Optimization of the Secondary Drying Step in Freeze Drying Using TDLAS Technology

The secondary drying phase in freeze drying is mostly developed on a trial-and-error basis due to the lack of appropriate noninvasive process analyzers. This study describes for the first time the application of Tunable Diode Laser Absorption Spectroscopy, a spectroscopic and noninvasive sensor for monitoring secondary drying in laboratory-scale freeze drying with the overall purpose of targeting intermediate moisture contents in the product. Bovine serum albumin/sucrose mixtures were used as a model system to imitate high concentrated antibody formulations. First, the rate of water desorption during secondary drying at constant product temperatures (−22°C, −10°C, and 0°C) was investigated for three different shelf temperatures. Residual moisture contents of sampled vials were determined by Karl Fischer titration. An equilibration step was implemented to ensure homogeneous distribution of moisture (within 1%) in all vials. The residual moisture revealed a linear relationship to the water desorption rate for different temperatures, allowing the evaluation of an anchor point from noninvasive flow rate measurements without removal of samples from the freeze dryer. The accuracy of mass flow integration from this anchor point was found to be about 0.5%. In a second step, the concept was successfully tested in a confirmation experiment. Here, good agreement was found for the initial moisture content (anchor point) and the subsequent monitoring and targeting of intermediate moisture contents. The present approach for monitoring secondary drying indicated great potential to find wider application in sterile operations on production scale in pharmaceutical freeze drying.     

Effect of freezing and freeze-drying on the viability and storage of Lilium longiflorum L. and Zea mays L. pollen

The freeze-preservation of pollen is dependent on the interaction of several factors such as freezing rate, thawing rate, freeze-drying temperature and duration, storage temperature and environment and rehydration rates. Changes in any of these variables affects the others directly or indirectly.Rapid freezing of pollen at rates of approximately 200 °C/min maintains the highest degree of viable pollen in combination with rapid thawing rates of 218 °C/min. Rapid cooling and slow rewarming resulted in a substantial loss of pollen viability. This might indicate that intracellular ice crystals formed during rapid cooling perhaps grow into larger ice masses during slow rewarming or storage at temperatures above ?50 °C.The germinability of pollen freeze-dried at temperatures below ?50 °C was also prolonged over that of the controls. Germination values for unfrozen pollen stored for 30 days at 0–5 °C averaged 50% for lily and 20% for corn. Freeze-dried pollen stored for 30 days at the same temperature yielded considerably higher viability percentages for both lily and corn pollen. Drying time is an important factor, perhaps indicating that residual moisture is critical. Freeze-dried pollen can be stored at higher temperatures than frozen and control pollen. Freeze-dried material stored for five months at 0–5 °C, upon slow rehydration yielded intact grains which has average germination percentages of 25 for lily and 15 for corn. The same pollen upon rapid rehydration showed rupturing of 20–40% of the cells and practically no germination.     

Storage of ethyl methanesulphonate-treated barley seeds with low moisture contents

Barley seeds were treated with ethyl methanesulphonate (EMS) for 3 h at 25° C, washed with tap water for 24 h at 25° C, redried at 40° C to different moisture contents below 15% and stored at 25° C in desiccators or in sealed plastic bags. The criteria used for expressing the effect of storage were the M₁ seedling height and the frequency of chromosomal aberrations. With 14·9% seed moisture a strong increase of biological injury occurred in the course of a 2-week storage, while storage of seeds having an initial moisture content of 11·7% led to a significant increase of injury only after 6 weeks. Superdry EMS-treated seeds with 5% or less moisture can be stored at 25° C without any changes in the biological effects. A method is recommended to avoid the EMS-storage effects.     

Reduced leaching of the herbicide MCPA after bioaugmentation with a formulated and stored Sphingobium sp.

The use of pesticides on sandy soils and on many non-agricultural areas entails a potentially high risk of water contamination. This study examined leaching of the herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) after bioaugmentation in sand with differently formulated and stored Sphingobium sp. T51 and at different soil moisture contents. Dry formulations of Sphingobium sp. T51 were achieved by either freeze drying or fluidised bed drying, with high initial cell viability of 67–85 %. Storage stability of T51 cells was related to formulation excipient/carrier and storage conditions. Bacterial viability in the fluidised bed-dried formulations stored at 25 °C under non-vacuum conditions was poor, with losses of at least 97 % within a month. The freeze-dried formulations could be stored substantially longer, with cell survival rates of 50 %, after 6 months of storage at the same temperature under partial vacuum. Formulated and long-term stored Sphingobium cells maintained their MCPA degradation efficacy and reduced MCPA leaching as efficiently as freshly cultivated cells, by at least 73 % when equal amounts of viable cells were used. The importance of soil moisture for practical field bioaugmentation techniques is discussed.     

Freeze-dried plasma proteins are stable at room temperature for at least 1 year

Thirty human EDTA plasma samples from male and female subjects ranging in age from 24 to 74 years were collected on ice, processed ice cold and stored frozen at ?80 °C, in liquid nitrogen (LN2), or freeze dried and stored at room temperature in a desiccator (FDRT) or freeze dried and stored at ?20 °C for 1 year (FD-20). In a separate experiment, EDTA plasma samples were collected onto ice, processed ice cold and maintained on ice ± protease inhibitors versus incubated at room temperature for up to 96 h. Random and independent sampling by liquid chromatography and tandem mass spectrometry (LC–ESI–MS/MS), as correlated by the MASCOT, OMSSA, X!TANDEM and SEQUEST algorithms, showed that tryptic peptides from complement component 4B (C4B) were rapidly released in plasma at room temperature. Random sampling by LC–ESI–MS/MS showed that peptides from C4B were undetectable on ice, but peptides were cleaved from the mature C4B protein including NGFKSHALQLNNR within as little as 1 h at room temperature. The frequency and intensity of precursors within ± 3 m/z of the C4B peptide NGFKSHALQLNNR was confirmed by automated targeted analysis where the precursors from MS/MS spectra that correlated to the target sequence were analyzed in SQL/R. The C4B preproprotein was processed at the N terminus to release the mature chain that was cleaved on the carboxyl side of the isoprene C2 domain within a polar C terminal sequence of the mature C4B protein, to reveal the thioester reaction site, consistent with LC–ESI–MS/MS and Western blot. Random sampling showed that proteolytic peptides from complement component C4B were rarely observed with long term storage at ? 80 °C in a freezer or in liquid nitrogen (LN2), freeze drying with storage at ? 20 °C (FD-20 °C) or freeze drying and storage at room temperature (FDRT). Plasma samples maintained at room temperature (RT) showed at least 10-fold to 100-fold greater frequency of peptide correlation to C4B and measured peptide intensity compared to samples on ice for up to 72 h or stored at ? 80 °C, LN2, FDRT or FD-20 °C for up to a year.     

New Method for Monitoring the Process of Freeze Drying of Biological Materials

A capacitive sensor was proposed and tested for the monitoring and control of a freeze drying process of a vaccine against the Newcastle disease of birds. The residual moisture of the vaccine was measured by the thermogravimetric method. The vaccine activity was determined by titration in chicken embryos. It was shown that, at the stages of freezing and primary drying, a capacitive sensor measured the fraction of unfrozen liquid phase in a material and allowed one to control the sublimation stage of drying in an optimal way. This prevented the foaming of the material and shortened the total drying time approximately twice. The control range at the sublimation stage of drying expanded up to −70°C. It was found at the final stage of drying that the signal of a capacitive sensor passed through a maximum value. We supposed that this maximum corresponds to the minimum of intramolecular mobility of biological macromolecules and hence to the optimal residual moisture of the material, which ensures long-term preservation of its activity. We also suppose that using the capacitive sensor at the final stage of drying allows one to more precisely detect the time when the residual moisture of dried material reaches the optimal value.KEY WORDS: biological materials, capacitive sensor, freeze drying, optimal residual moistureAt present, most biological materials containing live viruses or bacteria are exposed to lyophilization (i.e., drying from the frozen state); this ensures long-term preservation of their activity. Typically, this process consists of preliminary freezing and subsequent freeze drying. The latter process, in turn, consists of two stages: primary drying and secondary drying. During primary drying or sublimation, frozen water is removed from a biological product under vacuum and at temperatures below 0°C. At this stage, the drying rate is limited because of the foaming of a product that occurs due to its high temperature and the excess amount of liquid phase in it. The secondary drying, or final stage, begins after the end of the sublimation stage and occurs at temperatures above 0°C. The goal of the secondary drying is to bring the residual moisture of a biological product to an optimum level, which provides long-term preservation of its activity. Note that the moisture content both above and below the optimum value reduces the effective life of biological materials (1,2)To increase the shelf life of biological products, the following should be investigated: (1) the influence of the composition of the dried biological product and the residual moisture on the change in its activity over the time (3); (2) it is needed to optimize the sublimation drying process for different types of biological products (4). For the investigation of the of the state of water in the dried biologic drugs and the influence of the humidity of the biological on the change in their activity during shelf life, different physical methods are used such as neutron scattering (5), nuclear magnetic resonance (NMR) (6,7), Raman spectroscopy (8), infrared spectroscopy, differential scanning calorimetry, thermal activity monitor (9), and gravimetric sorption analysis (10). The investigations using these methods allow to find an optimum composition of a protective medium for biologics and to determine its optimal residual moisture.At all stages of the freeze drying, the parameters of the material and the parameters of the drying process (temperature of a material, the shelf temperature, the condenser temperature, the pressure in the sublimation chamber, etc.) are also monitored. According to these data, the mode of the process is selected to conduct him for the minimum time and get the best product quality (11). Usually during the drying process, the temperature is measured in several vials with biologic located on different shelves. The sharp increase of the temperature indicates the end of primary drying and the beginning of the secondary drying. The finish of the sublimation stage is revealed by a sharp decrease of the partial pressure of water vapor in the sublimation chamber (12,13). Note that the partial pressure of water vapor in the sublimation chamber does not characterize the state of the biological product to be dried and it is an indirect parameter. For monitoring and controlling the process of freeze drying, it is important to use the own properties of biological materials. In (14), a resistivity sensor placed in a frozen biological material was proposed to control the primary stage of freeze drying. A disadvantage of this method is that one cannot establish an unambiguous relationship between the amount of liquid phase in the frozen material and the value of resistivity: the resistance of the sensor depends not only on the amount of liquid phase but also on the concentration of dissolved salts. Another disadvantage of the resistivity sensor is that, when the temperature decreases, the resistivity of the material sharply increases to values that are difficult to measure, which makes impossible the control of the sublimation stage with this sensor.In (15,16), the interesting methods for determining the moisture of biological materials during secondary drying were proposed. These methods are based on the measurement of the partial pressure of water vapors in the sublimation chamber by NIR spectroscopy or Raman spectroscopy. Note that this method is indirect and requires laborious calibration to establish a correspondence between the current moisture of the biological material in vials and the pressure of water vapor in the sublimation chamber.It should be noted that one has to carry out a series of long-term experiments to find the optimal residual moisture of a biological product. These experiments result in the lifetimes of biological samples with various residual moistures. As the optimal residual moisture of a biological product, one takes the value that provides the longest term preservation of its activity.However, finding the optimal conditions of freeze drying has traditionally been a process of trial and error and required several experimental runs (17). Note also that the freeze drying process is time-consuming and labor intensive.A promising method for the investigation of the properties of biological materials is dielcometry (18,19). This method is relatively simple and very informative since it gives information about the structure of biological macromolecules and the state and role of water in the biological material, etc. This method was used in (20–22) for monitoring biological materials at the primary stage of freeze drying. In (20), authors had found an anomalous low-frequency dispersion of the dielectric permittivity in the biological under study and explain this phenomenon by the proton transfer among water molecules, connected by hydrogen bonds The dielectric relaxation time turned out to be sensitive to the loss of moisture content in the product, and the authors suggested to use of this phenomenon to determine the end point of the freeze drying process. The authors mounted the electrodes of the capacitive sensor on the outer surface of vials with the material to be dried. This approach allows monitoring the sublimation rate and determining the end of the primary stage of freeze drying. Unfortunately, the sensitivity of the capacitive sensor of this design is not enough for the reliable monitoring of the stage of secondary drying.In this paper, a new design of a capacitive sensor and measurement technique are proposed that enable monitoring all stages of the drying process: the freezing stage, the sublimation stage, and the final stage. During freezing and the sublimation stages, the sensor monitors the amount of liquid phase in the frozen material. This allows an optimal control during the whole sublimation stage which prevents the foaming of the material and significantly reduces the total drying time. The sensor also fixes the end of the sublimation stage and the beginning of the final stage of drying. At this stage, the high sensitivity of the measuring system enables one to discover that there is a certain time interval when the signal of the capacitive sensor passes through a maximum. We believe that this maximum corresponds to the minimum of the molecular mobility of biological macromolecules and the optimal residual moisture of the material to be dried.     

Viability of rape (Brassica napus L.) seeds following selection on the basis of newly-emerged radicles then subsequent drying and storage

Germinating rape seeds selected on the basis of newly-emerged radicles (1 ± 0.5 mm) were dried to an equilibrium moisture content (c. 11%) in air at 20°C and 80% relative humidity without loss of viability. Storage life of these low-moisture-content germinating (LMCG) seeds at 15°C was limited to 7 days before viability was significantly reduced. However, viability of LMCG seeds was maintained for 84 days in storage at -20°C. Longer periods in store reduced viability, but 96% of seeds still remained viable after 336 days at - 20°C. Increasing periods of storage at -20°C reduced the subsequent seed longevity at 15°C, indicating a reduction in vigour during storage. Storage under reduced pressure or in a nitrogen atmosphere had little significant effect on seed longevity. Reduction of moisture content below 11% using vacuum drying at a range of temperatures reduced seed vigour.     

Preservation of Bacteria by Circulating-Gas Freeze Drying

Water-washed Serratia marcescens and Escherichia coli were freeze dried in a circulating-gas system at atmospheric pressure. This convective procedure resulted in a substantially higher survival of organisms than could be obtained by the vacuum method of freeze drying. There was little or no decrease in cell viability during convective drying when the residual moisture content was 15% or higher. Below this level, survival declined with decreasing moisture content. A detailed comparison of the convective and vacuum methods indicated that the advantage gained by freeze drying bacteria in air accrues in the early period of sublimation, at which time cells were found to be sensitive to vacuum drying but insensitive to air drying. An explanation for this difference is proposed, based upon the kinetics of water removal in the two processes. In brief, it is suggested that the convective method permits samples to be dried more uniformly; and regional over-drying, which may be deleterious even if transient, is thus avoided in achieving the optimal level of moisture.     

Delayed onset of adult antifreeze activity in juveniles of the Antarctic icefish <Emphasis Type="Italic">Chaenocephalus aceratus</Emphasis>

Many Antarctic notothenioid species endemic to the Seasonal Pack-ice Zone have converged on adult blood serum freezing points that are several tenths of a degree above the freezing point of seawater. While these fishes share high adult serum freezing points, the development of their freeze avoidance during ontogeny has not been studied. We investigated this in wild caught juveniles of one such species, Chaenocephalus aceratus (family Channichthyidae), using blood serum antifreeze activity as a proxy for their freeze avoidance. Juvenile serum antifreeze activity was significantly below that of adults through the oldest year 2+ specimens collected. This increased at an estimated rate of 0.368 × 10⁻³ ± 0.405 × 10⁻⁴°C day⁻¹ which, if sustained, would leave C. aceratus below their adult serum antifreeze activity levels of 0.57 ± 0.08°C until 4.2 years after hatching. Underlying the 2.7-fold increase in their serum antifreeze activity from late year 0+ juveniles to adults was an even greater 10.4-fold increase in the concentration of their serum antifreeze glycopeptides, which increased proportionally across all of their serum AFGP size isoforms. With insufficient antifreeze activity to avoid freezing in the ice-laden surface waters, both adult and juvenile C. aceratus are most likely restricted to the year round ice-free waters where a metastable supercooled state can be maintained.     

Efficient solvothermal wet in situ transesterification of Nannochloropsis gaditana for biodiesel production

In situ transesterification of wet microalgae is a promising, simplified alternative biodiesel production process that replaces multiple operations of cell drying, extraction, and transesterification reaction. This study addresses enhanced biodiesel production from Nannochloropsis gaditana at elevated temperatures. Compared with the previously reported in situ transesterification process of conducting the reaction at a temperature ranging from 95 to 125 °C, the present work employs higher temperatures of at least 150 °C. This relatively harsh condition allows much less acid catalyst with or without co-solvent to be used during this single extraction-conversion process. Without any co-solvent, 0.58% (v/v) of H₂SO₄ in the reaction medium can achieve 90 wt% of the total lipid conversion to biodiesel at 170 °C when the moisture content of wet algal paste is 80 wt%. Here, the effects of temperature, acid catalyst, and co-solvent on the FAEE yield and specification were scrutinized, and the reaction kinetic was investigated to understand the solvothermal in situ transesterification reaction at the high temperature. Having a biphasic system (water/chloroform) during the reaction also helped to meet biodiesel quality standard EN 14214, as Na⁺, K⁺, Ca²⁺, Mg²⁺ cations and phosphorus were detected only below 5 ppm. With highlights on the economic feasibility, wet in situ transesterification at the high temperature can contribute to sustainable production of biodiesel from microalgae by reducing the chemical input and relieve the burden of extensive post purification process, therefore a step towards green process.

     

冻干人用狂犬病纯化疫苗稳定剂的筛选

取同一批人用狂犬病纯化疫苗纯化液,分别加入筛选出的A、B、C和D稳定剂,按已确定的适宜冻干曲线进行冻干后,分别检测其安全、效力、外观、水分等,效力结果显示,使用A、B、D稳定剂的制品在37℃放置3个月,效价降低了48%～60%;使用C稳定剂的制品在37℃放置3个月效价仅下降了16.1%。表明C稳定剂为冻干人用狂犬病纯化疫苗适宜的稳定剂配方。     

Temperature preferences of the mite, Alaskozetes antarcticus, and the collembolan, Cryptopygus antarcticus from the maritime Antarctic

Abstract. The thermal preferences of Alaskozetes antarcticus (Acari, Cryptostigmata) and Cryptopygus antarcticus (Collembola, Isotomidae) were investigated over 6 h within a temperature gradient (?3 to +13 °C), under 100% relative humidity (RH) conditions. After 10 days of acclimation at ?2 or +11 °C, individual supercooling points (SCP) and thermopreferences were assessed, and compared with animals maintained for 10 days under fluctuating field conditions (?6 to +7 °C). Acclimation at ?2 °C lowered the mean SCP of both A. antarcticus (?24.2 ± 9.1) and C. antarcticus (?14.7 ± 7.7) compared to field samples (?19.0 ± 9.0 and ?10.7 ± 5.2, respectively). Acclimation at +11 °C increased A. antarcticus mean SCP values (?13.0 ± 8.5) relative to field samples, whereas those of C. antarcticus again decreased (?16.7 ± 9.1). Mites acclimated under field conditions or at +11 °C selected temperatures between ?3 and +1 °C. After acclimation at ?2 °C, both species preferred +1 to +5 °C. Cryptopygus antarcticus maintained under field conditions preferred +5 to +9 °C, whereas individuals acclimated at +11 °C selected +9 to +13 °C. For A. antarcticus, thermopreference was not influenced by its cold hardened state. The distribution of field specimens was further assessed within two combined temperature and humidity gradient systems: (i) 0–3 °C/12% RH, 3–6 °C/33% RH, 6–9 °C/75% RH and 9–12 °C/100% RH and (ii) 0–3 °C/100% RH, 3–6 °C/75% RH, 6–9 °C/33% RH and 9–12 °C/12% RH. In gradient (i), C. antarcticus distributed homogeneously, but, in gradient (ii), C. antarcticus preferred 0–3 °C/100% RH. Alaskozetes antarcticus selected temperatures between 0 and +6 °C regardless of RH conditions. Cryptopygus antarcticus appears better able than A. antarcticus to opportunistically utilize developmentally favourable thermal microclimates, when moisture availability is not restricted. The distribution of A. antarcticus appears more influenced by temperature, especially during regular freeze‐thaw transitions, when this species may select low temperature microhabitats to maintain a cold‐hardened state.     

Desiccation studies in relation to the storage of Araucaria seed

Relationships between seed moisture content (fresh weight basis) and germination were examined for nine Araucaria species by desiccation under mild environmental conditions. The lowest safe moisture content, below which germination percentage begins to decline, was estimated in each case. Seeds can be grouped into three moisture content categories: the first group (including A. araucana, A. angustifolia, A. hunsteinii and A. bidwillii) cannot be safely dried to below 25–40%; the second group (including A. columnaris, A. rulei, A. nemorosa and A. scopulorum) cannot be dried to below about 12% without damage; the third category contains A. cunninghamii, which can be dried to 2% without damage. Seeds in the first group should be stored at 0–5 °C with moisture contents above the lowest-safe value. Provided freezing damage does not exceed 10%, seeds in the second group should be kept at - 18°C or lower with about 7% moisture content for long-term storage and at 0–5 °C with about 12% moisture content in the short term. Seed of A. cunninghamii is best retained at near 5% moisture content and in -18°C or lower. The lowest-safe moisture content was found to be associated with seed size and weight, higher moisture content values coinciding with greater size and weight of seed. Food reserve materials also differed among the groups; seeds of the first group were mainly starchy, whilst those in the other categories possessed a high lipid content.     

Produktion von Ochratoxin A und B durch<Emphasis Type="Italic">Penicillium verrucosum</Emphasis> auf Gerste in Abhängigkeit der Parameter Wasseraktivität,Wassergehalt und Temperatur

The ochratoxin A and B (OTA, OTB) production by a toxigenic isolate ofPenicillium verrucosum grown on brewing barley up to six weeks was studied at a storage temperature of 25 °C and different moisture and water activity conditions. Sorption isothermes for barley were prepared at temperatures of 10°C, 15°C and 25°C. OTA was produced after 2 weeks of storage at moisture contents of ≥19%, which is equivalent to water activities (a_w) of 0.83 (adsorptive) and 0.82 (desorptive) at 25 °C. Increased OTA concentrations (5.8-fold and 16.1-fold) were noticed when the moisture contents were adjusted to 20% (a_{w [ads]} 25 °C=0.86) and 21% (a_{w [ads]} [ 25 °C=0.88), respectively. An increase was also shown during storage of 4 and 6 weeks (1.2-fold and 2.4-fold, respectively). Production of OTB was shown to occur at moisture contents ≥18% (a_{w [ads]} 25 °C=0.81). The findings document that OTA and OTB are not produced byP. verrucosum grown on barley stored below 18% moisture content.     

Impact of freezing on pH of buffered solutions and consequences for monoclonal antibody aggregation

Freezing of biologic drug substance at large scale is an important unit operation that enables manufacturing flexibility and increased use‐period for the material. Stability of the biologic in frozen solutions is associated with a number of issues including potentially destabilizing pH changes. The pH changes arise from temperature‐associated change in the pK_as, solubility limitations, eutectic crystallization, and cryoconcentration. The pH changes for most of the common protein formulation buffers in the frozen state have not been systematically measured. Sodium phosphate buffer, a well‐studied system, shows the greatest change in pH when going from +25 to ?30°C. Among the other buffers, histidine hydrochloride, sodium acetate, histidine acetate, citrate, and succinate, less than 1 pH unit change (increase) was observed over the temperature range from +25 to ?30°C, whereas Tris‐hydrochloride had an ～1.2 pH unit increase. In general, a steady increase in pH was observed for all these buffers once cooled below 0°C. A formulated IgG2 monoclonal antibody in histidine buffer with added trehalose showed the same pH behavior as the buffer itself. This antibody in various formulations was subject to freeze/thaw cycling representing a wide process (phase transition) time range, reflective of practical situations. Measurement of soluble aggregates after repeated freeze–thaw cycles shows that the change in pH was not a factor for aggregate formation in this case, which instead is governed by the presence or absence of noncrystallizing cryoprotective excipients. In the absence of a cryoprotectant, longer phase transition times lead to higher aggregation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Collapse temperature of solutions important for lyopreservation of living cells at ambient temperature

In this study, the collapse temperature was determined using the freeze‐drying microscopy (FDM) method for a variety of cell culture medium‐based solutions (with 0.05–0.8 M trehalose) that are important for long‐term stabilization of living cells in the dry state at ambient temperature (lyopreservation) by freeze‐drying. Being consistent with what has been reported in the literature, the collapse temperature of binary water‐trehalose solutions was found to be similar to the glass transition temperature (T′_g ～ ?30°C) of the maximally freeze‐concentrated trehalose solution (～80 wt% trehalose) during the freezing step of freeze‐drying, regardless of the initial concentration of trehalose. However, the effect of the initial trehalose concentration on the collapse temperature of the cell culture medium‐based trehalose solutions was identified to be much more significant, particularly when the trehalose concentration is less than 0.2 M (the collapse temperature can be as low as ?65°C). We also determined that cell density from 1 to 10 million cells/mL and ice seeding at high subzero temperatures (?4 and ?7°C) have negligible impact on the solution collapse temperature. However, ice seeding does significantly affect the ice crystal morphology formed during the freezing step and therefore the drying rate. Finally, bulking agents (mannitol) could significantly affect the collapse temperature only when trehalose concentration is low (<0.2 M). However, improving the collapse temperature by using a high concentration of trehalose might be preferred to the addition of bulking agents in the solutions for freeze‐drying of living cells. We further confirmed the applicability of the collapse temperature measured with small‐scale (2 µL) samples using the FDM system to freeze‐drying of large‐scale (1 mL) samples using scanning electron microscopy (SEM) data. Taken together, the results reported in this study should provide useful guidance to the development of optimal freeze‐drying protocols for lyopreservation of living cells at ambient temperature for easy maintenance and convenient wide distribution to end users, which is important to the eventual success of modern cell‐based medicine. Biotechnol. Bioeng. 2010;106: 247–259. © 2010 Wiley Periodicals, Inc.     

Cryopreservation and microencapsulation of a probiotic in alginate-chitosan capsules improves survival in simulated gastrointestinal conditions

The aim of the present study was to focus on the impact of two different methods and the effects of cryoprotectants on the survival of a probiotic bacterium, Streptococcus phocae PI80, during storage. For the protection of freeze dried cells, the optimal storage conditions were determined with a high survival rate. After the freeze drying process, all cryoprotectants exhibited a protective effect on cell viability at all storage temperatures. High relative cell viability was observed when cells were incubated at ?20°C, which was optimum for the protection of S. phocae PI80. Trehalose was the most promising cryoprotectant at all temperatures during the storage period of bacterial cells. The combination of trehalose + skim milk showed more than 85% survivability compared to other combinations at ?20°C for 60 days. In addition, encapsulation of probiotic cells into alginate-chitosan gel capsules showed better survival of S. phocae cells (5.468 ± 0.15 LogCFU/mL) with high bacteriocin activity at ?20°C for six months. The cell-loaded microcapsules remained stable when treated with simulated gastric and intestinal fluids. After 6 h in vivo treatment, the capsules were found to be broken, releasing the probiotic cells directly into the intestinal system of rats. Therefore, microencapsulation was found to be the most efficient technique, which not only protected the cells for a longer time but also released the cells into the in vivo intestinal system.     

State transitions and physicochemical aspects of cryoprotection and stabilization in freeze-drying of Lactobacillus rhamnosus GG (LGG)

Aims: The frozen and dehydrated state transitions of lactose and trehalose were determined and studied as factors affecting the stability of probiotic bacteria to understand physicochemical aspects of protection against freezing and dehydration of probiotic cultures. Methods and Results: Lactobacillus rhamnosus GG was frozen (–22 or –43°C), freeze‐dried and stored under controlled water vapour pressure (0%, 11%, 23% and 33% relative vapour pressure) conditions. Lactose, trehalose and their mixture (1 : 1) were used as protective media. These systems were confirmed to exhibit relatively similar state transition and water plasticization behaviour in freeze‐concentrated and dehydrated states as determined by differential scanning calorimetry. Ice formation and dehydrated materials were studied using cold‐stage microscopy and scanning electron microscopy. Trehalose and lactose–trehalose gave the most effective protection of cell viability as observed from colony forming units after freezing, dehydration and storage. Enhanced cell viability was observed when the freezing temperature was ?43°C. Conclusions: State transitions of protective media affect ice formation and cell viability in freeze‐drying and storage. Formation of a maximally freeze‐concentrated matrix with entrapped microbial cells is essential in freezing prior to freeze‐drying. Freeze‐drying must retain a solid amorphous state of protectant matrices. Freeze‐dried matrices contain cells entrapped in the protective matrices in the freezing process. The retention of viability during storage seems to be controlled by water plasticization of the protectant matrix and possibly interactions of water with the dehydrated cells. Highest cell viability was obtained in glassy protective media. Significance and Impact of the Study: This study shows that physicochemical properties of protective media affect the stability of dehydrated cultures. Trehalose and lactose may be used in combination, which is particularly important for the stabilization of probiotic bacteria in dairy systems.     

Factors affecting the stability of cryogenically preserved granulocytes

Human granulocytes free of other cell types were obtained by counterflow centrifugation, cryogenically preserved, and studied for stability and function after thawing.Isolation of granulocytes by counterflow centrifugation was optimal at reduced temperatures (4–10 °C) in phosphate-buffered saline (or Ca²⁺-free buffers) at pH 7.1. A stabilizing protein, or HES was required. Routinely, 1.2% human or bovine serum albumin was used. Hyperosmolar (310 m0sm) buffers and post isolation handling in ice water baths was optimal for cryogenic preservation. Addition of DMSO at 22 °C produced transient shrinkage initially which depended on the rate of addition, concentration, and temperature. Within 10–15 min granulocytes returned to volume, but continued to swell, equilibrating for 1 hr at 20% larger volume. Ethidium uptake gradually increased. After 24 hr, extreme swelling, lysis, and ethidium uptake was observed at the highest concentration (10%) of DMSO. DMSO-induced swelling was prevented with HES.Granulocytes (30 × 10⁶ ? 50 × 10⁶) were frozen in 2.0-ml volumes in plastic tubes. The combination of 5% DMSO, 6% HES, 4% albumin, 0.056 M glucose in NormosolR at pH 7.1 produced the best yields. Granulocytes were first cooled to 4 °C, then to ?80 °C (approx rate 4 °C per min) in a mechanical freezer and finally stored in liquid nitrogen. Storage varied from days to months. Granulocytes were thawed at 42 °C by manually twirling the freezing tubes and they were subsequently maintained in ice water. They were diluted 3:1 dropwise with a room temperature solution of 7% HES, 1.2% albumin, and 0.026 M glucose in Normosol. Particle ingestion tests were conducted by incubation at room temperature for forty minutes with yeast or zymosan opsonized with autologous serum. Particles ingested were counted by microfluorimetry after two washings at 150g.Granulocytes could not be cryogenically preserved in plasma or serum. Heating or prefreezing of serum was ineffective, but dialysis or addition of EDTA overcame the destructive effect of serum. Neither treatment was an improvement over the standard freeze procedure using buffered albumin and cryoprotective components. β-mercaptoethanol added to the freezing medium caused the production of a single homogeneous population of osmotically inert, nonviable, ethidium-reactive granulocytes. This suggests that osmoregulation by granulocyte membranes is a critical requirement for cryopreservation.Preservation efficiency is species dependent, increasing in the order of human, baboon, guinea pig, and dog. Dog granulocytes can be stored for at least 8 months in liquid nitrogen with small loss of cells and functionality.The present efficiency of preservation of human granulocytes for 3–4 weeks of liquid nitrogen storage is 90–100% morphological and 40% functional recovery. Attempts to increase stability of thawed granulocytes with other additions to our current procedure have so far proved fruitless. These have consisted of inosine, adenine, pyruvate, gluconate, vitamin C, β-mercaptoethanol, para-phenylmethyl-sulfonylfluoride, and mannitol.     

Proceedings: A method for preservation of bacteria and bacteriophages by drying in vacuo

An efficient and practical method was established to preserve bacterial strains and bacteriophages. The method is characterized by drying without freezing and by use of a cotton wool plug (nonabsorbent) to prevent contamination. Drying conditions were examined by measuring temperature, vacuum, and residual moisture of the samples. From the measurement, it was found that the cotton wool plug acts as a buffer and a desiccant. Thus, the specimens reached optimal conditions during storage. Another point of advantage is that the temperature of the specimen during the drying procedure was 2–5 °C; therefore, the evaporation of the water is rapid and the time of completion is shorter than that during lyophilization.