Scenedesmus rubescens Heterotrophic Production Strategies for Added Value Biomass.

Microalgae attract interest worldwide due to their potential for several applications. Scenedesmus is one of the first in vitro cultured algae due to their rapid growth and handling easiness. Within this genus, cells exhibit a highly resistant wall and propagate both auto- and heterotrophically. The main goal of the present work is to find scalable ways to produce a highly concentrated biomass of Scenedesmus rubescens in heterotrophic conditions. Scenedesmus rubescens growth was improved at the lab-scale by 3.2-fold (from 4.1 to 13 g/L of dry weight) through medium optimization by response surface methodology. Afterwards, scale-up was evaluated in 7 L stirred-tank reactor under fed-batch operation. Then, the optimized medium resulted in an overall productivity of 8.63 g/L/day and a maximum biomass concentration of 69.5 g/L. S. rubescens protein content achieved approximately 31% of dry weight, similar to the protein content of Chlorella vulgaris in heterotrophy.

Membrane Bioreactor for Simultaneous Synthesis and Fractionation of Oligosaccharides.

Galacto-oligosaccharides (GOS) are prebiotic sugars obtained enzymatically from lactose and used in food industry due to their nutritional advantages or technological properties. Selective mass transport and enzymatic synthesis were integrated and followed using a membrane bioreactor, so that selective removal of reaction products may lead to increased conversions of product-inhibited or thermodynamically unfavorable reactions. GOS syntheses were conducted on lactose solutions (150 g·L-1) at 40 °C and 10 Uß-galactosidase.mL-1, and sugar fractionation was performed by cellulose acetate membranes. Effects of pressure (20; 24 bar) and crossflow velocity (1.7; 2.0; 2.4 m·s-1) on bioreactor performance were studied. Simultaneous GOS synthesis and production fractionation increased GOS production by 60%, in comparison to the same reactions promoted without permeation. The presence of a high-molecular-weight solute, the enzyme, in association with high total sugar concentration, leads to complex selective mass transfer characteristics. Without the enzyme, the membrane presented tight ultrafiltration characteristics, permeating mono- and disaccharides and retaining just 25% of trisaccharides. During simultaneous synthesis and fractionation, GOS-3 were totally retained, and GOS-2 and monosaccharides were retained at 80% and 40%, respectively. GOS synthesis-hydrolysis evolution was strongly dependent on crossflow velocity at 20 bar but became fairly independent at 24 bar.

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%.

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.

Monoclonal Antibodies Production Platforms: An Opportunity Study of a Non-Protein-A Chromatographic Platform Based on Process Economics.

Monoclonal antibodies currently dominate the biopharmaceutical market with growing sales having reached 80 billion USD in 2016. As most top-selling mAbs are approaching the end of their patent life, biopharmaceutical companies compete fiercely in the biosimilars market. These two factors present a strong motivation for alternative process strategies and process optimization. In this work a novel purification strategy for monoclonal antibodies comprising phenylboronic acid multimodal chromatography for capture followed by polishing by ion-exchange monolithic chromatography and packed bed hydrophobic interaction chromatography is presented and compared to the traditional protein-A-based process. Although the capital investment is similar for both processes, the operation cost is 20% lower for the novel strategy. This study shows that the new process is worthwhile investing in and could present a viable alternative to the platform process used by most industrial players.

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.

An overview on the development of a bio-artificial pancreas as a treatment of insulin-dependent diabetes mellitus.

This paper presents the concept and most of the research undertaken all over the world for the development of a bio-artificial pancreas (BAP) device over the last 30 years. The devices studied, meant to mimic the insulin secretion of the natural organ, were diverse and have been reviewed. Allogeneic or xenogeneic cells or cell clusters have been separated from the host's immune system by synthetic biocompatible semipermeable membranes to prevent the need, of the host, for immune-suppressing regimens. The biocompatible polymer used as a barrier and its intrinsic characteristics, the cell immobilization or suspension media, the existence or not of co-immobilized molecules or cells, the number of devices used and the implantation site, were addressed.