Protein and solute distribution in drug substance containers during frozen storage and post-thawing: a tool to understand and define freezing-thawing parameters in biotechnology process development

Active pharmaceutical ingredient for biotechnology-based drugs, commonly known as drug substance (DS), is often stored frozen for longer shelf-life. Freezing DS enhances stability by slowing down reaction rates that lead to protein instability, minimizes the risk of microbial growth, and eliminates the risk of transport-related stress. High density polyethylene bottles are commonly used for storing monoclonal antibody DS due to good mechanical stress/strain resistant properties even at low temperatures. Despite the aforementioned advantages for frozen storage of DS, this is not devoid of risks. Proteins are known to undergo ice-water surface denaturation, cryoconcentration, and cold denaturation during freezing. A systematic investigation was performed to better understand the protein and solute distribution along with potential of aggregate formation during freeze and thaw process. A significant solute and protein concentration gradient was observed for both frozen and thawed DS bottles. In case of thawed DS, cryoconcentration was localized in the bottom layer and a linear increase in concentration as a function of liquid depth was observed. On the other hand, for frozen DS, a "bell shaped" cryoconcentration distribution was observed between the bottom layers and centre position. A cryoconcentration of almost three-fold was observed for frozen DS in the most concentrated part when freezing was conducted at -20 and -40 °C and 2.5-fold cryoconcentration was observed in the thawed DS before mixing. The information obtained in this study is critical to design freeze thaw experiments, storage condition determination, and process improvement in manufacturing environment.     

Periplasmically located α-santonin binding factor in <Emphasis Type="Italic">Sphingomonas paucimobilis</Emphasis> strain S ATCC 43388

A marked reduction in uptake of α-santonin, accompanied by loss of ability of cells to transform the substrate, is observed on shocking Sphingomonas paucimobilis strain S ATCC 43388 cells by freeze — thaw method. The shock fluid shows a 26% quench in fluorescence at 350nm on incubation with the substrate. Addition of shock fluid to the freeze thawed cells restores both uptake as well as transformation of α-santonin to near normal.     

In situ detection and localization of lipid peroxidation in individual bovine sperm cells

Reactive oxygen species (ROS) have been implicated in many pathologies, including sub- and infertility. Freeze/thawing of sperm samples is routinely performed in the cattle breeding industries in order to perform artificial insemination. This freeze/thaw procedure is known to induce ROS in sperm samples. Lipid peroxidation in fresh and frozen/thawed sperm cells was assessed by mass spectrometric analysis of the main endogenous phospholipid class, phosphatidylcholine, and by fluorescence techniques using the lipid peroxidation reporter probe C11-BODIPY(581/591). Peroxidation as reported by the fluorescent probe, clearly corresponded with the presence of hydroxy- and hydroperoxyphosphatidylcholine in the sperm membranes, which are early stage products of lipid peroxidation. This allowed us, for the first time, to correlate endogenous lipid peroxidation with localization of this process in living sperm cells. Lipid peroxidation was particularly strong in the midpiece and tail of frozen/thawed spermatozoa and significantly less intense in the head. Induction of peroxidation in fresh sperm cells with the lipid soluble ROS tert-butylhydroperoxide gave an even more pronounced effect, demonstrating antioxidant activity in the head of fresh sperm cells. Furthermore, we were able to show that spontaneous peroxidation was not a result of cell death, as only a pronounced subpopulation of living cells showed peroxidation after freeze/thawing.     

The effect of rice aging on the freeze–thaw stability of rice flour gels

This study investigated the effect of aging rice on the freeze–thaw stability of rice flour gels since repeated freeze–thaw cycles can lead to reduced food quality. A rice grain cultivar called ‘Khoa Dawk Mali 105’ was aged for three different time periods, ranging from 0 to 12 months. Rice gels made from the aged rice were then freeze–thawed for up to 5 cycles. Repeated freeze–thaw cycles lead to an increase in syneresis values and hardness with increasing rice aging. Differential scanning calorimetry showed an increase in the enthalpy of melting of the amylose–lipid complex after 5 freeze–thaw cycles and an increase in peak gelatinization temperature and gelatinization enthalpy with longer rice aging. Moreover, aging length of the rice correlated significantly with a decrease in peak viscosity and breakdown but also an increase in final viscosity and setback. These results demonstrate that aging the rice reduced the freeze–thaw stability of the rice flour gels.     

Cryobiology of neurospora crassa. I. Freeze response of Neurospora crassa conidia

Neurospora crassa conidia were frozen and thawed in water suspensions at various rates and with different minimum temperatures. Colony counts of the experimental conidia were compared with those of controls, which were taken as 100% survival. The data revealed that (1) survivals were near 100% after fast thaw (400 °/min) regardless of the freeze rate, (2) percentage of survival was inversely related to freeze rate when combined with slow thaw, (3) slow thaw (0.5 °/min) was damaging, and (3) the rates of freeze-thaw affected the system only in the −5 to −20 ° interval. The damaging freeze conditions were those which favor ice crystal growth. It is suggested that rupture of the membrane by ice crystals seems to be the plausible mechanism of damage in freezing and thawing N. crassa conidia.     

Trehalose uptake and dehydration effects on the cryoprotection of CHO–K1 cells expressing TRET1

Chinese hamster ovary cells (CHO–K1 cells) in which the trehalose transporter (TRET1) is expressed can have greater cryoprotection than ordinary CHO–K1 cells. This study examines the uptake characteristics of trehalose into cells via TRET1 and determines the influence of intracellular trehalose on the freeze–thaw viabilities. In our experiments, the intracellular trehalose concentration is controlled by the extracellular trehalose concentration and the immersion time in a freezing solution. In this freezing solution, both kinds of CHO–K1 cells are independently dispersed with various amount of trehalose, and then put into the CO₂ incubator for 0–6 h. After a set immersion time, the cell-suspended sample is cooled to 193 K, stored for 1 week, then quickly thawed at 310 K and its viability measured. The uptake amount of intracellular trehalose is measured before freezing. We find an upper limit for the uptake amount of trehalose when the extracellular trehalose concentration is about 400 mM, at which the freeze–thaw viability is the highest. When the extracellular trehalose concentration exceeds 400 mM, shorter immersion times are needed to obtain the maximum freeze–thaw viability. Also, longer immersion weakens the cells. Our analyses indicate that when the extracellular trehalose-concentration is less than 400 mM, the trehalose uptake occurs more slowly with less dehydration, resulting in less stress on the cell. When the extracellular trehalose concentration exceeds the saturation level, the cell is stressed by the excess dehydration due to the remaining osmotic pressure, with apoptosis occurring before freezing.     

Response of the cell membrane-cytoskeleton complex to osmotic and freeze/thaw stresses

In order to develop successful cryopreservation protocols a better understanding of the freeze- and dehydration-induced changes occurring in the cell membrane and its underlying support, the actin cytoskeleton, is required. In this study, we compared the biophysical response of model mammalian cells (human foreskin fibroblasts) to hyperosmotic stress and freeze/thaw. Transmitted light, infrared spectroscopy, fluorescence- and cryo-microscopy were used to investigate the changes in the cell membrane and the actin cytoskeleton. We observed that a purely hyperosmotic challenge at room temperature resulted in bleb formation. A decrease in temperature abrogated the blebbing behavior, but was accompanied by a decrease in viability. These results suggested that cell survival depended on the availability of the membrane material to accommodate the volumetric expansion back to the original cell volume at isotonic conditions. Our data also showed that freeze/thaw stresses altered the cell membrane morphology resulting in a loss of membrane material. There was also a significantly lower incidence of blebbing after freeze/thaw as compared to isothermal osmotic stress experiments at room temperature. Significant depolymerization of the actin cytoskeleton was seen in cells whose membranes had been compromised by freeze/thaw stresses. Actin depolymerization using cytochalasin D affected the stability of the membrane against mechanical stress at isothermal conditions. This study shows that both the membrane and cytoskeleton, as a system, are involved in the osmotic and freeze/thaw-induced responses of the mammalian cells.     

Evaluating of influence of repeated thaw/freeze cycles on IgG and IgM stability

The purpose of this study was evaluating influence of repeated cycles of thawing and freezing serum samples on IgG and IgM stability. Serum positive samples for anti-cytomegalovirus and antimeasles virus IgG and/or IgM were aliquoted. Tubes containing 100 microl aliquot were frozen at -20 degrees C. Samples were repetitively thawed and froze in one to ten cycles. Antibodies presence was examined with commercially available ELISA tests. All samples reminded positive even after ten repeated thaw/freeze cycles.     

Changes in acrosome morphology during cooling and freezing of dog semen

Sixteen semen samples, collected from purebred beagles over a period of 12 months, were diluted, frozen and thawed. In an attempt to monitor any changes occurring to the acrosome during processing, smears made at various stages of the handling procedure were stained with Spermac stain and examined under 1000X magnification. Most acrosomal damage appeared to occur during the freeze/thaw processes. Sperm motility did not correlate well with acrosomal integrity.     

The effect of freeze/thaw cycles on the stability of compounds in DMSO

A diverse set of 320 compounds from the Procter & Gamble Pharmaceuticals organic compound repository was prepared as 20-mM DMSO solutions and stored at 4 degrees C under argon in pressurized canisters to simulate a low-humidity environment. The plates were subjected to 25 freeze/thaw cycles while being exposed to ambient atmospheric conditions after each thaw to simulate the time and manner by which compound plates are exposed to the atmosphere during typical liquid-handling and high-throughput screening processes. High-performance liquid chromatography-mass spectrometry with evaporative light-scattering detection was used to quantitate the amount of compound remaining after every 5th freeze/thaw cycle. Control plates were stored either at room temperature under argon or at 4 degrees C under argon without freeze/thaw cycling and were evaluated at the midpoint and the endpoint of the study. The study was conducted over a short time period (i.e., 7 weeks) to minimize the effect of compound degradation over time due to the exposure of the compounds to DMSO. The results from this study will be used to determine the maximum number of freeze/thaw cycles that can be achieved while maintaining acceptable compound integrity.     

Freezing Injury in Onion Bulb Cells: II. Post-thawing Injury or Recovery

Onion (Allium cepa L.) bulbs were subjected for 12 days to either a moderate freeze (−4 C) or a severe freeze (−11 C). They were then thawed slowly over ice. During 7 to 12 days following the thaw, the injury progressed with time in the severely frozen bulbs, but appeared completely repaired in the moderately frozen bulbs. This was shown by the following post-thawing changes.     

Maintenance of steroid metabolism and hormone responsiveness in cryopreserved dog, monkey and human hepatocytes.

The efficient and effective use of hepatocytes from larger species and rare human material requires a reliable storage method for cells not needed on the day of preparation. Cryopreservation would seem to be the only viable alternative. In this study the suitability of a published cryopreservation technique on dog, monkey and human hepatocytes has been examined and the cells were tested for functionality directly after thawing and subsequent to culture using steroid metabolism and hormone responsiveness of glycogen phosphorylase a. Monkey and human hepatocytes appear to survive the freezing and thawing process better than dog cells-the latter losing the ability to respond to adrenergic stimuli and their ability to maintain steroid metabolism in culture. Although monkey and human cells do preserve their steroid metabolising capacity after freeze/thawing, there is not the significant increase in enzyme activity seen during culturing freshly isolated cells. It would appear, therefore, that some damage has occurred to the cells during the freeze/thaw process. As previously noted, Williams' medium E is superior to Ham's F-10 in maintaining enzyme activities in culture. It is suggested that cryopreservation is the way forward for the development of stockpiles of viable hepatocytes for biomedical and toxicological research and development but that further modifications to the process are still necessary to optimise the maintenance of liver-specific functions in the thawed cells.     

Introduction of Impermeant Molecules into Synaptosomes Using Freeze/Thaw Permeabilization

Brief freezing as a means of transiently permeabilizing synaptosomes was explored. Rat brain synaptosomes frozen and thawed in the presence of 5% dimethyl sulfoxide, a cryoprotectant, were shown to release, in a calcium-dependent manner, previously accumulated [3H]norepinephrine and [14C]acetylcholine in response to elevated [K+]. In addition, synaptosomes subjected to freeze/thaw were shown to retain their ability to exhibit resting protein phosphorylation, as well as stimulated protein phosphorylation occurring in response to calcium influx. Brief freezing of synaptosomes in the presence of [gamma-32P]ATP and either the catalytic subunit of cyclic AMP-dependent protein kinase or calcium/calmodulin-dependent protein kinase II rendered the synaptosomal interior accessible to these agents, as reflected by the phosphorylation of substrate proteins, such as synapsin I, which reside within the nerve terminal. Inclusion of inhibitors of these protein kinases during freeze/thaw blocked synaptosomal protein phosphorylation, indicating that the inhibitors were also introduced. After freezing, the synaptosomes resealed rapidly and spontaneously, as shown by the inability of any of the agents to elicit an effect on phosphorylation when added at the end of the freezing period. The permeabilization procedure should contribute to an understanding of the functional roles of phosphoproteins, and of their associated protein kinases and protein phosphatases, in nerve terminals.     

Comparative analyses of cell disruption methods for mitochondrial isolation in high-throughput proteomics study

One of the most crucial steps in mitochondrial isolation is disruption of intact cells to denude intracellular organelles, but the yield and purity of different disruption protocols have not been well addressed. In the present study, MDCK cells were disrupted by mechanical (sonication and homogenization), physical (repeated freeze/thaw cycles and hypoosmotic burst), and chemical (using Triton X-100, NP-40, or CHAPS) methods. Efficacy of cell disruption was evaluated by trypan blue staining and mitochondria were subsequently isolated by standardized differential centrifugation. The yield of isolation was also determined by measuring protein concentrations, whereas the purity was examined by Janus green B staining, Western blot analyses of markers for mitochondria (COX-4) and other subcellular organelles/locales (i.e., nucleus, cytoplasm, endoplasmic reticulum, and lysosome), transmission electron microscopy, two-dimensional electrophoresis, and Q-TOF MS and/or MS/MS analyses. Our data demonstrated that sonication is the method of choice for disruption of cells prior to mitochondrial isolation for proteome analysis.     

Mechanisms of interaction of amino acids with phospholipid bilayers during freezing

In this study we compare the ability of various amino acids to protect small unilamellar vesicles against damage during freeze/thaw. Liposomes were composed of 75% palmitoyloleoyl phosphatidylcholine and 25% phosphatidylserine. Damage to liposomes frozen in liquid nitrogen and thawed at 20 degrees C was assessed by resonance energy transfer. Cryoprotection by numerous amino acids was compared in the presence and absence of 350 mM NaCl. The majority of amino acids with hydrocarbon side chains increased membrane damage during freeze/thaw regardless of the presence of salt. However, amino acids with hydrocarbon side chains of less than three carbons long, e.g. glycine, alanine, and 2-aminobutyric acid, were cryoprotective only in the presence of salt. We suggest that NaCl selectively increases the solubility of such amino acids, allowing them to act as cryoprotectants. In contrast, amino acids with side chains containing charged amine groups were cryoprotective regardless of the presence of salt. The degree of charge on the second amine group is shown to be important for cryoprotection by these molecules. We present evidence that suggests an interaction between the positively charged, second amine group of the amino acid, and the negatively charged phospholipid headgroup.     

The effect of initial tonicity on freeze/thaw injury to human red cells suspended in solutions of sodium chloride

Human red blood cells, suspended in solutions of sodium chloride, have been frozen to temperatures between -2 and -14 degrees C and thawed, and the extent of hemolysis was measured. In parallel experiments, red cells were exposed to similar cycles of change in the composition of the suspending solution, but by dialysis at 21 degrees C. The tonicity of the saline in which the cells were initially suspended was varied between 0.6x isotonic and 4x isotonic; some samples from each experimental treatment were returned to isotonic saline before hemolysis was measured. It was found that the tonicity of the saline used to suspend the cells for the main body of the experiment affected the amount of hemolysis measured: raising the tonicity from 0.6x to 1x to 2x reduced hemolysis, both in the freezing and in the dialysis experiments, whereas raising the tonicity further to 4x reversed that trend. There was little difference between the freeze/thaw and the dialysis treatments for the cells suspended in 1x or 2x saline, whether or not the cells were returned to isotonic conditions. However, the cells suspended in 0.6x saline showed greater damage from freezing and thawing than from the comparable change in the composition of the solution, whether or not they were returned to isotonic conditions. Cells that were suspended in 4x saline and exposed to changes in salt concentration by dialysis showed less hemolysis when they were assayed in the 4x solution than cells that had received the comparable freezing/thaw treatment, but when the experiment included a return to isotonicity, the two treatments gave similar results. Returning the cells to isotonic saline had a negligible affect on the cells in 0.6x and 1x saline, but caused considerable hemolysis in the 2x and 4x samples, more so after dialysis than after freezing and thawing. We conclude that cells suspended in 0.6x and 4x saline behave differently from cells suspended in 1x and 2x saline and hence that cells suspended in a range of solutions of differing initial tonicity should not be treated as a homogeneous population. We argue that an effect of the unfrozen fraction of water (U) cannot be distinguished, within the framework of these freeze/thaw experiments alone, from an effect of initial tonicity, and that the biphasic nature of the correlation between haemolysis and U makes a causal connection improbable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Freeze and Thaw of CD4+CD25+Foxp3+ Regulatory T Cells Results in Loss of CD62L Expression and a Reduced Capacity to Protect against Graft-versus-Host Disease

The adoptive transfer of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in murine models of allogeneic hematopoietic cell transplantation (HCT) has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD) and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L), which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies.     

冻融交替后不同尺度黑土结构变化特征

冻融交替是改变黑土结构、加剧土壤侵蚀的重要因子。以典型黑土区耕作土壤为研究对象,采用野外季节性冻融循环与室内模拟冻融循环相结合、X射线计算机断层摄影(CT)与扫描电子显微镜(SEM)相结合的方法,通过水分物理性质、团聚体破坏率、孔隙数目、孔隙面积、孔隙成圆率、孔隙Feret直径的测定与分析,研究了冻融交替后0—40 cm、40—80 cm和120—160 cm3个土层以及田间季节性冻融环刀、室内模拟冻融CT扫描和室内模拟冻融SEM3种方式下黑土结构特征的变化规律。结果表明:冻融交替能够对不同土层和不同尺度的耕地黑土结构产生不同程度的影响。季节性冻融后,表层土壤容重升高,非毛管孔隙度和持水能力显著降低(P0.05),40—80 cm土层团聚体破坏率增加40.97%(P0.05),土壤抗蚀性有所削弱,120—160 cm土壤没有受到季节性冻融的显著影响。CT扫描尺度上,3个土层均以1—2 mm径级的孔隙数目为最多,形状也相对规则、接近圆形;冻融循环没有对表层土壤大孔隙结构产生影响,却能够显著降低40—80 cm土层范围内大孔隙面积以及Feret直径(P0.05)。SEM扫描显示冻融后土壤表面粗糙度增加,颗粒松散、脱离,孔壁断裂,证明了冻融交替对土壤微结构的破坏作用;同时结合电子能谱的元素分析可知冻融交替能够改变土壤颗粒表面化学特征。     

Identification and preliminary characterization of a mutant defective in the bacteriophage T4-induced unfolding of the Escherichia coli nucleoid.

The nucleoids of Escherichia coli S/6/5 cells are rapidly unfolded at about 3 min after infection with wild-type T4 bacteriophage or with nuclear disruption deficient, host DNA degradation-deficient multiple mutants of phage T4. Unfolding does not occur after infection with T4 phage ghosts. Experiments using chloramphenicol to inhibit protein synthesis indicate that the T4-induced unfolding of the E. coli chromosomes is dependent on the presence of one or more protein synthesized between 2 and 3 min after infection. A mutant of phage T4 has been isolated which fails to induce this early unfolding of the host nucleoids. This mutant has been termed "unfoldase deficient" (unf-) despite the fact that the function of the gene product defective in this strain is not yet known. Mapping experiments indicate that the unf- mutation is located near gene 63 between genes 31 and 63. The folded genomes of E. coli S/6/5 cells remain essentially intact (2,000-3,000S) at 5 min after infection with unfoldase-, nuclear disruption-, and host DNA degradation-deficient T4 phage. Nuclear disruption occurs normally after infection with unfoldase- and host DNA degradation-deficient but nuclear disruption-proficient (ndd+), T4 phage. The host chromosomes remain partially folded (1,200-1,800S) at 5 min after infection with the unfoldase single mutant unf39 x 5 or an unfoldase- and host DNA degradation-deficient, but nuclear disruption-proficient, T4 strain. The presence of the unfoldase mutation causes a slight delay in host DNA degradation in the presence of nuclear disruption but has no effect on the rate of host DNA degradation in the absence of nuclear disruption. Its presence in nuclear disruption- and host DNA degradation-deficient multiple mutants does not alter the shutoff to host DNA or protein synthesis.