Leda R. Castilho

Virus-like particles in vaccine development

Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome, potentially yielding safer and cheaper vaccine candidates. A handful of prophylactic VLP-based vaccines is currently commercialized worldwide: GlaxoSmithKline’s Engerix® (hepatitis B virus) and Cervarix® (human papillomavirus), and Merck and Co., Inc.’s Recombivax HB® (hepatitis B virus) and Gardasil® (human papillomavirus) are some examples. Other VLP-based vaccine candidates are in clinical trials or undergoing preclinical evaluation, such as, influenza virus, parvovirus, Norwalk and various chimeric VLPs. Many others are still restricted to small-scale fundamental research, despite their success in preclinical tests. This article focuses on the essential role of VLP technology in new-generation vaccines against prevalent and emergent diseases. The implications of large-scale VLP production are discussed in the context of process control, monitorization and optimization. The main up- and down-stream technical challenges are identified and discussed accordingly. Successful VLP-based vaccine blockbusters are briefly presented concomitantly with the latest results from clinical trials and the recent developments in chimeric VLP-based technology for either therapeutic or prophylactic vaccination.

Lipase production by Penicillium restrictum in solid-state fermentation using babassu oil cake as substrate

Abstract Growth and enzyme production in SSF by a Brazilian strain of Penicillium restrictum was studied. Solid waste from the babassu oil industry was used as the basic nutrient source and was supplemented with peptone, olive oil or starch at different C/N ratios. The highest lipase activity (30.3 U/g initial dry weight) was achieved after 24 h of cultivation with 2% olive oil enrichment. Lipase activity was very sensitive to the kind and the level of supplementation, and decreased as protease level and pH in the media increased. Maximal levels of glucoamylase and protease were obtained with 4% starch enrichment, indicating that the type of carbon source supplemented to the basal medium determines the major enzymes produced.

Production and extraction of pectinases obtained by solid state fermentation of agroindustrial residues with Aspergillus niger

In this work soy and wheat bran were employed as raw materials for the production of pectinases by Aspergillus niger through solid-state fermentation. Several fermentation and recovery parameters were studied. The kinetics of enzyme synthesis was investigated in the range from 13 to 96 h with moisture contents varying from 25% to 70% (w/w). A medium moisture content of 40% and a fermentation time of 22 h were selected, as these conditions resulted in high pectolytic activity and enhanced polygalacturonase productivity. In order to optimise the recovery step, the best combination of temperature of extraction, contact time and solvent type was investigated. Acetate buffer (pH 4.4), 35°C and 30 min provided the best recovery. The present results show that optimising the extraction conditions is a simple way of obtaining more concentrated enzyme extracts and could be a useful instrument to extract more selectively a desired biomolecule from fermented solids.

Economic analysis of lipase production by Penicillium restrictum in solid-state and submerged fermentations

In the present work an economic analysis of the production of Penicillium restrictum lipase in both submerged (SF) and solid state fermentations (SSF) was performed. For a production scale of 100 m 3 lipase concentrate per year, total capital investment needed for the submerged process was 78% higher than that needed for the solid-state fermentation process. The submerged process proved to be economically unfeasible, as unitary product cost was 68% higher than the product selling price. Contrastingly, the solid-state fermentation process turned out to be very attractive from an economic point of view. Also for a scale of 100 m 3 /year, SSF unitary product cost was 47% lower than the selling price, payback time was 1.5 years, return on investment was 68% and internal return rate was 62% for a 5-year-project life. Furthermore, the profitability of this process remained high even with eventual increases of 40% in product concentration or total capital investment, or decreases of 20% in product price. The great advantage of the SSF process is the extremely cheap raw material it uses as main substrate. ©2000 Elsevier Science S.A. All rights reserved.

Production of polyhydroxyalkanoates (PHAs) from waste materials and by-products by submerged and solid-state fermentation.

Polyhydroxyalkanoates are biodegradable polymers produced by prokaryotic organisms from renewable resources. The production of PHAs by submerged fermentation processes has been intensively studied over the last 30 years. In recent years, alternative strategies have been proposed, such as the use of solid-state fermentation or the production of PHAs in transgenic plants. This paper gives an overview of submerged and solid-state fermentation processes used to produce PHAs from waste materials and by-products. The use of these low-cost raw materials has the potential to reduce PHA production costs, because the raw material costs contribute a significant part of production costs in traditional PHA production processes.

Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system

Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder- and Matrigel-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.

Production of an acidic and thermostable lipase of the mesophilic fungus Penicillium simplicissimum by solid-state fermentation.

The production of a lipase by a wild-type Brazilian strain of Penicillium simplicissimum in solid-state fermentation of babassu cake, an abundant residue of the oil industry, was studied. The enzyme production reached about 90 U/g in 72 h, with a specific activity of 4.5 U/mg of total proteins. The crude lipase showed high activities at 35-60 degrees C and pH 4.0-6.0, with a maximum activity at 50 degrees C and pH 4.0-5.0. Enzyme stability was enhanced at pH 5.0 and 6.0, with a maximum half-life of 5.02 h at 50 degrees C and pH 5.0. Thus, this lipase shows a thermophilic and thermostable behavior, what is not common among lipases from mesophilic filamentous fungi. The crude enzyme catalysed the hydrolysis of triglycerides and p-nitrophenyl esters (C4:0-C18:0), preferably acting on substrates with medium-chain fatty acids. This non-purified lipase in addition to interesting properties showed a reduced production cost making feasible its applicability in many fields.

Cell retention devices for suspended-cell perfusion cultures.

Perfusion cultures of animal cells have several advantages over batch or fed-batch cultures. They give, for instance, higher productivities and a consistent product quality, and allow steady state operation and better cell physiology control. However, one of the main aspects limiting performance and scale-up of perfusion processes is the need for an adequate cell retention device. The devices currently in use for stirred perfusion bioreactors are continuous centrifuges, tangential flow membrane filters, dynamic filters, spin-filters, ultrasonic and dielectrophoretic separators, gravity settlers and, more recently, hydrocyclones. The advantages and disadvantages of each of these methods will be discussed.

Recovery of pectolytic enzymes produced by solid state culture of Aspergillus niger

Abstract Pectinases are enzymes with a wide range of applications in the food and drink industries. In the present work, the extraction of pectinases produced by Aspergillus niger in a solid state fermentation system was investigated. The purpose was to reduce enzyme losses in the fermented solids and at the same time obtain a crude extract as concentrated as possible. Initially the performances of stirred tank and fixed bed extractors were compared. Polygalacturonase activity and viscosity reducing capacity obtained in the stirred tank system were 105% and 15% superior, respectively. Repeated extractions and multiple stage countercurrent extraction were studied, employing stirred tanks. It was possible to observe that three stages were enough for total recovery of the enzymes contained in the solids. The final enzyme extract obtained by counter-current extraction with three stages showed a polygalacturonase activity 81% higher than the one obtained by one-stage extraction.

Lipase production by solid-state fermentation: cultivation conditions and operation of tray and packed-bed bioreactors.

The production of lipase by Penicillium simplicissimum in solid-state fermentation was studied using babassu cake as the basal medium. Tray-type and packed-bed bioreactors were employed. In the former, the influence of temperature; content of the medium, and medium supplementation with olive oil, sugarcane molasses, corn steep liquor, and yeast hydrolysate was studied. For all combinations of supplements, a temperature of 30°C, a moisture content of 70%, and a concentration of carbon source of 6.25% (m/m, dry basis) provided optimum conditions for lipase production. When used as single supplements olive oil and molasses also were able to provide high lipase activities (20 U/g). Using packed-bed bioreactors and molasses-supplemented medium, optimum conditions for enzyme production were air superficial velocities above 55 cm/min and temperatures below 28°C. The lower temperature optimum found for these reactors is probably related to radial heat gradient formation inside the packed bed. Maximum lipase activities obtained in these bioreactors (26.4 U/g) were 30% higher than in tray-type reactors.