Conceptual design of a cellular filter to remove trace viral contaminants in blood.

Various process alternatives and designs of using a filter containing cellular adsorbents to remove trace viral contaminants from blood and other protein solutions have been studied. Sterilization charts have been developed that can be used to estimate the filter size required to achieve a desired sterilization criterion. A parametric study was carried out to identify various process parameters that may affect this physical trace removal process. It has been demonstrated that the adsorption rate constant is a critical parameter in the design of an efficient cellular filter for viral contaminant removal. This constant is characteristic of the virus-cell system under consideration and is shown to be particularly sensitive to the cell surface receptor density, adsorbent diameter, and fluid flow rate. Higher log titer reduction in virus concentrations can be achieved with low flow rates and no recycle. Preliminary analyses indicate the feasibility of using a magnetically stabilized fluidized filter (MSFF) reactor design for effective virus removal from these complex solutions.     

Recombinant antibody production by perfusion cultures of rCHO cells in a depth filter perfusion system

Recombinant Chinese hamster ovary cells, producing recombinant antibody against the human platelet, were cultivated in a depth filter perfusion system (DFPS). When perfusion cultures with working volume of 1 L were operated at perfusion rates of 5/d and 6/d, volumetric antibody productivities reached values 28 and 34 times higher than that of batch suspension culture in Erlenmeyer flasks and 43 and 53 times higher than that of batch culture in a controlled stirred tank reactor, respectively. Perfusion cultures in the DFPS showed stable antibody production over the whole culture period of up to 20 days. In the DFPS, inoculated cells in suspension were entrapped in a few hours within the depth filter matrix by medium circulation and retained there until the void space of the filter matrix was saturated by the cultured cells. After cells in the depth filter matrix reached saturation, overgrown viable cells at a perfusion rate of 5/d or 6/d were continuously collected into waste medium at a density of 2-4 x 10(5) cells/mL, which resulted in stable operation at high perfusion rates, maintaining values of process parameters such as glucose/lactate concentration, pH, and dissolved oxygen concentration. Because the DFPS overcomes most drawbacks observed with conventional perfusion systems, it is preferable to be used as a key culture system to produce monoclonal antibody stably for a long culture period.     

Growth and production of some important species of fish in a Mexican coastal lagoon system

In terms of biomass, the three dominant fish species in the Huizache-Caimanero lagoon system during 1975–76 were the mullet Mugil curema , the anchovy, Anchoa panamensis and the catfish, Galeichthys caerulescens . Peaks in the abundance of the secondary predators occurred at the end of the wet season (September-October), while pelagic forms and members of the centropomid-gerreid association (Warburton 1968a ) were most common during November-February and December-February respectively. Twelve species, including the ten having the highest overall mean biomass, were selected for growth and production studies. Lagoon-specific differences in the patterns of growth and recruitment of several species were observed. The total estimated annual fish production in Caimanero lagoon was 34·48 g m⁻² y⁻¹, with M. curema contributing 9·36 g m⁻², A. panamensis 9·24 g m⁻² and G. caerulescens 6·15 g m⁻² (maximum estimate). Mean turnover ratios (annual production: mean biomass) were 3·25 (demersal species), 8·44 (pelagic species) and 4·48 (all species). Implications for possible fishery and aquacultural expansion are discussed.     

The use of regenerable, affinity ligand-based surfaces for immunosensor applications.

The regeneration of antibody-binding surfaces is of major importance for re-usable sensor formats such as required for direct 'real-time' biosensing technologies and is often difficult to achieve. Antibodies commonly bind the antigen with high avidity and may themselves be sensitive to regeneration conditions. The interaction of polyclonal anti-chlorpyriphos antibody with an immobilised chlorpyriphos-ovalbumin (chlor-oval) conjugate and the interaction of soluble recombinant CD4 with covalently immobilised anti-CD4 IgG are presented in order to highlight these difficulties. Affinity-capture is suggested as an alternative format as it facilitates surface regeneration, directed immobilisation and the attainment of interaction progress curves that conform to the ideal pseudo-first-order kinetic interaction model. Protein A, protein G and polyclonal anti-mouse Fe-coated surfaces were used to observe the interaction of captured anti-GST monoclonal antibody with glutathione-s-transferase (GST). It was shown that a protein A affinity-capture surface produced ideal interaction progress curves while both protein G and polyclonal anti-mouse Fe resulted in systemic deviations.     

Continuous production of tissue plasminogen activator from recombinant CHO cells in a depth filter perfusion system

Summary A depth filter perfusion system (DFPS), equipped with a 40-m polypropylene depth filter for cell immobilization, was used for the continuous production of tissue plasminogen activator (t-PA) from recombinant Chinese hamster ovary cells. Final cell density in the DFPS with oxygen control was 1.8×10⁷ cells/mL of the total working volume and maximum t-PA productivity was 2.63 mg/L/day. Dissolved oxygen concentration in the filter matrix was successfully controlled by air sparging and stable operation was possible for more than 20 days.     

The design, construction and use of a radio-tracking system for some British mammals

Radio-tracking equipment designed by the late G. E. Ashwell is described. Detailed instructions are given for building transmitters suitable for foxes and rats so that the circuits can be made by people with only limited electronic knowledge. Constructional details are also provided for receiver aerials. The address of a firm that can supply receivers is given. The function and performance of each part of the equipment is described and details provided of the methods used by the authors to track mammals. The handling of mammals and attachment of transmitters is covered briefly. Some comments are included on the limitations of radio-tracking as a means of studying wild mammals.     

Treatment of volatile organic contaminants in a vertical flow filter: Relevance of different removal processes

Vertical flow filters and vertical flow constructed wetlands are established wastewater treatment systems and have also been proposed for the treatment of contaminated groundwater. This study investigates the removal processes of volatile organic compounds in a pilot-scale vertical flow filter. The filter is intermittently irrigated with contaminated groundwater containing benzene, MTBE and ammonium as the main contaminants. The system is characterized by unsaturated conditions and high contaminant removal efficiency. The aim of the present study is to evaluate the contribution of biodegradation and volatilization to the overall removal of benzene and MTBE. Tracer tests and flow rate measurements showed a highly transient flow and heterogeneous transport regime. Radon-222, naturally occurring in the treated groundwater, was used as a gas tracer and indicated a high volatilization potential. Radon-222 behavior was reproduced by numerical simulations and extrapolated for benzene and MTBE, and indicated these compounds also have a high volatilization potential. In contrast, passive sampler measurements on top of the filter detected only low benzene and MTBE concentrations. Biodegradation potential was evaluated by the analysis of catabolic genes involved in organic compound degradation and a quantitative estimation of biodegradation was derived from stable isotope fractionation analysis. Results suggest that despite the high volatilization potential, biodegradation is the predominant mass removal process in the filter system, which indicates that the volatilized fraction of the contaminants is still subject to subsequent biodegradation. In particular, the upper filter layer located between the injection tubes and the surface of the system might also contribute to biodegradation, and might play a crucial role in avoiding the emission of volatilized contaminants into the atmosphere.     

大型除菌过滤器在重组干扰素生产中的应用

为适应大规模生产的需要,用Millipore293型滤器取代常用的赛氏滤器对重组干扰素α1b及γ进行除菌过滤,此种大型滤器不仅滤膜面积大,而且单位面积过滤量也有明显增加,因此,过滤速度显著加快,可适应大规模生产的应用,用此二种滤器作除菌过滤对干扰素活性无影响,过滤制品前用2%白蛋白预滤可减少滤膜吸附干扰素作用,Millipore和赛氏滤器除菌过滤后热原质均能达到肌肉注射标准,且大部分能达到静脉注射标准,有个别制品用赛氏滤器过滤后热原质达至不到静脉注射标准者,用Millipore滤器复滤后,可以合格,上述结果表明用Milli-pore293型滤器制备重组干扰素α1b及γ是切实可行的。     

The application of kudzu as a medium for the adsorption of heavy metals from dilute aqueous wastestreams

This study assessed the use of kudzu (Pueraria lobata ohwi) as a medium for the capture of copper, cadmium, and zinc from low concentration solutions. The rate and extent of uptake was studied using a system of standardized batch adsorbers under steady-state and transient-rate conditions. All plant components were tested. Residual metals analyses were performed on an ICP-AES/OES (Optima 3000 DV). The Langmuir, Freundlich, and Redlich-Peterson isotherms were determined; the Langmuir isotherm was found to best represent the data for copper and cadmium uptake. The Redlich-Peterson best represented the data for zinc. Kudzu was determined to be an effective adsorbent for removal of heavy metals. Though its capacity for metals removal is less than commercial grade ion exchange resins, it could be used at much lower cost, and may find application in the treatment of dilute mixed-matrix metal wastestreams, such as urban runoff, where the application of resins would be expensive and subject to premature poisoning by interfering contaminants.     

An Escherichia coli plasmid vector system for production of streptavidin fusion proteins: expression and bioselective adsorption of streptavidin-beta-galactosidase

Covalently immobilized biotin was used as a biospecific adsorbant to investigate the application of streptavidin as an affinity domain for simultaneous purification and immobilization of recombinant proteins. A streptavidin-beta-galactosidase fusion protein was constructed and tested as a model system. The gene for streptavidin from Streptomyces avidinii was modified by polymerase chain reaction to mutate the stop codon and to facilitate cloning into an Escherichia coli expression vector yielding a versatile plasmid with 37 unique restriction enzyme sites at the 3' end. E. coli beta-galactosidase was cloned in-frame to the streptavidin gene. Analysis of lysates of induced recombinant E. coli cells by SDS-PAGE and Western blots indicated that the 133.6-kDa fusion protein was expressed. Sulfosuccinimidyl-6-(biotinamido) hexanoate was covalently immobilized on 3-aminopropyl-controled-pore glass beads. Exposure of recombinant cell lysates to this support indicated that streptavidin-beta-galactosidase was bioselectively adsorbed. The resulting biocatalyst contained 300 mg protein per gram of beads and exhibited a specific activity of 306 betamol/min per milligram protein with o-nitrophenyl-beta-D-galactopyranoside as substrate corresponding to approximately 50% of that observed for commercially pure E. coli beta-galactosidase. (c) 1994 John Wiley & Sons, Inc.     

Perspectives for production and application of resveratrol

The polyphenol trans-resveratrol (3,5,4′-trihydroxy-trans-stilbene) is one of the best known plant secondary metabolites. The number of articles devoted to trans-resveratrol has been steadily increasing. Trans-resveratrol is a molecule that is beneficial to human health; this explains the high level of interest in trans-resveratrol among different research groups. Therefore, it is important to develop an effective method to produce this compound commercially. The applicability of biotechnology for trans-resveratrol extraction is still uncertain. This review describes and compares the available biotechnological methods of trans-resveratrol production, focusing on their advantages and disadvantages.     

Characterization and evaluation of Aspergillus oryzae lactase coupled to a regenerable support

A derivative of crosslinked Sepharose, p-(N-acetyl-L-tyrosine azo) benzamidoethyl-CL-Sepharose 4B, was synthesized and used for the selective immobilization of thermostable lactase from Aspergillus oryzae.Preparations of soluble and immobilized lactase were evaluated under initial velocity conditions in a batch process. Immobilization had no significant effect on the pH optimum at 50 degrees C or kinetic parameters at pH 4.5 or pH 6.5 and 50 degrees C. At pH 4.5, the soluble enzyme possessed maximum activity at 60 degrees C and the immobilized at 55 degrees C; at pH 6.5 both showed maximum activity at 55 degrees C. The activation energy, entropy, and enthalpy decreased significantly with immobilization at pH 4.5 but not at pH 6.5. When the immobilized enzyme was placed in a packed-bed reactor, the effect of temperature on activity was altered as reflected by a marked decrease in the thermodynamic parameters of activation at both pH levels. Upon immobilization there was also a dramatic increase in the apparent thermal stability of the lactase, and the mean half-life at 50 degrees C was increased from 7.2 to 13 days at pH 4.5 and from 3.8 to 16 days at pH 6.5.     

A new efficient expression system for Bacillus and its application to production of recombinant phytase

A new expression system was developed for Bacillus subtilis.This system uses a shuttle vector (B. subtilis– Eschericia coli) carrying a phosphate starvation-inducible promoter (pst) and on a fed-batch cultivation strategy. The pst-promoter proved to be very strong and retain its tight regulation also when present on a multi-copy plasmid. The expression system developed showed promising results when applied to the production of recombinant Bacillusphytase – phytase activity at the end of cultivation reached 28.7 U ml^–1.     

The production and application of single-chain antibody fragments

This review discusses methods for the single-chain antibody fragment ($cFv) generation and scFv expression systems, and describes potential applications of scFv in the therapy of viral diseases and cancer, with emphasis on intracellularly expressed scFvs (intrabodies), application of scFvs in detection and diagnostics, and their use in proteomics.     

A regenerable flow-through affinity sensor for label-free detection of proteins and DNA

Label-free monitoring of biomolecular reactions in real-time is of great interest since it can provide valuable information about binding kinetics and equilibrium constants. In this report, a sensor based on White Light Reflectance Spectroscopy (WLRS) is presented that is capable of real-time monitoring of biomolecular reactions taking place on top of a polymer covered silicon dioxide reflective surface. The optical set-up consists of a visible–near infrared light source, a bifurcated optical fiber and a spectrometer. The outer part of the optical fiber guides the light vertically onto the surface where the biomolecular reactions occur, whereas the reflected light is driven from the central part of the fiber to the spectrometer. A microfluidic module in combination with a pump supplies the reagents at a constant rate. The biomolecular interactions are monitored as shifts of the wavelength of the interference minimum. The proposed methodology was applied for real-time and label-free monitoring mouse gamma-globulins binding onto immobilized anti-mouse IgG antibody. Mouse gamma-globulins at concentrations down to 150 pM were detected in reaction times of 1-min. Regeneration of immobilized antibody was accomplished up to seven times without loss of its activity. In addition, real-time monitoring of hybridization reaction between complementary oligonucleotides was accomplished. The proposed sensor provides a simple, fast, low cost approach for label-free monitoring of biomolecular interactions and therefore it should by suitable for a wide range of analytical applications.     

Enhancement of somatic embryogenesis of Ipomoea batatas in solid eultures and production of mature somatic embryos in liquid cultures for application to a bioreactor production system

Somatic embryos of Ipomoea batatas Lam. (sweet potato cv. White Star) were produced in an airlift bioreactor. This work describes the optimization of the embryogenic system on semisolid medium, followed by transfer of the system to liquid cultures and ultimately to the airlift bioreactor. The physiological age of embryogenic callus influenced the number and overall morphology of the embryo population in both semisolid and liquid medium. Maximum mature embryo production (35 embryos 10 mg^-1 inoculum) was obtained from six-week-old callus at 30°C. Somatic embryogenesis occurred in liquid cultures containing 20 mM NH₄NO₃ and 30 mM KCl. Globular embryos formed and continued development in suspension producing viable, mature cotyledonary embryos by day 14. Embryo formation and development was limited in the bioreactor. Although shear stress was responsible for some embryogenic damage, the effect of purging the system with fresh air needed to be investigated. To isolate aeration effects from shear stress effects, atmospheric determinations were performed on shaker flask cultures. Initially the gas composition within the Erlenmeyer headspace was that of room air. Ethylene increased to a maximum of 6.4 ppm (day 16), maximum CO₂, 21.2%, was also evident on day 16, and oxygen was depleted to a minimum of 8.1% by day 14. Purging the cultures with fresh air reduced the number of embryos formed; however, they were significantly longer than those formed in closed flasks. The gas response model of Ipomoea batatas will enable atmosphere replenishment in the bioreactor mimicking that of the shaker flask environment. Once the damaging effects of shear stress have been overcome, the regulation of bioreactor gasses should allow somatic embryo formation in the bioreactor comparable to that in shaker flasks.