Rapid and inexpensive purification of adenovirus vectors using an optimised aqueous two-phase technology.

Adenoviruses (AdVs) are used as gene therapy vectors to treat human diseases and as vaccines against COVID-19. AdVs are produced by transfecting human embryonic kidney 239 (HEK293) or PER.C6 virus producer cells with AdV plasmid vectors or infecting these cells withcell lysates containing replication-defective AdV. Cell lysates can be purified further by caesium chloride or chromatographic protocols to research virus seed stocks (RVSS) for characterisation to high quality master virus seed stocks (MVSS) and working virus seed stocks (WVSS) before downstream production of pure, high titre AdV. Lysates are poorly infectious, block filtration columns and have limited storage capability. Aqueous two-phase systems (ATPS) are an alternative method for AdV purification that rapidly generates cleaner RVSS for characterisation to MVSS. After testing multiple ATPS formulations, an aqueous mixture of 20 % PEG 600 and 20 % (NH4)2SO4 (w/w) was found most effective for AdV partitioning, producing up to 97+3% yield of high-titre virus that was devoid of aggregates both effective in vitro and in vivo with no observable cytotoxicity. Importantly, AdV preparations stored at -20 °C or 4 °C show negligible loss of titre and are suitable for downstream processing to clinical grade to support the need for AdV vaccines.

Practical aspects of the automated preparation of aqueous two phase systems for the analysis of biological macromolecules.

A robust strategy for the automated preparation of aqueous two-phase systems (ATPS) using a liquid handling sample processor was developed using gravimetric methods: to determine the accuracy of preparation. The major robotic control parameters requiring adjustment were; speed of aspiration and dispense; delay times following aspiration and dispense alongside measures to control cross-contamination during phase sampling. In general mixture compositions of both polymer/polymer and polymer/salt mixtures could be prepared with a target bias accuracy of less than 5%. However, we found that the bias accuracy with which systems of defined TLL and MR could be constructed was highly dependent on the tie line length of the ATPS and the geometrical form of the ATPS co-existence curve. For systems with a very low degree of curvature (PEG/salt systems here) increases in bias (accuracy) are appreciable at relatively long tie line lengths. Where the degree of curvature is more pronounced (PEG/dextran systems) closer approach to the critical point was possible without major effect on bias/accuracy. Application of the strategy to the measurement of the partitioning of phosphorylated and dephosphorylated forms of the model protein ovalbumin are reported. Differences in partition of phosphorylated (native) forms and dephosphorylated forms could be demonstrated. In a PEG/salt system this was manifest as a substantial decrease in solubility based on overall protein recovery derived from accurate knowledge of the system mass ratio. In a PEG/dextran system differences in partition coefficient could be demonstrated between phosphorylated and dephosphorylated forms.

The opposite effect of temperature on polyethylene glycol-based aqueous biphasic systems versus aqueous biphasic extraction chromatographic resins.

Variation in operational temperatures has revealed differences in the partitioning behavior of probe solutes between the phases in aqueous biphasic systems (ABS) and the related aqueous biphasic extraction chromatographic resin (ABEC). This difference has been studied using the hydrophobic anion, 99TcO4-, as a probe and (NH4)2SO4 as the kosmotropic salt. Distribution of the hydrophobic anion 99TcO4- to the PEG-rich phase in a MePEG-5000/(NH4)2SO4 ABS increases with increasing temperature, but decreases are observed in batch uptakes of this anion to ABEC resins from (NH4)2SO4 solutions. Phase diagrams were constructed at five different temperatures from 10 to 50 degreesC using cloud point titration for the ABS and a correlation between the phase divergence, measured in terms of tie line length (TLL), and the temperature of the partitioning system was verified. Thermodynamic parameters (deltaHdegrees,deltaSdegrees, deltaGdegrees, ) as a function of temperature were calculated for the various systems studied and the results imply thermodynamic differences between partitioning in ABS versus ABEC.

Application of polyethylene glycol-based aqueous biphasic reactive extraction to the catalytic oxidation of cyclic olefins.

Glutaric acid and 1,2,3,4-butanetetracarboxylic acid (BTCA) have been synthesized by sodium tungstate catalyzed oxidation of the cyclic olefins: cyclopentene and 1,2,3,6-tetrahydrophthalic anhydride (THPA), using hydrogen peroxide in a polyethylene glycol (PEG)-2000/NaHSO(4) aqueous biphasic system (PEG-ABS). The production of glutaric acid and BTCA was found to increase from the monophasic to the biphasic regimes, and was found to be greatest at short tie-line lengths (TLLs), close to the system's critical point, yielding glutaric acid and BTCA in 73.1 and 82.5% yield, respectively. The results imply that mutual mixing or contact of the components is important, because the product dicarboxylic acids were found to increase from the monophasic side to the critical point and decrease from the critical point to more divergent regimes. The two reactions were compared with adipic acid synthesis from cyclohexene in terms of the cyclic olefin structure, and the partitioning of the dicarboxylic acid product in the ABS.

Theoretical scales of hydrogen bond acidity and basicity for application in QSAR/QSPR studies and drug design. Partitioning of aliphatic compounds.

Phenomenological analysis of existing hydrogen bond (HB) donor and acceptor scales and apparent physical considerations have enabled the establishment of new quantitative scales of hydrogen bond basicity and acidity. Chemical structures represented by molecular graphs and the orbital electronegativities of Hinze and Jaffe are utilized as an input data. The scales obtained correlate well with several experimental solvent polarity scales such as and, pK(HB), and E(T)(30). To demonstrate the applicability of the new quantities, we have applied them to seven equilibrium partitioning data sets: octanol-water, hexadecane-water, chloroform-water, gas-water, gas-octanol, gas-hexadecane, and gas-chloroform partition coefficients. The hydrogen bond descriptors when supplemented by a cavity-forming term and a dipolarity term show high performance in correlations of the partition coefficients of aliphatic compounds. These new HB descriptors can be used in studying hydrogen bonding and fluid phase equilibria as well as scoring functions in ligand docking and descriptors in ADME evaluations.

Comparison of an empirical and a theoretical linear solvation energy relationship applied to the characterization of solute distribution in a poly(ethylene) glycol-salt aqueous biphasic system.

A Linear Solvation Energy Relationship (LSER) developed by Abraham has been applied to the partition of organic solutes in an Aqueous Biphasic System (ABS) and to published values of their 1-octanol/water partition coefficients. The results are compared to the application of a conceptually similar Theoretical Linear Solvation Energy Relationship (TLSER) developed by Famini and co-workers. Differences and similarities in the molecular properties highlighted by each LSER are discussed. The theoretical relationship of Famini, although modeled on the approach of Abraham, seems not to encapsulate molecular properties in the same way as the Abraham's empirical relationship. However, this theoretical approach has the great merit of computational simplicity, and it is concluded that efforts directed toward its improvement could be rewarding.

Aqueous biphasic systems. Partitioning of organic molecules: a QSPR treatment.

The partitioning of 29 small organic probes in a PEG-2000/(NH4)2SO4 biphasic system was investigated using a quantitative structure-property relationship (QSPR) approach. A three-descriptor equation with the squared correlation coefficient (R2) of 0.97 for the partition coefficient (log D) was obtained. All descriptors were derived solely from the chemical structure of the compounds. Using the same descriptors, a three-parameter model was also obtained for log P (octanol/water, R2=0.89); predicted log P values were used as an external descriptor for modeling log D.

Controlling the aqueous miscibility of ionic liquids: aqueous biphasic systems of water-miscible ionic liquids and water-structuring salts for recycle, metathesis, and separations.

Hydrophilic ionic liquids can be salted-out and concentrated from aqueous solution upon addition of kosmotropic salts forming aqueous biphasic systems as illustrated by the phase behavior of mixtures of 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) and K3PO4.