Side-by-side comparability of batch and continuous downstream for the production of monoclonal antibodies

Continuous processing is the future production method for monoclonal antibodies (mAbs). A fully continuous, fully automated downstream process based on disposable equipment was developed and implemented inside the MoBiDiK pilot plant. However, a study evaluating the comparability between batch and continuous processing based on product quality attributes was not conducted before. The work presented fills this gap comparing both process modes experimentally by purifying the same harvest material (side-by-side comparability). Samples were drawn at different time points and positions in the process for batch and continuous mode. Product quality attributes, product-related impurities, as well as process-related impurities were determined. The resulting polished material was processed to drug substance and further evaluated regarding storage stability and degradation behavior. The in-process control data from the continuous process showed the high degree of accuracy in providing relevant process parameters such as pH, conductivity, and protein concentration during the entire process duration. Minor differences between batch and continuous samples are expected as different processing conditions are unavoidable due to the different nature of batch and continuous processing. All tests revealed no significant differences in the intermediates and comparability in the drug substance between the samples of both process modes. The stability study of the final product also showed no differences in the stability profile during storage and forced degradation. Finally, online data analysis is presented as a powerful tool for online-monitoring of chromatography columns during continuous processing.     

Relationship between epipelon,epiphyton and phytoplankton in two limnological phases in a shallow tropical reservoir with high Nymphaea coverage

Macrophytes and phytoplankton are recognized as having roles in determining alternative stable states in shallow lakes and reservoirs, while the role of periphyton has been poorly investigated. Temporal and spatial variation of phytoplankton, epipelon and epiphyton was examined in a shallow reservoir with high abundance of aquatic macrophytes. The relationships between algae communities and abiotic factors, macrophyte coverage and zooplankton density were also analyzed. Monthly sampling was performed in three zones of the depth gradient of the reservoir. Two phases of algal dominance were found: a phytoplankton phase and epipelon phase. The phase of phytoplankton dominance was characterized by high macrophyte coverage. Rotifera was the dominant zooplankton group in all the zones. Flagellate algae were dominant in phytoplankton, epipelon and epiphyton. Macrophyte coverage was found to be a predictor for algal biomass. Changes in biomass and species composition were associated with macrophyte cover variation, mainly the Nymphaea. In addition to the abiotic factors, the macrophyte coverage was a determining factor for changes to the algal community, contributing to the alternation between dominance phases of phytoplankton and epipelon. The macrophyte–phytoplankton–periphyton relationship needs to be further known in shallow reservoirs, especially the role of epipelon as an alternate stable state.

     

Exploitation of dihydroorotate dehydrogenase (DHODH) and p53 activation as therapeutic targets: A case study in polypharmacology

The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, dihydroorotate dehydrogenase (DHODH), and the blockage of uridine transport into cells. These findings hold a 3-fold significance: first, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; second, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibition as this is a major contributor to the mechanism of action of the most widely used tenovins; and finally, that small changes in the structure of a small molecule can lead to a dramatic change in the target profile of the molecule even when the phenotypic readout remains static.     

In Vitro Evaluation of the Effect of Stimulation with Magnetic Fields on Chondrocytes

Magnetic fields (MFs) have been used as an external stimulus to increase cell proliferation in chondrocytes and extracellular matrix (ECM) synthesis of articular cartilage. However, previously published studies have not shown that MFs are homogeneous through cell culture systems. In addition, variables such as stimulation times and MF intensities have not been standardized to obtain the best cellular proliferative rate or an increase in molecular synthesis of ECM. In this work, a stimulation device, which produces homogeneous MFs to stimulate cell culture surfaces was designed and manufactured using a computational model. Furthermore, an in vitro culture of primary rat chondrocytes was established and stimulated with two MF schemes to measure both proliferation and ECM synthesis. The best proliferation rate was obtained with an MF of 2 mT applied for 3 h, every 6 h for 8 days. In addition, the increase in the synthesis of glycosaminoglycans was statistically significant when cells were stimulated with an MF of 2 mT applied for 5 h, every 6 h for 8 days. These findings suggest that a stimulation with MFs is a promising tool that could be used to improve in vitro treatments such as autologous chondrocyte implantation, either to increase cell proliferation or stimulate molecular synthesis. Bioelectromagnetics. 2020;41:41–51 © 2019 Bioelectromagnetics Society     

Different assembly of acid and salt tolerance response in two dairy Listeria monocytogenes wild strains

A lack on the association between acid tolerance response (ATR) and osmotolerance response (OTR) among Listeria monocytogenes dairy isolates was found. In order to evaluate how wild L. monocytogenes isolates mount tolerance responses under a sub-lethal pH and a low sodium chloride concentration (pH 5.5 and 3.5 % [w/v] NaCl), a proteomic approach was used. The ATR and OTR of two L. monocytogenes cheese dairy isolates (strain T8, serotype 4b and A9, serotype 1/2b or 3b) were determined. The proteomes of the adapted and non-adapted cultures were evaluated by 2-DE. One strain displayed an ATR, but not an OTR and the other displayed an OTR, but not an ATR. The ATR positive strain showed the over-production of proteins related with protein synthesis, protein folding, attainment of reduction power, ribose production and cell wall. In contrast, in the OTR-positive-strain proteins related with glycolysis, general stress and detoxification were identified.     

Local NO synthase inhibition produces histological and functional recovery in Achilles tendon of rats after tenotomy

Repair of injured tendon is a very slow process and involves the release of many molecules, including nitric oxide. We investigate the influence of local nitrergic inhibition in histological and functional recovery of injured Achilles tendon. A standard murine model of tendon injury by rupture was used. The animals were divided into three experimental groups: control, injury + vehicle (normal saline) and injury + Nω-nitro-L-arginine methyl ester (L-NAME). The products were injected into the paratendinous region every 2 days and body weight gain and Achilles functional index (AFI) were evaluated on days 0, 7, 14 and 21 after tendon injury. On day 21 post-injury, the animals were killed to evaluate nitric oxide production and tissue organization. We observed that tendon surgical division led to increased tissue nitrite levels, which were reduced in L-NAME-treated rats. The AFI revealed functional recovery of L-NAME-treated animals on day 21 post-injury, which was not observed in the saline-treated group. Microscopic analysis of hematoxylin-eosin staining and collagen autofluorescence showed that L-NAME-treated rats had more aligned areas of collagen fibers and that the diameter of newly organized collagen in this group was also greater than that in the vehicle-treated one. We demonstrate that local treatment with L-NAME significantly improves the functional parameters and accelerates histomorphological recovery.     

Exploring the concept of interaction computing through the discrete algebraic analysis of the Belousov–Zhabotinsky reaction

Interaction computing (IC) aims to map the properties of integrable low-dimensional non-linear dynamical systems to the discrete domain of finite-state automata in an attempt to reproduce in software the self-organizing and dynamically stable properties of sub-cellular biochemical systems. As the work reported in this paper is still at the early stages of theory development it focuses on the analysis of a particularly simple chemical oscillator, the Belousov–Zhabotinsky (BZ) reaction. After retracing the rationale for IC developed over the past several years from the physical, biological, mathematical, and computer science points of view, the paper presents an elementary discussion of the Krohn–Rhodes decomposition of finite-state automata, including the holonomy decomposition of a simple automaton, and of its interpretation as an abstract positional number system. The method is then applied to the analysis of the algebraic properties of discrete finite-state automata derived from a simplified Petri net model of the BZ reaction. In the simplest possible and symmetrical case the corresponding automaton is, not surprisingly, found to contain exclusively cyclic groups. In a second, asymmetrical case, the decomposition is much more complex and includes five different simple non-abelian groups whose potential relevance arises from their ability to encode functionally complete algebras. The possible computational relevance of these findings is discussed and possible conclusions are drawn.     

Liposomal temocene (m-THPPo) photodynamic treatment induces cell death by mitochondria-independent apoptosis

Background

The cell death pathway activated after photodynamic therapy (PDT) is controlled by a variety of parameters including the chemical structure of the photosensitizer, its subcellular localization, and the photodynamic damage induced. The present study aims to characterize a suitable m-THPPo liposomal formulation, to determine its subcellular localization in HeLa cells and to establish the cell death mechanisms that are activated after photodynamic treatments.

Methods

Liposomes containing m-THPPo were prepared from a mixture of DPPC and DMPG at a 9:1 molar ratio. In order to procure the best encapsulation efficiency, the m-THPPo/lipid molar ratio was considered. HeLa cells were incubated with liposomal m-THPPo and the subcellular localization of m-THPPo was studied. Several assays such as TUNEL, annexin V/propidium iodide and Hoechst-33258 staining were performed after photodynamic treatments. The apoptotic initiation was assessed by cytochrome c and caspase-2 immunofluorescence.

Results

m-THPPo encapsulated in liposomes showed a decrease of the fluorescence and singlet oxygen quantum yields, compared to those of m-THPPo dissolved in tetrahydrofuran. Liposomal m-THPPo showed colocalization with LysoTracker® and it induced photoinactivation of HeLa cells by an apoptotic mechanism. In apoptotic cells no relocalization of cytochrome c could be detected, but caspase-2 was positive immediately after photosensitizing treatments.

Conclusions

Photodynamic treatment with liposomal m-THPPo leads to a significant percentage of apoptotic morphology of HeLa cells. The activation of caspase-2, without the relocalization of cytochrome c, indicates a mitochondrial-independent apoptotic mechanism.

General significance

These results provide a better understanding of the cell death mechanism induced after liposomal m-THPPo photodynamic treatment.