Design of a periodic counter-current chromatography process for efficient oncolytic virus purification

Virus-based biologicals are one of the most promising biopharmaceuticals of the 21st century medicine and play a significant role in the development of innovative therapeutic, prophylactic and clinical applications. These biologicals share between them a high degree of complexity and offer various challenges requiring innovative technologies for their manufacturing. Oncolytic virus manufacturing scale can range from 5L in research and development up to 50L for clinical studies and reach hundreds of liters for commercial scale. The inehrent productivity and high integration potential of periodic counter-current chromatography offers a transversal solution to decrease equipment footprint and the reduction of several non-value-added unit operations. The work to be reported focus on the design of a periodic counter-current chromatography process applied to the intermediate purification of oncolytic adenovirus. Moving away from single-column batch operation towards continuous or semi-continuous, multi-column chromatography creates the opportunity to benefit from synergies of solvent gradients, recycling chromatography, and simulated counter-current movement of the adsorbent and fluid phases, providing substantial reductions in chromatographic resin volume and buffer consumption. The developed ion exchange chromatographic purification method was carried out using a four-column setup, supported by mechanistic mathematical modeling. Obtained virus recoveries (\u3e 60%) and impurity reductions (\u3e 80% DNA, and \u3e 70% total protein) match or overcome batch purification. The impact of column cycling on column capacity will be presented and the steps taken to minimize it will be discussed, highlighting the optimization of the cleaning-in-place step and the need to include organic solvents to promote the stripping of tighter-adsorbing impurities. Moreover, the robustness of the dynamic control strategy and its ability to overcome perturbations originated in precedent stages will be demonstrated using feeds with different impurity profiles and titers, showing that it is possible to generate elution pools with consistent quality and traceability. Additionally, due to the wealth of data generated through the cycling operations, such as historic columns breakthrough and elution peak profiles, a deeper insight on product quality and process knowledge is gained. Moreover, process automation enables the minimization of errors, maximizing process efficiency, uptime, repeatability, and process replication

Allometry of the Duration of Flight Feather Molt in Birds

Replacement of flight feathers takes disproportionately more time for large birds than it does for small birds, because feather length increases with body size almost twice as fast as feather growth rate increases

Alkaline hydrolysis combined with stir-bar sorptive extraction or sonication for the determination of banned azo dyes in leather

Two approaches based on alkaline hydrolysis, in combination with either stir-bar sorptive extraction (SBSE) or sonication, were developed for the determination of banned azo dyes in bovine leather. All dyes were determined indirectly by measuring their corresponding harmful amines, formed after chemical reduction. Quantification was performed by employing standard addition methodology to compensate for analyte losses due to sample preparation (e.g. incomplete extraction). The mean recoveries (n=3) obtained by employing the SBSE and sonication procedures were 100 percent (RSD=26 percent) and 114 percent (RSD=11 percent), respectively. The linearity of the standard addition curves obtained was reasonable [SBSE (mean R-2=0.980) and sonication (mean R-2=0.997)]. Both procedures were very simple to perform and provided clean LC-UV chromatograms. Taking into account the repeatability, sonication was considered the preferred alternative

Membrane techniques for analysis, sampling and speciation in environmental measurements

Abstract is not availabl

Data from: The genetics of extreme microgeographic adaptation: an integrated approach identifies a major locus underlying leaf trichome divergence in Yellowstone Mimulus guttatus

Microgeographic adaptation provides a particularly interesting context for understanding the genetic basis of phenotypic divergence, and may also present unique empirical challenges. In particular, plant adaptation to extreme soil mosaics may generate barriers to gene flow or shifts in mating system that confound simple genomic scans for adaptive loci. Here, we combine three approaches - QTL mapping of candidate intervals in controlled crosses, population resequencing (PoolSeq), and analyses of wild recombinant individuals - to investigate one trait associated with Mimulus guttatus (yellow monkeyflower) adaptation to geothermal soils in Yellowstone National Park. We mapped a major QTL causing dense leaf trichomes in thermally-adapted plants to a < 50kb region of Linkage Group 14 (Tr14) previously implicated in trichome divergence between independent M. guttatus populations. A PoolSeq scan of Tr14 region revealed a cluster of six genes, co-incident with the inferred QTL peak, with high allele frequency differences sufficient to explain observed phenotypic differentiation. One of these, the R2R3 MYB transcription factor Migut.N02661, is a plausible functional candidate, and was also strongly-associated (r2 = 0.27) with trichome phenotype in analyses of wild-collected admixed individuals. Although functional analyses will be necessary to definitively link molecular variants in Tr14 with trichome divergence, our analyses are a major step in that direction. They point to a simple, and parallel, genetic basis for one axis of Mimulus guttatus adaptation to an extreme habitat, suggest a broadly conserved genetic basis for trichome variation across flowering plants, and pave the way for further investigations of this challenging case of microgeographic incipient speciation

Development of methods for the determination of vitamins A, E and beta-carotene in processed foods based on supercritical fluid extraction: a collaborative study.

New methodologies based on supercritical fluid extraction (SFE) have been developed for the determination of fat-soluble vitamins in processed foods. The results obtained so far indicate that SFE is well suited to extraction of fat-soluble vitamins from food products, although validation work is required to establish accuracy and precision. The vitamins investigated were A, E and beta-carotene, and the processed foods were UHT milk, milk powder, minced meat, liver paste, infant formula, canned baby food and margarine. Extraction equipment employed analyte collection on either a solid-phase trap or in a solvent. After extraction, the samples were saponified and the vitamins determined using reversed-phase liquid chromatography with ultraviolet or fluorescence detection. Sample throughput was at least 12 samples day(-1), i.e. at least twice the number achievable with a conventional extraction methodology. The detection limits for the vitamins in different processed foods were well below 0.1 microg g(-1). Recoveries (in comparison with vitamin levels obtained using conventional solvent extraction) were close to 100% for experienced personal with access to modern automatic equipment. To reach this level, it was necessary to protect the vitamins with an antioxidant during the different steps of the analysis procedure, to add methanol or ethanol to the extraction cell to facilitate the analyte extraction from the food matrix, and when using a solid-phase trap, to employ a fractionated extraction-elution procedure to prevent breakthrough losses. The developed methods were tested in a validation exercise between five laboratories, which had taken part in the method development, and in an intercomparison between 10 laboratories including laboratories with less experience of vitamin determination. The within-laboratory RSD was generally </= 11%. The average of the between-laboratory relative standard deviation (RSD) was about 23% in the validation, and increased to about 40% in the intercomparison. Ruggedness tests performed at different steps of the project showed that different types and models of equipment did not give large differences in recoveries. Thus, the increasing RSD can largely be ascribed to differences in experience in vitamin analysis of the participants

Data for MEC-16-0039R1

The excel file contains 4 sheets with datasets from this paper.This file contains datasets analyzed in Hendrick et al. 2016 paper on trichome variation in Yellowstone Mimulus guttatus 1. SdtrichQTL - phenotypes and marker genotypes from an outbred F2 mapping population (AHQT x AHQNT), grown under short daylengths and cool temperature conditions 2. AHQF2_2015 - pehnotypes and genotypes from an inbred line F2 (AHQT x AHQNT) grown under long daylength conditions in greenhouse 3. AHQgradient_markerpopgen - STRUCTURE input file for AHQT (plots 1-6), AHQNT (plots 14-17), AND AHQ_Canyon (plots 7-13) populations, marker information is in Lekberg et al. 2012 4. AHQG15-trichadmixture - marker genotypes and phenotypes for wild Tr14-recombinant AHQNT AND AHQ_Canyon individuals grown under common garden condition