Development of a pre-screening method for bDtBPP, a cytotoxic leachable from single-use bioprocessing containers

Extractable and leachable compounds are a major obstacle in the implementation of single-use technologies. Recent studies show that a widely used trisarylphosphite-based processing stabilizer in plastic films, Irgafos 168®, can breakdown from gamma irradiation. One of the breakdown compounds, bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP) has been found to leach out of single-use containers and suppress CHO cell growth. Due to its cytotoxic effect on bioprocessing cell lines, a targeted test for bDtBPP would be highly beneficial for pre-screening single-use bioprocessing containers and bags. In this study, an ultra-performance liquid chromatography method was developed for the separation and detection of bDtBPP and other extractable compounds related to Irgafos 168® (such as 2,4-di-tert-butylphenol, 1,3-di-tert-butylbenzene, 2,4-di-tert-butylphenylphosphate and oxidised Irgafos 168®). The method was applied to an extractables study of three single-use plastic films, where bDtBPP and the oxidised form of Irgafos 168® were detected and quantified in all three films. Extraction methods using either a whole bag or a small piece of film were evaluated, and the latter shown to be more suitable for finding concentration per area of film and/or estimating the total load of extractables in the entire bag. The feasibility of monitoring bDtBPP during the bioprocess was investigated with a Waters PATROL UPLC System with a Flownamics® sampling probe as the sampling interface. bDtBPP spiked in cell culture medium was only detected in unfiltered or at-line injections, indicating that with the current sampling interface, monitoring of extractables and leachables is limited to at-line sampling

Large-scale assessment of Extractables and Leachables in single-use bags for biomanufacturing using ultra high performance liquid chromatography coupled to quadrupole-orbitrap mass spectrometry

Single-use technologies (SUTs) are increasingly used in biopharmaceutical manufacturing processes. Despite their advantages, these plastic assemblies draw concern because they are a potential source of contamination due to extractable and leachable compounds (E&Ls) that result from residual polymeric fragments and different additives used in their manufacture1. Characterizing E&Ls from such materials is a necessary step in establishing their suitability for use. There is evidence of some cytotoxic compounds that leach out of single-use bags into cell culture media in concentrations are deleterious to CHO cell growth, even at trace levels2. Given the potential for these substances to adversely affect biopharmaceutical production, this discovery clearly shows an urgent need for analytical techniques to identify and quantitatively assess compounds resulting from the extraction of SUS components. This study was focused specifically in single-use bags (SUBs), one important application of the disposable technologies, used for the production of therapeutic antibodies, proteins and vaccines. 34 single-use bags from different suppliers were extracted under conditions that are relevant for the characterisation of E&Ls. Extraction with different model solvents was intended to establish a comprehensive extractables profile: water for injection, isopropanol:H2O (1:1), 0.1 M H3PO4 and 50mM NH4CH3COO pH 9.5, while extraction with chemically defined cell culture media was used for leachable assessment. This analysis is challenging as leachables usually exist at trace levels within a very complex matrix. For this reason, a simple and fast analytical method based on a sample treatment by dispersive liquid-liquid microextraction (DLLME) was also developed and applied for analyte preconcentration and matrix elimination. Then, the extracts were analysed by LC-Orbitrap-MS, with high mass resolution performance and exceptional mass accuracy for the detection and identification of non-volatile E&Ls in SUBs. 130 E&Ls were identified, with many of these compounds being polymer additives or their degradation products. Interestingly, some leachables were not found during extractables analysis, suggesting that they might be produced by the interaction of components of the media with compounds from bags. Multivariate analysis performed on the analytical data established significant correlations between the type and concentration of compounds and bags features as brand, manufacturing date and type of polymer. New production techniques have allowed to develop new types of polymers and the advent of regulatory issues that limit/ban the use of certain raw materials and additives has produced a change in the nature of E&Ls. Consequently, it is necessary to provide practical and versatile guidelines for confident determination of these substances that would enable early identification of non-satisfactory films for control and improvement of SUBs. The analytical workflow that is presented has all the necessary features to be used as part of the quality control in bags manufacturing. 1 Gao, Y.; Allison, N. Extractables and leachables issues with the application of single use technology in the biopharmaceutical industry. J Chem Technol Biotechnol. 2016, 91, 289–295. 2 Hammond, M.; Nunn, H.; Rogers, G.; Lee, H.; Marghitoiu, A.-L.; Perez, L.; Nashed-Samuel, Y.; Anderson, C.; Vandiver, M.; Kline, S. Identification of a Leachable Compound Detrimental to Cell Growth in Single-Use Bioprocess Containers. PDA J. Pharm. Sci. Technol. 2013, 67, 123–134

Secondary polymer layered impregnated tile

A low density organic polymer impregnated preformed fibrous ceramic article includes a plurality of layers. A front layer includes ceramic fibers or carbon fibers or combinations of ceramic fibers and carbon fibers, and is impregnated with an effective amount of at least one organic polymer. A middle layer includes polymer impregnated ceramic fibers. A back layer includes ceramic fibers or carbon fibers or combinations of ceramic fibers and carbon fibers, and is impregnated with an effective amount of at least one low temperature pyrolyzing organic polymer capable of decomposing without depositing residues

Multilayer Impregnated Fibrous Thermal Insulation Tiles

The term "secondary polymer layered impregnated tile" ("SPLIT") denotes a type of ablative composite-material thermal- insulation tiles having engineered, spatially non-uniform compositions. The term "secondary" refers to the fact that each tile contains at least two polymer layers wherein endothermic reactions absorb considerable amounts of heat, thereby helping to prevent overheating of an underlying structure. These tiles were invented to afford lighter-weight alternatives to the reusable thermal-insulation materials heretofore variously used or considered for use in protecting the space shuttles and other spacecraft from intense atmospheric-entry heating

Using the posterior distribution of deviance to measure evidence of association for rare susceptibility variants

Aitkin recently proposed an integrated Bayesian/likelihood approach that he claims is general and simple. We have applied this method, which does not rely on informative prior probabilities or large-sample results, to investigate the evidence of association between disease and the 16 variants in the KDR gene provided by Genetic Analysis Workshop 17. Based on the likelihood of logistic regression models and considering noninformative uniform prior probabilities on the coefficients of the explanatory variables, we used a random walk Metropolis algorithm to simulate the distributions of deviance and deviance difference. The distribution of probability values and the distribution of the proportions of positive deviance differences showed different locations, but the direction of the shift depended on the genetic factor. For the variant with the highest minor allele frequency and for any rare variant, standard logistic regression showed a higher power than the novel approach. For the two variants with the strongest effects on Q1 under a type I error rate of 1%, the integrated approach showed a higher power than standard logistic regression. The advantages and limitations of the integrated Bayesian/likelihood approach should be investigated using additional regions and considering alternative regression models and collapsing methods

Lentiviral Vector Delivery of Human Interleukin-7 (hIL-7) to Human Immune System (HIS) Mice Expands T Lymphocyte Populations

Genetically modified mice carrying engrafted human tissues provide useful models to study human cell biology in physiologically relevant contexts. However, there remain several obstacles limiting the compatibility of human cells within their mouse hosts. Among these is inadequate cross-reactvitiy between certain mouse cytokines and human cellular receptors, depriving the graft of important survival and growth signals. To circumvent this problem, we utilized a lentivirus-based delivery system to express physiologically relevant levels of human interleukin-7 (hIL-7) in Rag2-/-γc-/- mice following a single intravenous injection. hIL-7 promoted homeostatic proliferation of both adoptively transferred and endogenously generated T-cells in Rag2-/-γc-/- Human Immune System (HIS) mice. Interestingly, we found that hIL-7 increased T lymphocyte numbers in the spleens of HIV infected HIS mice without affecting viral load. Taken together, our study unveils a versatile approach to deliver human cytokines to HIS mice, to both improve engraftment and determine the impact of cytokines on human diseases

Phenolic Impregnated Carbon Ablators (PICA) as Thermal Protection Systems for Discovery Missions

This paper presents the development of the light weight Phenolic Impregnated Carbon Ablators (PICA) and its thermal performance in a simulated heating environment for planetary entry vehicles. The PICA material was developed as a member of the Light Weight Ceramic Ablators (LCA's), and the manufacturing process of this material has since been significantly improved. The density of PICA material ranges from 14 to 20 lbm/ft(exp 3), having uniform resin distribution with and without a densified top surface. The thermal performance of PICA was evaluated in the Ames arc-jet facility at cold wall heat fluxes from 375 to 2,960 BtU/ft(exp 2)-s and surface pressures of 0.1 to 0.43 atm. Heat loads used in these tests varied from 5,500 to 29,600 BtU/ft(exp 2) and are representative of the entry conditions of the proposed Discovery Class Missions. Surface and in-depth temperatures were measured using optical pyrometers and thermocouples. Surface recession was also measured by using a template and a height gage. The ablation characteristics and efficiency of PICA are quantified by using the effective heat of ablation, and the thermal penetration response is evaluated from the thermal soak data. In addition, a comparison of thermal performance of standard and surface densified PICA is also discussed

FLORA: a novel method to predict protein function from structure in diverse superfamilies

Predicting protein function from structure remains an active area of interest, particularly for the structural genomics initiatives where a substantial number of structures are initially solved with little or no functional characterisation. Although global structure comparison methods can be used to transfer functional annotations, the relationship between fold and function is complex, particularly in functionally diverse superfamilies that have evolved through different secondary structure embellishments to a common structural core. The majority of prediction algorithms employ local templates built on known or predicted functional residues. Here, we present a novel method (FLORA) that automatically generates structural motifs associated with different functional sub-families (FSGs) within functionally diverse domain superfamilies. Templates are created purely on the basis of their specificity for a given FSG, and the method makes no prior prediction of functional sites, nor assumes specific physico-chemical properties of residues. FLORA is able to accurately discriminate between homologous domains with different functions and substantially outperforms (a 2–3 fold increase in coverage at low error rates) popular structure comparison methods and a leading function prediction method. We benchmark FLORA on a large data set of enzyme superfamilies from all three major protein classes (α, β, αβ) and demonstrate the functional relevance of the motifs it identifies. We also provide novel predictions of enzymatic activity for a large number of structures solved by the Protein Structure Initiative. Overall, we show that FLORA is able to effectively detect functionally similar protein domain structures by purely using patterns of structural conservation of all residues

Event-related alpha suppression in response to facial motion

This article has been made available through the Brunel Open Access Publishing Fund.While biological motion refers to both face and body movements, little is known about the visual perception of facial motion. We therefore examined alpha wave suppression as a reduction in power is thought to reflect visual activity, in addition to attentional reorienting and memory processes. Nineteen neurologically healthy adults were tested on their ability to discriminate between successive facial motion captures. These animations exhibited both rigid and non-rigid facial motion, as well as speech expressions. The structural and surface appearance of these facial animations did not differ, thus participants decisions were based solely on differences in facial movements. Upright, orientation-inverted and luminance-inverted facial stimuli were compared. At occipital and parieto-occipital regions, upright facial motion evoked a transient increase in alpha which was then followed by a significant reduction. This finding is discussed in terms of neural efficiency, gating mechanisms and neural synchronization. Moreover, there was no difference in the amount of alpha suppression evoked by each facial stimulus at occipital regions, suggesting early visual processing remains unaffected by manipulation paradigms. However, upright facial motion evoked greater suppression at parieto-occipital sites, and did so in the shortest latency. Increased activity within this region may reflect higher attentional reorienting to natural facial motion but also involvement of areas associated with the visual control of body effectors. © 2014 Girges et al