Combined metabolomics and proteomics reveals hypoxia as a cause of lower productivity on scale‐up to a 5000‐liter CHO bioprocess

Large‐scale bioprocessing is key to the successful manufacturing of a biopharmaceutical. However, cell viability and productivity are often lower in the scale‐up from laboratory to production. In this study, we analyzed CHO cells, which showed lower percent viabilities and productivity in a 5‐KL production scale bioreactor compared to a 20‐L bench‐top scale under seemingly identical process parameters. An increase in copper concentration in the media from 0.02 µM to 0.4 µM led to a doubling of percent viability in the production scale albeit still at a lower level than the bench‐top scale. Combined metabolomics and proteomics revealed the increased copper reduced the presence of reactive oxygen species (ROS) in the 5‐KL scale process. The reduction in oxidative stress was supported by the increased level of glutathione peroxidase in the lower copper level condition. The excess ROS was shown to be due to hypoxia (intermittent), as evidenced by the reduction in fibronectin with increased copper. The 20‐L scale showed much less hypoxia and thus less excess ROS generation, resulting in little to no impact to productivity with the increased copper in the media. The study illustrates the power of 'Omics in aiding in the understanding of biological processes in biopharmaceutical production.     

Human metapneumovirus Induces Reorganization of the Actin Cytoskeleton for Direct Cell-to-Cell Spread

Paramyxovirus spread generally involves assembly of individual viral particles which then infect target cells. We show that infection of human bronchial airway cells with human metapneumovirus (HMPV), a recently identified paramyxovirus which causes significant respiratory disease, results in formation of intercellular extensions and extensive networks of branched cell-associated filaments. Formation of these structures is dependent on actin, but not microtubule, polymerization. Interestingly, using a co-culture assay we show that conditions which block regular infection by HMPV particles, including addition of neutralizing antibodies or removal of cell surface heparan sulfate, did not prevent viral spread from infected to new target cells. In contrast, inhibition of actin polymerization or alterations to Rho GTPase signaling pathways significantly decreased cell-to-cell spread. Furthermore, viral proteins and viral RNA were detected in intercellular extensions, suggesting direct transfer of viral genetic material to new target cells. While roles for paramyxovirus matrix and fusion proteins in membrane deformation have been previously demonstrated, we show that the HMPV phosphoprotein extensively co-localized with actin and induced formation of cellular extensions when transiently expressed, supporting a new model in which a paramyxovirus phosphoprotein is a key player in assembly and spread. Our results reveal a novel mechanism for HMPV direct cell-to-cell spread and provide insights into dissemination of respiratory viruses.     

Balancing economic costs and ecological outcomes of passive and active restoration in agricultural landscapes: the case of Brazil

Forest restoration requires strategies such as passive restoration to balance financial investments and ecological outcomes. However, the ecological outcomes of passive restoration are traditionally regarded as uncertain. We evaluated technical and legal strategies for balancing economic costs and ecological outcomes of passive versus active restoration in agricultural landscapes. We focused in the case of Brazil, where we assessed the factors driving the proportion of land allocated to passive and active restoration in 42 programs covering 698,398 hectares of farms in the Atlantic Forest, Atlantic Forest/cerrado ecotone and Amazon; the ecological outcomes of passive and active restoration in 2955 monitoring plots placed in six restoration programs; and the legal framework developed by some Brazilian states to balance the different restoration approaches and comply with legal commitments. Active restoration had the highest proportion of land allocated to it (78.4%), followed by passive (14.2%) and mixed restoration (7.4%). Passive restoration was higher in the Amazon, in silviculture, and when remaining forest cover was over 50 percent. Overall, both restoration approaches showed high levels of variation in the ecological outcomes; nevertheless, passively restored areas had a smaller percentage canopy cover, lower species density, and less shrubs and trees (dbh > 5 cm). The studied legal frameworks considered land abandonment for up to 4 years before deciding on a restoration approach, to favor the use of passive restoration. A better understanding of the biophysical and socioeconomic features of areas targeted for restoration is needed to take a better advantage of their natural regeneration potential.     

Gastroretentive Matrix Tablets of Boswellia Oleogum Resin: Preparation,Optimization, <Emphasis Type="Italic">In Vitro</Emphasis> Evaluation,and Cytoprotective Effect on Indomethacin-Induced Gastric Ulcer in Rabbits

Currently available anti-ulcer drugs suffer from serious side effects which limited their uses and prompted the need to search for a safe and efficient new anti-ulcer agent. Boswellia gum resin (BR) emerged as a safe, efficient, natural, and economic potential cytoprotective agent. Thus, it is of medical importance to develop gastroretentive (GR) formulations of BR to enhance its bioavailability and anti-ulcer efficacy. Early attempts involved the use of organic solvents and non-applicability to large-scale production. In this study, different tablet formulations were prepared by simple direct compression combining floating and bioadhesion mechanisms employing hydroxypropyl methylcellulose (HPMC), sodium carboxymethyl cellulose (SCMC), pectin (PC), and/or carbopol (CP) as bioadhesive polymers and sodium bicarbonate (SB) as a gas former. The prepared tablets were subjected for assessment of swelling, floating, bioadhesion, and drug release in 0.1 N HCl. The optimized GR formulation was examined for its protective effect on the gastric ulcer induced by indomethacin in albino rabbits compared with lactose tablets. The obtained results disclosed that swelling, floating, bioadhesion, and drug release of the GR tablets of BR depend mainly on the nature of the matrix and the ratio of polymer combinations. Moreover, a combination of SCMC-CP in a ratio of 2:1 (SCP21) exhibited desirable floating, bioadhesion, swelling, and extended drug release. Also, a 6-h pretreatment with SCP21 tablets decreased the severity of inflammation and number of bleeding spots among ulcer-induced rabbits in comparison to those treated with lactose tablets.     

Investigation of Thermal and Viscoelastic Properties of Polymers Relevant to Hot Melt Extrusion,IV: Affinisol™ HPMC HME Polymers

Most cellulosic polymers cannot be used as carriers for preparing solid dispersion of drugs by hot melt extrusion (HME) due to their high melt viscosity and thermal degradation at high processing temperatures. Three HME-grade hydroxypropyl methylcelluloses, namely Affinisol™ HPMC HME 15 cP, Affinisol™ HPMC HME 100 cP, and Affinisol™ HPMC HME 4 M, have recently been introduced by The Dow Chemical Co. to enable the preparation of solid dispersion at lower and more acceptable processing temperatures. In the present investigation, physicochemical properties of the new polymers relevant to HME were determined and compared with that of Kollidon^® VA 64. Powder X-ray diffraction (PXRD), modulated differential scanning calorimetry (mDSC), thermogravimetric analysis (TGA), moisture sorption, rheology, and torque analysis by melt extrusion were applied. PXRD and mDSC showed that the Affinisol™ polymers were amorphous in nature. According to TGA, the onset of degradation for all polymers was >220°C. The Affinisol™ polymers exhibited less hygroscopicity than Kollidon^® VA 64 and another HPMC polymer, Methocel™ K100LV. The complex viscosity profiles of the Affinisol™ polymers as a function of temperature were similar. The viscosity of the Affinisol™ polymers was highly sensitive to the shear rate applied, and unlike Kollidon^® VA 64, the viscosity decreased drastically when the angular frequency was increased. Because of the very high shear rate encountered during melt extrusion, Affinisol™ polymers showed capability of being extruded at larger windows of processing temperatures as compared to that of Kollidon^® VA 64.KEY WORDS: Affinisol™ HPMC HME, hot melt extrusion, hydroxypropyl methylcellulose, solid dispersion, thermal analysis, viscosity     

Short-term variability of airway caliber-a marker of asthma?

Variability in airway caliber is a characteristic feature of asthma. Previous studies reported that the variability in respiratory system impedance (Zrs), measured by the forced oscillation technique during several minutes of tidal breathing, is increased in asthma and may be a marker of inherent instability of the airways. The aims of this study were to determine if short-term variability in impedance correlates with peak expiratory flow (PEF) variability or airway hyperresponsiveness (AHR). The SD of log-transformed impedance (lnZrsSD) was measured as a marker of short-term variability and compared with the diurnal variability of PEF over 2 wk in 28 asthmatic and 7 nonasthmatic subjects and with AHR to histamine in a cohort of 17 asthmatic and 82 nonasthmatic subjects. In addition, lnZrsSD was measured in eight nonasthmatic subjects before and after methacholine challenge in the upright and supine positions. There were no significant differences in lnZrsSD between asthmatic and nonasthmatic subjects (P = 0.68). Furthermore, in asthmatic subjects, lnZrsSD did not correlate with diurnal variability of PEF (rs = -0.12 P = 0.54) or with AHR to histamine (r = 0.10, P = 0.71). Neither methacholine challenge nor supine posture caused any significant change in lnZrsSD. We conclude that our findings do not support previous reports about the utility of short-term variability of impedance. Our findings suggest that, using standard methods for forced oscillometry, impedance variability does not provide clinically useful information about the severity of asthma.