Case study: Relevance of ASTM and ISTA standard shipping simulation studies for the validation of real world shipping of drug substances

Today’s global bio-manufacturing networks require that high value drug substances transfer between traditional production centers, CMOs and offshore facilities. These transfers increasingly performed in single-use bags require a thorough validation to demonstrate their integrity and safety for the drug substances. ASTM and ISTA both provide standardized validation methods using a variety of simulated shipping hazards. These tests are qualitative by nature with simple pass fail criteria and unknown factors of safety. The paper first describes an integrated validation approach that establishes the safety factor for the standardized test methods vs. real world shipping conditions in single-use bags. Normalized ASTM D4169 and ISTA and real shipping tests were performed using liquid filled bags in their containers. Results on vibrations and shock are discussed and compared to establish the robustness, the worst case nature and the safety factor of the standard protocol. The author then presents the data from a 4-step qualification study using the ASTM D4169 conditions at 2 different temperatures. The study contributes to the robustness of the overall supplier qualification and provides extensive data that end-users can leverage to support their own process qualification for liquid shipping in single-use systems

New approach for qualifying liquid handling in single-use bags

The technical and economic benefits of Single-use in bio-manufacturing are well known and the industry is broadening their adoption. The increasing manual handling of bags in the biotech process involves higly trained operators and specific guidelines in order to secure the process, especially for bulk drug substances or even for drug products handling. Please click Additional Files below to see the full abstract

Community post impact assessment - rapid appraisal: Tropical Cyclone Debbie, Whitsunday Coast, QLD, Australia 5-8 April 2017

A rapid social research appraisal of residents from the Whitsunday's coastal communities directly impacted by Tropical Cyclone Debbie, 2017. Investigating issues of hazard awareness and preparedness; sheltering and evacuation behaviour; and initial recovery progress

The 2-Ga peraluminous magmatism of the Jacobina-Contendas Mirante belts (Bahia, Brazil) : geologic and isotopic constraints on the sources

L'étude pétrographique des intrusions granitiques du craton Sao Francisco (Bahia, Brésil) permet de définir la chronologie absolue des épisodes magmatiques et d'identifier les sources magmatiques ainsi que les processus géodynamiques se produisant lors de l'orogenèse transamazonienn

Conformational Changes of Calpain from Human Erythrocytes in the Presence of Ca2

Small angle x-ray scattering has been used to monitor calpain structural transitions during the activation process triggered by Ca(2+) binding. The scattering pattern of the unliganded enzyme in solution does not display any significant difference with that calculated from the crystal structure. The addition of Ca(2+) promotes the formation of large aggregates, indicating the exposure of hydrophobic patches on the surface of the protease. In contrast, Ca(2+) addition in the presence of the thiol proteinase inhibitor E64 or of the inhibitor leupeptin causes a small conformational change with no dissociation of the heterodimer. The resulting conformation appears to be slightly more extended than the unliganded form. From the comparison between ab initio models derived from our data with the crystal structure, the major observable conformational change appears to be localized at level of the L-subunit and in particular seems to confirm the mutual movement already observed by the crystallographic analysis of the dII (dIIb) and the dI (dIIa) domains creating a functional active site. This work not only provides another piece of supporting evidence for the calpain conformational change in the presence of Ca(2+), but actually constitutes the first experimental observation of this change for intact heterodimeric calpain in solution

22q11 Deletion Syndrome and Urogenital Manifestations: A Clinicopathological Case Report.

Deletion in the chromosomal region 22q11 results from the abnormal development of the third and fourth pharyngeal pouches during embryonic life and presents an expansive phenotype with more than 180 clinical features described that involve every organ and system. A 23-year-old African woman presented for the first trimester echography, which revealed an isolated anechoic structure suggesting a ureteral dilatation. The suspicion of a malposition of great arteries in the second trimester indicated an amniocentesis leading to a diagnosis of 22q11 deletion. At 32 weeks, the patient was admitted for premature rupture of membranes and gave birth 2 weeks later to a male newborn who presented a respiratory distress syndrome and probably died secondary to a tracheal stenosis. Necropsy revealed typical clinical features of 22q11 deletion associated with left renal agenesis, hypospadias, and penile hypoplasia. We report a case of 22q11 deletion syndrome with typical clinical features associated with urogenital manifestations suspected at the first trimester ultrasound

Characterization of Monomeric Intermediates during VSV Glycoprotein Structural Transition

Entry of enveloped viruses requires fusion of viral and cellular membranes, driven by conformational changes of viral glycoproteins. Crystal structures provide static pictures of pre- and post-fusion conformations of these proteins but the transition pathway remains elusive. Here, using several biophysical techniques, including analytical ultracentrifugation, circular dichroïsm, electron microscopy and small angle X-ray scattering, we have characterized the low-pH-induced fusogenic structural transition of a soluble form of vesicular stomatitis virus (VSV) glycoprotein G ectodomain (Gth, aa residues 1–422, the fragment that was previously crystallized). While the post-fusion trimer is the major species detected at low pH, the pre-fusion trimer is not detected in solution. Rather, at high pH, Gth is a flexible monomer that explores a large conformational space. The monomeric population exhibits a marked pH-dependence and adopts more elongated conformations when pH decreases. Furthermore, large relative movements of domains are detected in absence of significant secondary structure modification. Solution studies are complemented by electron micrographs of negatively stained viral particles in which monomeric ectodomains of G are observed at the viral surface at both pH 7.5 and pH 6.7. We propose that the monomers are intermediates during the conformational change and thus that VSV G trimers dissociate at the viral surface during the structural transition

Fibrinogen species as resolved by HPLC-SAXS data processing within the UltraScan Solution Modeler ( US-SOMO ) enhanced SAS module

Fibrinogen is a large heterogeneous aggregation/degradation-prone protein playing a central role in blood coagulation and associated pathologies, whose structure is not completely resolved. When a high-molecular-weight fraction was analyzed by size-exclusion high-performance liquid chromatography/small-angle X-ray scattering (HPLC-SAXS), several composite peaks were apparent and because of the stickiness of fibrinogen the analysis was complicated by severe capillary fouling. Novel SAS analysis tools developed as a part of the UltraScan Solution Modeler ( US-SOMO ; http://somo.uthscsa.edu/), an open-source suite of utilities with advanced graphical user interfaces whose initial goal was the hydrodynamic modeling of biomacromolecules, were implemented and applied to this problem. They include the correction of baseline drift due to the accumulation of material on the SAXS capillary walls, and the Gaussian decomposition of non-baseline-resolved HPLC-SAXS elution peaks. It was thus possible to resolve at least two species co-eluting under the fibrinogen main monomer peak, probably resulting from in-column degradation, and two others under an oligomers peak. The overall and cross-sectional radii of gyration, molecular mass and mass/length ratio of all species were determined using the manual or semi-automated procedures available within the US-SOMO SAS module. Differences between monomeric species and linear and sideways oligomers were thus identified and rationalized. This new US-SOMO version additionally contains several computational and graphical tools, implementing functionalities such as the mapping of residues contributing to particular regions of P ( r ), and an advanced module for the comparison of primary I ( q ) versus q data with model curves computed from atomic level structures or bead models. It should be of great help in multi-resolution studies involving hydrodynamics, solution scattering and crystallographic/NMR data

Predicting translational diffusion of evolutionary conserved RNA structures by the nucleotide number

Ribonucleic acids are highly conserved essential parts of cellular life. RNA function is determined to a large extent by its hydrodynamic behaviour. The presented study proposes a strategy to predict the hydrodynamic behaviour of RNA single strands on the basis of the polymer size. By atom-level shell-modelling of high-resolution structures, hydrodynamic radius and diffusion coefficient of evolutionary conserved RNA single strands (ssRNA) were calculated. The diffusion coefficients D of 17–174 nucleotides (nt) containing ssRNA depended on the number of nucleotides N with D = 4.56 × 10−10 N−0.39 m2 s−1. The hydrodynamic radius RH depended on N with RH = 5.00 × 10−10 N0.38 m. An average ratio of the radius of gyration and the hydrodynamic radius of 0.98 ± 0.08 was calculated in solution. The empirical law was tested by in solution measured hydrodynamic radii and radii of gyration and was found to be highly consistent with experimental data of evolutionary conserved ssRNA. Furthermore, the hydrodynamic behaviour of several evolutionary unevolved ribonucleic acids could be predicted. Based on atom-level shell-modelling of high-resolution structures and experimental hydrodynamic data, empirical models are proposed, which enable to predict the translational diffusion coefficient and molecular size of short RNA single strands solely on the basis of the polymer size

Late orogenic carboniferous extensions in the Variscan French Massif Central

International audienceThe Variscan French Massif Central experienced two successive stages of extension from Middle Carboniferous to Early Permian. In the northern Massif Central, the first stage began in the late Visean, immediately after nappe stacking, and is well recorded by Namurian-Westphalian synkinematic plutonism. The Middle Carboniferous leucogranites widespread in the NW Massif Central (Limousin and Sioule area) were emplaced within a crust extending along a NE-SW direction. At the same time, the hanging wall or "Guéret extensional allochton" moved toward the SE. Several examples of the synextensional plutonism are also recognized in central Limousin: Saint Mathieu dome, La Porcherie, and Cornil leucogranites. These examples illustrate the relationship between granite emplacement and crustal scale deformation characterized by NW-SE stretching and NE-SW shortening. In the central and southern Massif Central (Cévennes, Châtaigneraie, and Margeride areas), plutonism is dominantly granodioritic and exhibits the same structural features: NW-SE maximum stretching and overturning to the SE. Middle Carboniferous (Namurian-Westphalian) extension was parallel to the Variscan belt both in the Massif Central and southern Armorican area. This extensional regime was active from the late Visean in the north, while compression dominated in the southernmost domains (Montagne Noire and Pyrenées). The second extensional stage occurred from Late Carboniferous to Early Permian. This event was responsible for the opening of intramontane coal basins, brittle deformation in the upper crust, and ductile normal faulting localized on the margin of cordierite granite-migmatite domes. Data from the coal basins show that the half-graben is the dominant structural style, except for basins located along submeridianal left-lateral faults which have pull-apart geometries. Late Carboniferous extension occurred along the NE-SW direction. The NE-SW maximum stretching direction can be found in the whole Massif Central but is more developed in the eastern part. The extensional direction is transverse to the general trend of the belt, and top-to-the-NE shearing is dominant. Correlations of these two extension directions with neighboring Variscan massifs are discussed