Clarification and capture of a CHO-derived monoclonal antibody through flocculation and AEX processes

Flocculants have been widely used in the food and chemical industry, as well as in wastewater clarification. More recently, studies have shown the applicability of these agents in the clarification of mammalian cell suspensions as an alternative e.g. to centrifugation and microfiltration1,2. Flocculants present several advantages such as low cost, process simplicity and facility to run under continuous mode. On the other hand, some flocculants have shown toxicity and thus their total clearance has to be demonstrated. In this work, the cationic flocculants chitosan and poly diallyldimethylammonium chloride (PDADMAC) have been investigated and compared for the clarification of suspensions of CHO cells producing a recombinant humanized monoclonal antibody. Anion exchange (AEX) adsorption has been then evaluated for its capacity to remove host cell protein (HCP) and host cell DNA (HCDNA), with the final aim of in the future developing an integrated train of clarification and AEX capture within a tangential flow filtration (TFF) device. CHO-DP12 cells (ATCC) were cultivated in batch mode in shake flasks at 37 ºC and 185 rpm using the animal component free medium TC-LECC (Xell AG, Germany), reaching approximately 10 million cells/mL and 0.2 g/L mAb. After cultivation, 20 mL of cell suspension were put in contact with flocculants under different conditions of pH and of flocculant concentration, for 30 min at 100 rpm at room temperature. The samples were then let to sediment for 90 min and the turbidity was measured for 90 min at 600 nm. The AEX resin Q-Sepharose (GE Healthcare, Sweden) was equilibrated at pH 6.5 prior to adsorption studies with the clarified supernatant from the flocculation step. Adsorption was carried out in 1.5-mL tubes by mixing the resin at a 1:10 volumetric ratio with different dilutions of the clarified supernatant for up to 120 min at room temperature under agitation at 1200 rpm. Samples were monitored for IgG (PrA-HPLC), total proteins (BCA) and DNA (picogreen). Regarding flocculation, when chitosan was used, no flocculation was observed in the concentration range of 0.01%-0.2% (10 to 200 pg per total cells), but did occur at lower flocculant concentrations. The best condition for chitosan was thus obtained at a concentration of 0.005% (5 pg per cell) and pH 6.5, resulting in 93.5% ± 1.1% IgG recovery and 98.5% ± 1.3% host cell DNA (HCDNA) removal. PDADMAC provided recoveries higher than 90% at concentrations between 0.0225% and 0.090%, with the latter concentration yielding an IgG recovery of 95.5% ± 2.4% and a HCDNA removal of 98.5%. For both agents, 30 min were sufficient to sediment all the flocculated particles under these best conditions. Under the selected conditions for chitosan flocculation (pH 6.5 and 5 pg per cell), AEX studies were performed in order to determine the adsorption capacity at equilibrium and the residence time needed to maximize removal of HCP and DNA from the clarified supernatant. Equilibrium was reached after 10 min, and adsorption capacity at equilibrium was 11 mg/mL for total proteins of and 15 mg/mL for DNA. Although at pH 6.5 IgG adsorption was not expected to occur, approximately 5 mg/mL were adsorbed (as measured by Protein A-HPLC). Further studies are ongoing to improve IgG recovery in the adsorption step, but these first results show that these simple, low-cost techniques can be used to obtain a cleaner material for further purification steps. The next steps of this work will focus on integrating flocculation, AEX and TFF to obtain a particulate free stream. 1. Riske, F. et al. The use of chitosan as a flocculant in mammalian cell culture dramatically improves clarification throughput without adversely impacting monoclonal antibody recovery. 128, 813–823 (2007). 2. McNerney, T. et al. PDADMAC flocculation of Chinese hamster ovary cells : Enabling a centrifuge-less harvest process for monoclonal antibodies. 862, 413-427 (2015)

Comparative phylogeography of northwest African Natrix maura (Serpentes: Colubridae) inferred from mtDNA sequences

While the comparative phylogeography of European fauna is relatively well understood, with Pleistocene climatic oscillations leading to ‘southern refugia’ for many species, the equivalent pattern has not been determined for North Africa. In this context variation within North African populations of the riverine snake Natrix maura were assessed using mitochondrial DNA sequences. Recent literature regarding North African phylogeographic studies of reptiles was compiled, and several surprising patterns emerged. The most interesting was the extensive movement of species across the Strait of Gibraltar during the Pleistocene. Another is the general pattern of deep genetic divergences between Tunisian and Moroccan populations, often at a level implying the existence of cryptic species. Natrix maura has three distinct lineages in North Africa, however, it apparently did not cross the Strait of Gibraltar during the Pleistocene, but probably did so during the Messinian salinity crisis

Rare coral under the genomic microscope: timing and relationships among Hawaiian Montipora

Background Evolutionary patterns of scleractinian (stony) corals are difficult to infer given the existence of few diagnostic characters and pervasive phenotypic plasticity. A previous study of Hawaiian Montipora (Scleractinia: Acroporidae) based on five partial mitochondrial and two nuclear genes revealed the existence of a species complex, grouping one of the rarest known species (M. dilatata, which is listed as Endangered by the International Union for Conservation of Nature - IUCN) with widespread corals of very different colony growth forms (M. flabellata and M. cf. turgescens). These previous results could result from a lack of resolution due to a limited number of markers, compositional heterogeneity or reflect biological processes such as incomplete lineage sorting (ILS) or introgression. Results All 13 mitochondrial protein-coding genes from 55 scleractinians (14 lineages from this study) were used to evaluate if a recent origin of the M. dilatata species complex or rate heterogeneity could be compromising phylogenetic inference. Rate heterogeneity detected in the mitochondrial data set seems to have no significant impacts on the phylogenies but clearly affects age estimates. Dating analyses show different estimations for the speciation of M. dilatata species complex depending on whether taking compositional heterogeneity into account (0.8 [0.05–2.6] Myr) or assuming rate homogeneity (0.4 [0.14–0.75] Myr). Genomic data also provided evidence of introgression among all analysed samples of the complex. RADseq data indicated that M. capitata colour morphs may have a genetic basis. Conclusions Despite the volume of data (over 60,000 SNPs), phylogenetic relationships within the M. dilatata species complex remain unresolved most likely due to a recent origin and ongoing introgression. Species delimitation with genomic data is not concordant with the current taxonomy, which does not reflect the true diversity of this group. Nominal species within the complex are either undergoing a speciation process or represent ecomorphs exhibiting phenotypic polymorphisms.info:eu-repo/semantics/publishedVersio

Three Li-rich K giants: IRAS 12327-6523, IRAS 13539-4153, and IRAS 17596-3952

We report on spectroscopic analyses of three K giants previously suggested to be Li-rich: IRAS 12327-6523, IRAS 13539-4153, and IRAS 17596-3952. High-resolution optical spectra and the LTE model atmospheres are used to derive the stellar parameters: ($T_{\rm eff}$, log $g$, [Fe/H]), elemental abundances, and the isotopic ratio $^{12}$C/$^{13}$C. IRAS 13539-4153 shows an extremely high Li abundance of $\log\epsilon$(Li) $\approx$ 4.2, a value ten times more than the present Li abundance in the local interstellar medium. This is the third highest Li abundance yet reported for a K giant. IRAS 12327-6523 shows a Li abundances of $\log\epsilon$(Li)$\approx$ 1.4. IRAS 17596-3952 is a rapidly rotating ($V{\sin i}$ $\approx$ 35 km s$^{-1}$) K giant with $\log\epsilon$(Li) $\approx$ 2.2. Infrared photometry which shows the presence of an IR excess suggesting mass-loss. A comparison is made between these three stars and previously recognized Li-rich giants.Comment: 17 pages, 6 figures, accepted for A

Models of Metal Poor Stars with Gravitational Settling and Radiative Accelerations: I. Evolution and Abundance Anomalies

Evolutionary models have been calculated for Pop II stars of 0.5 to 1.0$M_\odot$ from the pre-main-sequence to the lower part of the giant branch. Rosseland opacities and radiative accelerations were calculated taking into account the concentration variations of 28 chemical species, including all species contributing to Rosseland opacities in the OPAL tables. The effects of radiative accelerations, thermal diffusion and gravitational settling are included. While models were calculated both for Z=0.00017 and 0.0017, we concentrate on models with Z=0.00017 in this paper. These are the first Pop II models calculated taking radiative acceleration into account. It is shown that, at least in a 0.8$M_\odot$ star, it is a better approximation not to let Fe diffuse than to calculate its gravitational settling without including the effects of $g_{rad}(Fe)$. In the absence of any turbulence outside of convection zones, the effects of atomic diffusion are large mainly for stars more massive than 0.7$M_\odot$. Overabundances are expected in some stars with \teff \ge 6000K. Most chemical species heavier than CNO are affected. At 12 Gyr, overabundance factors may reach 10 in some cases (e.g. for Al or Ni) while others are limited to 3 (e.g. for Fe). The calculated surface abundances are compared to recent observations of abundances in globular clusters as well as to observations of Li in halo stars. It is shown that, as in the case of Pop I stars, additional turbulence appears to be present.Comment: 40 pages, 17 color figures, to appear in The Astrophysical Journal, April 2002 (paper with original high resolution figures can be found at http://www.cerca.umontreal.ca/~richer/Fichiersps/popII_1.ps

Effects of hydroxycinnamic acids on the glycolysis pathway

Glycolysis is a metabolic pathway vital to the production of energy and some organisms rely on it solely to meet their energy requirements. It is also a central pathway in the metabolism of carbohydrates and a source of therapeutic targets against diabetes and cancer. Caffeoylquinic acids (CQAs) have been extensively studied for their role in the treatment and prevention of diabetes (and cancer) but their mechanisms of action remain mostly unknown. As such, molecular docking was used to find possible targets of CQAs in the glycolysis pathway. The molecular docking assays showed that CQAs were docked preferably to the Rossman fold (nicotinamide adenine dinucleotide — NAD(H) binding site) of oxidoreductases, that use NAD(H) as a cofactor, than to any other site. In-vitro assays were then performed using two NAD(H) dependent oxidoreductases from glycolysis (alcohol dehydrogenase and L-lactate dehydrogenase) in order confirm if CQAs would compete with the cofactor to inhibit the reaction. The results from these assays indicate that CQAs can act as both inhibitors and activators of NAD(H) dependent oxidoreductases of the glycolysis pathway.info:eu-repo/semantics/publishedVersio

Evaluation of the inorganic content of six underused wild berries from Portugal: Potential new sources of essential minerals

The mineral content and levels of trace elements in six wild underused berries (Elaeagnus umbellata, Myrica faya, Rubus grandifolius, Sambucus lanceolata, Vaccinium padifolium and Vaccinium cylindraceum) have been determined by inductively coupled plasma-mass spectrometry after microwave digestion. The inorganic content of these foodstuffs has not been previously described in scientific literature (except for E. umbellata). Hence, this information is of high interest concerning the introduction of these noncommercial berries in the mainstream market. The analytical method has been validated analyzing a certified reference material and performing recovery experiments. The results obtained have been discussed using the Recommended Daily Allowance for minerals provided by the Commission of the European Community, and a comparison between the composition of the analyzed berries and different commercialised berries has been carried out. Data obtained in this study support the consumption of these less-known berries, in particular R. grandifolius and S. lanceolata, as potential novel sources of essential minerals.The authors are grateful to Esmeralda Meneses, Cândida Dias and to Funchal Ecological Park for the supply of plant material. E. J. Llorent-Martínez acknowledges the financial support from the UCLM Research Plan. CICT from University of Jaén is also acknowledged.info:eu-repo/semantics/publishedVersio

Antioxidant polyphenols of Madeira sorrel (Rumex maderensis): How do they survive to in vitro simulated gastrointestinal digestion?

In this work, we report the phytochemical profile and antioxidant activity of different morphological parts of Rumex maderensis Lowe (Polygonaceae), a wild leafy-vegetable growing in Madeira Island (Portugal). Methanol extracts from leaves, flowers, and stems were submitted to high-performance liquid chromatography with mass spectrometry detection to obtain the phytochemical profile, which allowed the identification of 86 polyphenols (about 70% C- and O-flavonoids) and 9 non-phenolic compounds. In vitro antioxidant activities were measured against ABTS, DPPH, nitric oxide and superoxide free radicals. Then, the samples were subjected to an in vitro digestion, observing a decrease of about 50% in both the content of phenolics and the antioxidant activity. However, relevant antioxidant capacity was still observed after the simulated digestion. Therefore, this study supports the consumption of R. maderensis as an interesting foodstuff and a dietary source of antioxidant phytochemicals that survive the gastrointestinal digestion process.V. Spínola acknowledges Fundação para a Ciência e a Tecnologia (FCT, Portugal) for a Ph.D. grant SFRH/BD/84672/2012. This research was supported by FCT with funds from the Portuguese Government (Project PEst-OE/QUI/UI0674/2013) and the Portuguese National Mass Spectrometry Network (Contract RNEMREDE/1508/REM/2005). Funding through the project M1420-01-0145-FEDER-000005 - Centro de Química da Madeira - CQM+ (Madeira 14-20) is also acknowledged.info:eu-repo/semantics/publishedVersio