused with either HbAo or alpha alpha-Hb exhibited similar 10% decreases in left ventricular developed pressure and rate of change in left ventricular developed pressure at PaO2 of 141 Torr compared with a 58% decrease with BSA. However, corresponding venous PO2's were lower with HbAo (20 Torr) than with alpha alpha-Hb (35 Torr), and oxygen uptake decreased by 36% with HbAo but remained constant with alpha alpha-Hb. These data suggest that although myocardial function can be sustained over a fairly broad range of hemoglobin oxygen affinities, tissue oxygen gradients and myocardial oxygen uptake are maintained better by cell-free hemoglobin with an oxygen affinity in the normal physiological range.     

Fungal growth on different insulation materials exposed to different moisture regimes

Commercially available paper, flax, glass wool and rock wool insulation materials were tested for sensitivity to moisture and the ability of fungi to grow on them under different moisture regimes. Three levels of moisture were used, ambient, simulated rain and water holding capacity. After wetting, the materials were inoculated with a mixture of fungi and incubated at 26°C in boxes with high moisture levels for 4 weeks. The greatest degree of fungal growth was observed on paper and flax insulation materials initially conditioned to ambient moisture levels. Paper and flax insulation were strongly affected by moisture, with all treatments showing losses in dry mass of approx. 20%, except for paper subjected to simulated rain treatment in which the loss was 39%. Glass and rock wools were not sensitive to moisture and were resistant to fungal degradation, showing only traces of fungal growth and minimal loss in dry mass over 4 weeks.     

Simplified elimination of rabbit anti-rat acute phase protein IgG that shows cross reactivity with albumin

Antibody preparations against rat acute phase proteins were tested for cross reactivity with other serum proteins, including rat albumin. Rabbit anti-rat a alpha1-acid glycoprotein and ceruloplasmin IgG purified on protein A-Sepharose did not show any cross reactivity with rat albumin, hemoglobin or transferrin. Rabbit anti-rat haptoglobin and -macroglobulin IgG purified on protein A-Sepharose showed a 39% and 30% cross reactivity with rat albumin and a 20% and 19% cross reactivity with rat hemoglobin. Because these proteins in whole serum were not adsorbed on Cibacron Blue F3-GA Sepharose, the albumin would be adsorbed on Cibacron Blue F3-GA Sepharose by the use of whole rat serum. Rabbit anti-rat haptoglobin and alpha2-macroglobulin IgG showing cross reactivity with albumin was simply eliminated.     

Response of bacteria in wastewater sludge to moisture loss by evaporation and effect of moisture content on bacterial inactivation by ionizing radiation.

Two studies were carried out to determine the influence of moisture content of the survival of bacteria in raw wastewater sludge. The first study involved the effect of water loss by evaporation on the bacterial population. The second used these dewatered samples to measure the effects of moisture content on the inactivation of bacteria sludge by ionizing radiation. Both studies involved survival measurements of six representative fecally associated bacteria grown separately in sterilized sludge as well as survival data on bacteria indigenous to sludge. Growth of bacteria was stimulated in sludge during the initial phase of moisture removal by evaporation, but the reduction of moisture content below about 50% by weight caused a proportional decrease in bacterial numbers. In comparison with the original sludge, this decrease reached about one-half to one order of magnitude in all dried samples except those containing Proteus mirabilis, which decreased about four orders of magnitude. The rates of inactivation of bacteria by ionizing radiation in sludge were usually modified to some degrees by variations in moisture content. Most bacteria were found to be somewhat protected from ionizing radiation at reduced moisture levels. The largest effect was found with Salmonella typhimurium, whose radiation resistance approximately doubled in dried sludge. However, no excessively large D10 values were found for any bacterial species tested.