Design and analysis of accelerated degradation tests for the stability of biological standards I. Properties of maximum likelihood estimators

The accelerated degradation test is commonly used to predict the stability of a biological standard during long-term storage at low temperatures. The analysis of complicated by the fact that the standard generally defines its own unit of activity, so only relative rates of degradation at different temperatures can be observed. A series of Monte Carlo simulation studies is described which has been carried out to investigate the accuracy and precision of estimates based on the statistical method and are of use in assessing the extent to which the size and design of the accelerated degradation test influence the precision of the estimate of the low temperature degradation rate.     

Predicting the stability of biological standards and products

A high level of stability is essential for any biological standard and is desirable in most other biological products. It is in general impossible to observe directly the rate of degradation of a biological standard since no independent scale of measurement is available. An indirect method is therefore required. The most common approach is the accelerated degradation test in which samples are stored for a time at elevated temperatures and then compared with samples stored continuously at low temperature. The relative degradation rates are used to fit the Arrhenius equation (relating degradation rate to temperature) and hence to predict stability under normal storage conditions. Previous statistical work on this problem is reviewed and a maximum likelihood ML approach is suggested which overcomes some of the limitations of the existing methodology. The accelerated degradation test also finds wide application in the shelf-life prediction of biological products where the same statistical methods are appropriate.     

The thermal stability of yellow fever vaccines

The assessment of yellow fever vaccine thermostability both in lyophilized form and after reconstitution were analyzed. Two commercial yellow fever vaccines were assayed for their thermal stability. Vaccines were exposed to test temperatures in the range of 8 degrees C to 45 degrees C. Residual infectivity was measured by a plaque assay using Vero cells. The titre values were used in an accelerated degradation test that follows the Arrhenius equation and the minimum immunizing dose was assumed to be 10(3) particles forming unit (pfu)/dose. Some of the most relevant results include that (i) regular culture medium show the same degradation pattern of a reconstituted 17D-204 vaccine; (ii) reconstituted YF-17D-204 showed a predictable half life of more than six days if kept at 0 degrees C; (iii) there are differences in thermostability between different products that are probably due to both presence of stabilizers in the preparation and the modernization in the vaccine production; (iv) it is important to establish a proper correlation between the mouse infectivity test and the plaque assay since the last appears to be more simple, economical, and practical for small laboratories to assess the potency of the vaccine, and (v) the accelerated degradation test appears to be the best procedure to quantify the thermostability of biological products.     

Design and analysis of accelerated degradation tests for the stability of biological standards II. A flexible computer program for data analysis

The accelerated degradation test is commonly used to predict the stability of a biological standard during long-term storage at low temperature. A flexible computer program is described which has been written to analyse degradation test results by the method of maximum likelihood. In addition to predicting the degradation rate at low temperature, the program furnishes estimates of statistical precision and it carried out a test of goodness of fit of the data to the assumed Arrhenius equation model.     

骨生物材料通过细胞途径降解的研究进展

生物材料作为移植物已广泛应用于骨组织修复,在应用生物材料时需要考虑材料各个方面的性能,如生物兼容性、力学强度、可塑性等。材料的可降解性也是骨修复材料不得不考虑的方面。既往研究表明,生物材料可以通过物理、化学和生物三种方式进行降解。在材料的生物降解过程中,经细胞途径降解是其中重要的一环。这种降解途径主要是通过巨噬细胞、破骨细胞的生物学行为及其所分泌的生物活性氧、酶、酸性代谢物等作用机制进行。认识细胞作用对生物材料的降解有助于更好地理解细胞的生物学行为,精准设计、制造更合理的骨修复材料,既利于材料植入时的初始稳定,也可以符合材料降解与新骨形成的匹配,促进骨再生和骨修复。     

Highly stable mutants of human fibroblast growth factor-1 exhibit prolonged biological action

Fibroblast growth factor 1 (FGF-1) shows strong angiogenic, osteogenic and tissue-injury repair properties that might be relevant to medical applications. Since FGF-1 is partially unfolded at physiological temperature we decided to increase significantly its conformational stability and test how such an improvement will affect its biological function. Using an homology approach and rational strategy we designed two new single FGF-1 mutations: Q40P and S47I that appeared to be the most strongly stabilizing substitutions among those reported so far, increasing the denaturation temperature by 7.8 deg. C and 9.0 deg. C, respectively. As our goal was to produce highly stable variants of the growth factor, we combined these two mutations with five previously described stabilizing substitutions. The multiple mutants showed denaturation temperatures up to 27 deg. C higher than the wild-type and exhibited full additivity of the mutational effects. All those mutants were biologically competent in several cell culture assays, maintaining typical FGF-1 activities, such as binding to specific cell surface receptors and activation of downstream signaling pathways. Thus, we demonstrate that the low denaturation temperature of wild-type FGF-1 is not related to its fundamental cellular functions, and that FGF-1 action is not affected by its stability. A more detailed analysis of the biological behavior of stable FGF-1 mutants revealed that, compared with the wild-type, their mitogenic properties, as probed by the DNA synthesis assay, were significantly increased in the absence of heparin, and that their half-lives were extensively prolonged. We found that the biological action of the mutants was dictated by their susceptibility to proteases, which strongly correlated with the stability. Mutants which were much more resistant to proteolytic degradation always displayed a significant improvement in the half-life and mitogenesis. Our results show that engineered stable growth factor variants exhibit enhanced and prolonged activity, which can be advantageous in terms of the potential therapeutic applications of FGF-1.     

Precision determination for the specific oxygen uptake rate (SOUR) method used for biological stability evaluation of compost and biostabilized products

This work represents the first attempt to evaluate the precision of the specific oxygen uptake rate method expressed in terms of repeatability (r) and reproducibility limits (R). Three laboratories were involved in an inter-laboratory test for the validation of respiration analyses on six biomass samples (three composts and three biostabilized products) having different degrees of biological stability. Both the maximum specific oxygen uptake rate peak (SOUR) and the cumulative oxygen demand after 12 h (OD(12)) and 20 h (OD(20)) of respiration test were investigated. Precisions expressed as the relative standard deviation were in the range of 9-41%. Linear regressions found for r and R, versus OD(12) and OD(20), enabled derivation of precision values (r and R) for all respirometric levels within the operating range. The OD(12) and OD(20) indices were found to be more adequate to indicate biological stability since they were less influenced by random errors than the SOUR index.     

The effect of temperature on the ensiling process of corn and wheat

AIMS: The purpose of this work was to study the effect of temperature on the ensiling process and aerobic stability of corn and wheat silages. METHODS AND RESULTS: The crops were ensiled in 1.5 l anaerobic jars, with and without an inoculant, at room or elevated temperatures (37-41 degrees C). After two months of ensiling, the silages were subjected to an aerobic stability test at room and elevated (33 degrees C) temperature. The results indicate that ensiling at elevated temperatures resulted in higher pH values, less lactic acid and higher losses. The silages which were stored at elevated temperatures were more susceptible to aerobic spoilage than those stored at room temperature, especially when the test was performed at elevated temperature. CONCLUSION: High temperatures are detrimental to both the ensiling process and the aerobic stability of silages. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the current study suggest that in a warm climate, special care should be taken during silage making and storage in order to avoid heating as much as possible. In addition, in a warm climate, silages are more susceptible to aerobic deterioration and therefore, special care should be taken during unloading.     

The Stability of Cellulose: A Statistical Perspective from a Coarse-Grained Model of Hydrogen-Bond Networks

A critical roadblock to the production of biofuels from lignocellulosic biomass is the efficient degradation of crystalline microfibrils of cellulose to glucose. A microscopic understanding of how different physical conditions affect the overall stability of the crystalline structure of microfibrils could facilitate the design of more effective protocols for their degradation. One of the essential physical interactions that stabilizes microfibrils is a network of hydrogen (H) bonds: both intrachain H-bonds between neighboring monomers of a single cellulose polymer chain and interchain H-bonds between adjacent chains. We construct a statistical mechanical model of cellulose assembly at the resolution of explicit hydrogen-bond networks. Using the transfer matrix method, the partition function and the subsequent statistical properties are evaluated. With the help of this lattice-based model, we capture the plasticity of the H-bond network in cellulose due to frustration and redundancy in the placement of H-bonds. This plasticity is responsible for the stability of cellulose over a wide range of temperatures. Stable intrachain and interchain H-bonds are identified as a function of temperature that could possibly be manipulated toward rational destruction of crystalline cellulose.     

Engineering proteins with tunable thermodynamic and kinetic stabilities

It is widely recognized that enhancement of protein stability is an important biotechnological goal. However, some applications at least, could actually benefit from stability being strongly dependent on a suitable environment variable, in such a way that enhanced stability or decreased stability could be realized as required. In therapeutic applications, for instance, a long shelf-life under storage conditions may be convenient, but a sufficiently fast degradation of the protein after it has performed the planned molecular task in vivo may avoid side effects and toxicity. Undesirable effects associated to high stability are also likely to occur in food-industry applications. Clearly, one fundamental factor involved here is the kinetic stability of the protein, which relates to the time-scale of the irreversible denaturation processes and which is determined to some significant extent by the free-energy barrier for unfolding (the barrier that "separates" the native state from the highly-susceptible-to-irreversible-alterations nonnative states). With an appropriate experimental model, we show that strong environment-dependencies of the thermodynamic and kinetic stabilities can be achieved using robust protein engineering. We use sequence-alignment analysis and simple computational electrostatics to design stabilizing and destabilizing mutations, the latter introducing interactions between like charges which are screened out at high salt. Our design procedures lead naturally to mutating regions which are mostly unstructured in the transition state for unfolding. As a result, the large salt effect on the thermodynamic stability of our consensus plus charge-reversal variant translates into dramatic changes in the time-scale associated to the unfolding barrier: from the order of years at high salt to the order of days at low salt. Certainly, large changes in salt concentration are not expected to occur in biological systems in vivo. Hence, proteins with strong salt-dependencies of the thermodynamic and kinetic stabilities are more likely to be of use in those cases in which high-stability is required only under storage conditions. A plausible scenario is that inclusion of high salt in liquid formulations will contribute to a long protein shelf-life, while the lower salt concentration under the conditions of the application will help prevent the side effects associated with high-stability which may potentially arise in some therapeutic and food-industry applications. From a more general viewpoint, this work shows that consensus engineering and electrostatic engineering can be readily combined and clarifies relevant aspects of the relation between thermodynamic stability and kinetic stability in proteins.     

The effect of cyclization on the enzymatic degradation of herpes simplex virus glycoprotein D derived epitope peptide.

One linear and three cyclic peptides corresponding to the 278-287 ((278)LLEDPVGTVA(287)) sequence of glycoprotein D (gD-1) of herpes simplex virus were synthesized for the analysis of the effect of cyclization on protection against enzymatic degradation. In this design, the turn-forming motif ((281)DPVG(284)) was positioned in the central part of the peptide and elongated by three amino acids at both termini. Cyclopeptide formation was achieved by the introduction of a peptide bond, a disulfide bridge or a thioether link. The stability of these peptides was compared in human serum and also in rat lysosomal preparations. The data obtained in 10% and 50% human serum show that all three types of cyclization enhanced the stability, but at different levels. Complete stability was only achieved by the introduction of a thioether link, while the presence of a disulfide or peptide bond resulted in improved, but partial resistance against hydrolytic decomposition. In lysosomal preparations the presence of cyclic primary structure provided full protection against enzymatic hydrolysis. Taken together, these findings indicate that by appropriate structural modification it is feasible to construct a synthetic antigen with high stability against enzymatic degradation in complex biological fluids. Further studies are in progress to identify enzymes responsible for degradation in diluted human sera as well as in the lysosomal preparations and to gain more detailed information on the mechanism of action.     

Analytical and biological characterization of supercoiled plasmids purified by various chromatographic techniques

Supercoiled plasmids are an important component of gene-based delivery vehicles. A number of production methods for clinical applications have been developed, each resulting in very high-quality product with low levels of residual contaminants. There is, however, no consensus on the optimal methods to characterize plasmid quality, and further, to determine if these methods are predictive of either product stability or biological activity. We have produced two plasmids using four production purification methodologies based on PolyFlo and hydrophobic interaction chromatography (HIC), either alone or in tandem processes. In each case, the product was analyzed using standard molecular biological methods. We also performed a number of biophysical analyses such as dynamic light scattering (DLS), circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). Minimal differences were detected among the preparations based on the more standard molecular biological methods. Some small differences were detected, however, using biophysical techniques, particularly FTIR and DSC, which may reflect small variations in plasmid tertiary structure and thermal stability. Stability after heat exposure at 60 degrees C, exposure to fetal bovine serum and long-term storage at 4 degrees C varied between plasmids. One plasmid showed no difference in stability depending on the production process, but the other showed significant differences. Evaluation in vivo in models for gene immunization and gene therapy showed significant differences in the response depending on the method of purification. Preparations using a tandem process of PolyFlo used in two separation modes provided higher biological activity compared to a tandem HIC/PolyFlo process or either resin used alone in a single column process. These data indicate that the process by which supercoiled plasmids are made can influence plasmid stability and biological activity and emphasize the need for more rigorous methods to evaluate supercoiled plasmids as gene-delivery vehicles.     

蛋白酶抗性人睫状神经营养因子突变体基因的构建及其在巴斯德毕赤酵母中的表达

AX15是一种比天然睫状神经营养因子具有更高的生物学活性、更好的稳定性和可溶性的hCNTF突变体。在巴斯德毕赤酵母中表达时AX15易发生降解。氨基酸序列分析表明降解位于由12和13位氨基酸残基组成的双碱性氨基酸之后。根据KEX2蛋白酶的底物专一性把双碱性氨基酸从RR变为RK,构建了KEX2抗性的AX15突变体AX15（R13K）。AX15（R13K）的稳定性得到了显著的提高,在诱导100 h后也未发生降解。利用超滤浓缩和凝胶过滤得到了纯度>90%的AX15（R13K）。TF-1细胞存活实验表明AX15（R13K）具有与AX15相同的生物学活性。蛋白酶抗性人睫状神经营养因子突变体可能具有更好的体内稳定性,在临床应用上具有潜在的优势。     

Basic principles of stability

An understanding of the principles of degradation, as well as the statistical tools for measuring product stability, is essential to management of product quality. Key to this is management of vaccine potency. Vaccine shelf life is best managed through determination of a minimum potency release requirement, which helps assure adequate potency throughout expiry. Use of statistical tools such a least squares regression analysis should be employed to model potency decay. The use of such tools provides incentive to properly design vaccine stability studies, while holding stability measurements to specification presents a disincentive for collecting valuable data. The laws of kinetics such as Arrhenius behavior help practitioners design effective accelerated stability programs, which can be utilized to manage stability after a process change. Design of stability studies should be carefully considered, with an eye to minimizing the variability of the stability parameter. In the case of measuring the degradation rate, testing at the beginning and the end of the study improves the precision of this estimate. Additional design considerations such as bracketing and matrixing improve the efficiency of stability evaluation of vaccines.     

Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay

Humanized monoclonal antibodies (mAbs) are the fastest growing class of biological therapeutics that are being developed for various medical indications, and more than 30 mAbs are already approved and in the market place. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important biological function attributed to the mechanism of action of several therapeutic antibodies, particularly oncology targeting mAbs. The ADCC assay is a complicated and highly variable assay. Thus, the use of an ADCC assay as a lot release test or a stability test for clinical trial batches of mAbs has been a substantial challenge to install in quality control laboratories. We describe here the development and validation of an alternate approach, an ADCC-reporter gene assay that is based on the key attributes of the PBMC-based ADCC assay. We tested the biological relevance of this assay using an anti-CD20 based model and demonstrated that this ADCC-reporter assay correlated well with standard ADCC assays when induced with the drugable human isotypes [IgG1, IgG2, IgG4, IgG4S > P (S228P) and IgG4PAA (S228P, F234A, L235A)] and with IgG1 isotype variants with varying amounts of fucosylation. This data demonstrates that the ADCC-reporter gene assay has performance characteristics (accuracy, precision and robustness) to be used not only as a potency assay for lot release and stability testing for antibody therapeutics, but also as a key assay for the characterization and process development of therapeutic molecules.     

Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay

Humanized monoclonal antibodies (mAbs) are the fastest growing class of biological therapeutics that are being developed for various medical indications, and more than 30 mAbs are already approved and in the market place. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important biological function attributed to the mechanism of action of several therapeutic antibodies, particularly oncology targeting mAbs. The ADCC assay is a complicated and highly variable assay. Thus, the use of an ADCC assay as a lot release test or a stability test for clinical trial batches of mAbs has been a substantial challenge to install in quality control laboratories. We describe here the development and validation of an alternate approach, an ADCC-reporter gene assay that is based on the key attributes of the PBMC-based ADCC assay. We tested the biological relevance of this assay using an anti-CD20 based model and demonstrated that this ADCC-reporter assay correlated well with standard ADCC assays when induced with the drugable human isotypes [IgG1, IgG2, IgG4, IgG4S > P (S228P) and IgG4PAA (S228P, F234A, L235A)] and with IgG1 isotype variants with varying amounts of fucosylation. This data demonstrates that the ADCC-reporter gene assay has performance characteristics (accuracy, precision and robustness) to be used not only as a potency assay for lot release and stability testing for antibody therapeutics, but also as a key assay for the characterization and process development of therapeutic molecules.     

Enhancement of the biological degradation of soils contaminated with oil by the addition of compost

The fundamentals of the biological treatment of contaminated soils were investigated in bioreactors with the aim to optimize the processes of biological soil treatment in order to achieve the highest possible degree of degradation within the shortest period of time. Preinvestigations using test systems at different scales have provided information about the possibilities of enhancing the decomposition processes which are dependent on various factors, such as milieu conditions, additives, etc., that must be known before remedial actions are taken. The investigations made so far have shown that compost is a favourable additive when oil-contaminated soils are biologically treated. The degradation of contaminants can be enhanced by the addition of compost. This positive effect is attributed to various mechanisms. In this paper, results of a variety of test systems at different scales are presented. In test series, different amounts of biocompost were added to investigate the influence on the degradation of diesel fuel. It was demonstrated that by increasing the compost content – the cumulative O₂ consumption caused by the degradation of the diesel fuel contaminants increased. It could be shown that the reduction of the diesel fuel contaminants in the soil was independent of the compost age and amounted to approximately 94% of the initial quantity. The addition of biocompost could also enhance the degradation of real contaminants. After a test period of 162 days in set-ups with compost addition, more than 75% of the lubricating oil contaminants disappeared, while less than 37% of the contaminants disappeared in set-ups without compost addition. Moreover, by the addition of compost, the formation of pellets during the dynamic treatment of soil materials could be reduced.     

Degradation of moist soil aggregates by rapid temperature rise under low intensity fire

Background and aims

Soil structure degradation by fire is usually attributed to qualitative and quantitative change of organic and inorganic binding agents, especially in high severity burns (>300 °C) that last for prolonged periods (> 1 hour). In contrast, controlled burns are typically managed to be low in intensity and severity. Such burns are considered benign to soil structural stability because organic matter and inorganic binding agents (e.g., gypsum) are relatively stable at such low temperatures. Recent observations at a controlled burn site in the eastern Great Basin (Nevada) showed soil aggregate breakdown found in shrub canopies where soil temperatures briefly exceeded 300 °C as well as interspaces between shrubs, where the temperatures were likely lower than beneath shrubs because of less surface biomass. These alterations cannot be explained in terms of thermal alteration of binding agents. This study was designed to test whether pressure created by rapidly vaporized pore water can cause aggregate breakdown.

Methods

We subjected three different sizes of aggregates (0.25–1, 1–2 and 2–4 mm) of soils derived from the eastern Great Basin burn site as well as from a forest and urban garden in California to rapid and slow (3 °C/min) heating rates. These treatments were conducted at 5 peak temperatures (75, 100, 125, 150 and 175 °C).

Results

Post-burn water stability of the aggregates showed that rapid heating rate caused more pronounced degradation of aggregate stability than slow heating. Moreover, the heating-rate dependent structural degradation increased with peak temperature. For the majority of the aggregates, the effect also increased with initial water content. In all the soils tested, there was no preferential loss of organic matter in the rapid-heating treatment that can explain the observed enhanced breakdown of aggregates.

Conclusions

Our observations indicate that soil structural degradation under low-intensity fire occurs as a result of mechanical stresses extorted by rapidly escaping steam from soil pores under rapid heating rate.