Marília Mateus

Microporous Hollow Fibres for Carbon Dioxide Absorption: Mass Transfer Model Fitting and the Supplying of Carbon Dioxide to Microalgal Cultures

A method of supplying to photosynthetic algal cultures was devel- CO 2 oped based on mass transfer measurements of through microporous hydro- CO 2 phobic hollow -bres for various gas and liquid Now rates. A mathematical model was derived to describe the mass transfer. The designed hollow -bre module led to overall mass transfer coefficient values ranging from 1E26 ) 10~3 to 2E64 ) 10~3 cm s~1. Higher efficiencies of the transmission were obtained CO 2 at high liquid Now rates and low gas Now rates. The use of microporous hydro- phobic hollow -bres enabled an enhancement of the carbon dioxide transfer per area of membrane surface by a factor of 10, in comparison to operation with silicone tubing. The hollow -bre module was operated in an external bypass to a 1d m 3microalgae culture vessel. In this system the algal growth pattern was similar to that obtained with a control culture where was bubbled. CO 2 However, the dissolved oxygen concentration was always lower in the vessel in which was supplied by the module. 1998 SCI. CO 2 ( J. Chem. T echnol. Biotechnol. 71 ,6 1 E70 (1998)

Hydrophobic interaction membrane chromatography for plasmid DNA purification: Design and optimization

Chromatography is one of the key operations in the downstream processing of plasmid DNA (pDNA). However, the increased demand for highly purified pDNA experienced in recent years has made clear the need for alternative processes capable of retaining the advantages of conventional chromatography, such as selectivity, while providing increased throughput at a lower cost. The work presented in this article outlines the development and optimization of an alternative hydrophobic interaction membrane chromatography process for the purification of pDNA. The studies included the modification of functionalized membrane supports with a linear alkyl chain ligand and the testing of chromatographic performance of these membranes. Three modification procedures were tested and the membranes were screened for their capacity and selectivity. The modified membranes could separate the model plasmid pVAX1-LacZ (6050 bp) from impurities in clarified Escherichia coli cell lysates (specifically RNA), with good resolution. Subsequent optimization of elution profiles with the best-performing modified membrane, resulted in a high purification factor of 4.7, competitive with its bead process counterpart, and a plasmid yield of 73%.

Horseradish peroxidase immobilized through its carboxylic groups onto a polyacrylonitrile membrane: comparison of enzyme performances with inorganic beaded supports.

A hydrophilic polyacrylonitrile (PAN) flat sheet membrane was aminated (8.5 μmol of NH2/mg of dry support) for covalent binding of horseradish peroxidase (HRP), mediated by the soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Silica microbeads derivatized by silanization, to yield an aminated support, and commercial aminated glass microbeads were also coupled to HRP with EDC or activated with glutaraldehyde. The immobilized enzyme activities were determined in a batch enzyme reactor with an external loop, the highest specific immobilized HRP activity being obtained on the glass support (55.8U/mg of protein). Continuous operational stability studies showed that hydrophilic PAN membrane led to the highest retention of HRP activity after an overall period of 35 h, with a normalized productivity of 59.5 μmol of H2O2 reduced/(h·Uimmob HRP).

Development of a polysulfone hollow fiber vascular bio-artificial pancreas device for in vitro studies.

A system for vascular hollow fiber bio-artificial pancreas development, optimization and in vitro testing was implemented and operated in a simple and fully described manner, allowing other researchers to test a variety of experimental conditions (different biomaterials, biologic tissue, addition of proteins or other adjuvants). In this work, a polysulfone hollow fiber was used as bioprotective material. Two different cell sources were co-immobilized with agarose microspheres in and experimented with the membrane device: rat islets of Langerhans and mouse beta-TC-3 insulinoma cells. The results obtained with islets of Langerhans were used as islet comparable insulin-release data. Beta-TC-3 cells were mainly used in these studies, due to higher control and reproducibility of cell number and behavior: addition of hemoglobin was beneficial for sustained cell viability, especially during cell insertion in the device (viability assessed by beta-TC-3 lactate dehydrogenase activity in the recirculating culture medium); cells did not adhere to the polysulfone membrane (assessed by SEM observation of membrane samples from dynamic cultures). Comparable device functionality and insulin-release results were attained with both cell types: device functionality was maintained for 7-9 days and maximum insulin-release during dynamic glucose challenges were 2.6 x 10(-3)+/-7 x 10(-5)microU/beta-cell x 8 h, with islets, and 9.3 x 10(-4)+/-2 x 10(-5)microU/beta-cell x 2 h, with beta-TC-3 cells.

Microfiltration of cutinase and Escherichia coli cell fragment suspensions: The role of the electrolyte environment on the development of dynamic cake layers

Abstract Stirred cell microfiltration of industrial relevant enzyme solutions, with and without cell debris, was studied. The biological system used was a recombinant intracellular cutinase in suspensions of Escherichia coli fragments. In NaCl solutions, cutinase forms large soluble protein aggregates that vary in size with the electrostatic microenvironment. Filtration shear forces applied on these aggregates break the interaction among monomers, allowing 100% transmission of cutinase to permeate. Pure enzyme solutions showed permeation flux reduction (10–34% of simple electrolyte filtration runs) caused by adsorption and aggregated protein molecules, leading to membrane fouling by formation of dynamic cake layers. Cell homogenates presented severe decrease on permeation flux (range of 93–97%), leading to similar flux values, still dependent on the colloidal microenvironment. The presence of cutinase led to cell debris concentration polarisation layers less resistant to flow. The experimental filtration results were evaluated in accordance to the aggregation behaviour of cutinase, average size of cell debris and zeta potential of the colloidal particles and of the membrane.

Development of a phenyl membrane chromatography-based process yielding pharmaceutical grade plasmid deoxyribonucleic acid for mammalian cells transfection

Production of plasmid DNA pharmaceuticals requires fast, robust and cost effective methodologies able to deliver high amounts of the target molecule in short periods of time. Membrane adsorbers can be tailored and operated to suit such criteria. This study focuses on the impact of pDNA samples produced by a membrane chromatography-based purification methodology on the transfection efficiency of CHO cells. Chromatographies were performed with 5mL of plasmid-containing clarified bacterial lysate each on a Sartorius® Phenyl 3mL spiral cartridge using a bind and elute mode to purify the GFP expressing pVAX1/GFP model plasmid. The developed methodology could deliver up to 285μg pDNA samples per run that were virtually RNA free (over 99% removal) and chromatographic step yields of 85%. The purified samples had a reduced content of OC pDNA (∼15% less in average). Additionally, robustness of the process was assessed up to nine chromatographic runs without noticing any relevant loss in chromatographic performance and transfection capabilities. The increase of productivity was also studied by increasing the flow rate 5 fold-a maximum productivity of 100μg pDNA/(hmL-BV) was achieved. The pDNA samples produced led to transfection efficiencies that were comparable among all experiments-72% and within 4% relative standard deviation when samples were produced using a lower throughput. Transfection efficiencies obtained by the membrane process were comparable to a combined HIC/SEC bead-based purification process, with values ranging within 74-113% of the values obtained from the latter.

Impact of plasmid size on the purification of model plasmid DNA vaccines by phenyl membrane adsorbers

Plasmid DNA (pDNA) offers a versatile platform for the development of new pharmaceuticals. This versatility also adds in variability among plasmid products most of the times sharing only the same basic molecular structure. Membrane chromatography experiments performed with a Sartorius(®) Phenyl 3 mL spiral cartridge and differently sized plasmids (3.70 kbp, 6.05 kbp and 10.4 kbp) show that the strength of interaction of pDNA isoforms with HIC membrane adsorbers depends on size. These differences in relative binding strength were explored using a stepwise elution strategy of decreasing buffer conductivities in order to increase the purity of supercoiled (SC) pDNA isoforms. The open circular (OC) isoforms of all plasmids eluted earlier at a similar conductivity of 190 mS/cm, independently of the hydrodynamic diameter (Dh). A drop in conductivity of 16.0 mS/cm, 23 mS/cm and 19 mS/cm had to be imposed to elute the supercoiled (SC) counterparts of the 3.70 kbp, 6.05 kbp and 10.4 kbp, respectively. This corresponds to relative binding strengths of the SC over OC isoforms of 1.09, 1.14 and 1.11. Unlike the OC isoforms, the behavior of SC isoforms was dependent of the Dh. The purified and pooled plasmid fractions were assayed and demonstrated high degree of purity, compliant with regulatory agencies criteria: over 99% RNA removal, endotoxin levels below 0.001 EU/μg pDNA and undetectable protein content by BCA assay.

TiO2:(Fe, S) Thin Films Prepared from Complex Precursors by CVD, Physical Chemical Properties, and Photocatalysis

The TiO 2 thin films were prepared using Ti(dpm) 2( O P r i ) 2 and Ti( O P r i ) 4 (dpm = 2,2,6,6-tetramethylheptane-3,5-dione, P r i = isopropyl) as the precursors. The volatile compounds Fe[(C 2H 5) 2NCS 2] 3 and [(CH 3)C] 2S 2 were used to prepare doped TiO 2 films. The synthesis was done in vacuum or in the presence of Ar and O 2. The pressure in the CVD chamber was varied between 1 . 2 × 1 0 − 4 mbar and 0.1 mbar, with the system working either in the molecular beam or gas flow regime. Physical, chemical, and photocatalytic properties of the (Fe, S)-doped TiO 2 films were studied. Those TiO 2:(Fe, S) films prepared from the Ti( O P r i ) 4 precursor show increased photocatalytic activities, very close to those of Degussa P25 powder in UV region.

Steroid recovery by a rotary membrane system

A rotary membrane system was used in the downstream processing of Arthrobacter simplex fermentation media. Concentration and diafiltration were performed for cell harvesting and washing and for product (6α-methylprednisolone) recovery, after a biotransformation step. This system was compared with other conventional modules for ultrafiltration.

Design and characterization of plasmids encoding antigenic peptides of Aha1 from Aeromonas hydrophila as prospective fish vaccines

The current investigation aimed at designing DNA vaccines against Aeromonas hydrophila infections. The DNA vaccine candidates were designed to express two antigenic outer membrane protein (Aha1) peptides and to be delivered by a nanoparticle-based delivery system. Gene sequences of conserved regions of antigenic Aha1 [aha1(211-381), aha1(211-381)opt, aha1(703-999) and aha1(703-999)opt] were cloned into pVAX-GFP expression vector. The selected DNA vaccine candidates were purified from E. coli DH5α and transfected into Chinese hamster ovary cells. The expression of the antigenic peptides was measured in cells along post-transfection time, through the fluorescence intensity of the reporter GFP. The lipofection efficiency of aha-pVAX-GFP was highest after 24h incubation. Formulated PLGA-chitosan nanoparticle/plasmid DNA complexes were characterized in terms of size, size distribution and zeta potential. Nanocomplexes with average diameters in the range of 150-170nm transfected in a similar fashion into CHO cells confirmed transfection efficiency comparable to that of lipofection. DNA entrapment and further DNase digestion assays demonstrated ability for pDNA protection by the nanoparticles against enzymatic digestion.