Cell‐free production of a therapeutic protein: Expression,purification, and characterization of recombinant streptokinase using a CHO lysate

The use of cell‐free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell‐free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation, and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFP) and streptokinase in a cell‐free system using instrumented mini‐bioreactors for highly reproducible protein production. We achieved recombinant protein production (～600 μg/ml of tGFP and 500 μg/ml streptokinase) in 2.5 hr of expression time, comparable to previously reported yields for cell‐free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare those to a tag‐free self‐cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose‐level production of therapeutic proteins at the point‐of‐care.     

Evaluation and applications of optical cell density probes in mammalian cell bioreactors

On-line optical cell density probes were implemented to continuously monitor the cell densities in mammalian cell bioreactor and to achieve advanced bioreactor controls. We tested cell density probes from six manufacturers in high cell density bioreactors. When externally calibrated, Aquasant and Ingold backscattering probes produced the most linear probe responses (PR) versus cell density (CD), followed by the ASR and Cerex laser probes. Monitek and Wedgewood transmission probes had lower resolutions. All probes were tested in two murine hybridoma fermentations. Cell densities varied between 1 x 10(6) cells/mL to 20 x 10(6) cells/mL and the bioreactors were operated for 5 to 7 weeks. For our bioreactors, Aquasant, Ingold, ASR, Wedgewood, and Monitek probes gave satisfactory responses. Little fouling was observed with any probe at the end of 2 weeks. Fouling was a possibility after 3 weeks in one bioreactor but its effect can be easily corrected. Cell density control and specific perfusion control of bioreactors based on the Aquasant probe were achieved. Implementation of cell density probe based perfusion control, instead of "step perfusion adjustments" based on manual hemacytometer control, will result in smoother operation, healthier cultures, increased medium delivery efficiency, and reduced operational excursions. (c) 1995 John Wiley & Sons, Inc.     

Protocol for determining bioavailability and biokinetics of organic pollutants in dispersed, compacted and intact soil systems to enhance in situ bioremediation

The development of effective in situ and on-site bioremediation technologies can facilitate the cleanup of chemically-contaminated soil sites. Knowledge of biodegradation kinetics and the bioavailability of organic pollutants can facilitate decisions on the efficacy of in situ and on-site bioremediation of contaminated soils and determine the attainable treatment end-points. Two kinds of compounds have been studied: (1) phenol and alkyl phenols, which represent hydrophilic compounds, exhibiting high water solubility and moderate to low soil partitioning; and (2) polycyclic aromatic hydrocarbons which are hydrophobic compounds with low water solubility and exhibit significant partitioning in soil organic carbon. Representative data are given for phenol and naphthalene. The results provide support for a systematic multi-level protocol using soil slurry, wafer and porous tube or column reactors to determine the biokinetic parameters for toxic organic pollutants. Insights into bioremediation rates of soil contaminants in compact soil systems can be attained using the protocol. Received 04 December 1995/ Accepted in revised form 31 January 1997     

An internal sedimentation bioreactor for laboratory-scale removal of toxic metals from soil leachates using biogenic sulphide precipitation

An internal-sedimentation bioreactor was employed to provide biomass feedback and process intensification in a laboratory-scale sulphide-bioprecipitation system for toxic metals (Cd, Cr, Cu, Mn, Ni, Zn) present in acid leachates from metal-contaminated soil. Biomass feedback was improved by addition of a cationic polymer flocculant and the activity of the sulphate-reducing bacterial culture was increased by the addition of cornsteep in addition to the ethanol used as carbon/energy substrate. A mass-balance was carried out for carbon and sulphur in the system. Sulphate reduction in the reactor was able to remove acidity at moderate sulphate concentrations up to 50 mM although it was insufficient at the highest levels tested. When presented with a simulated toxic metal-containing leachate, the reactor was able to precipitate metals efficiently under all of the conditions of sulphate concentration and pH tested, producing an effluent with metal concentrations suitable for environmental discharge. Received 29 October 1996/ Accepted in revised form 31 March 1997     

Long-term liver cell cultures in bioreactors and possible application for liver support

Hybrid artificial liver systems are being developed as a temporary extracorporeal liver support therapy. A short overview is given which emphasizes the development of hepatocyte culture models for bioreactors, subsequent in vitro studies, animal studies and the clinical application of hybrid liver support systems.An own bioreactor construction has been designed for the utilization of hepatocytes and sinusoidal endothelial cells. The reactor is based on capillaries for hepatocyte aggregate immobilization, coated with biomatrix. Four separate capillary membrane systems, each permitting a different function, are woven in order to create a three-dimensional network. Cells are perfused via independent capillary membrane compartments. Decentralized oxygen supply and carbon dioxide removal with low gradients is possible. There is a decentralized co-culture compartment for nonparenchymal liver cells. The use of identical parallel units to supply a few hepatocytes facilitates scale-up.     

The problems associated with high biomass levels in plant cell suspensions

The development of plant cell cultures as an alternative supply of phytochemicals has been difficult. Although there has been some very suitable targets, the yields of these compounds has remained low despite considerable efforts. One of the main constituents of a process is its productivity which is the sum of the process run time (growth rate), yield, and biomass levels. The effect of changes in all three of these components on productivity has been demonstrated.Of the three components making up productivity, biomass is perhaps the easiest to increase. However, high biomass levels will increase the viscosity, which will affect both mixing and oxygen supply. Therefore, this will require more vigorous mixing which may increase the shear within the bioreactor. All these parameters need further investigation at high biomass concentrations.     

New protocol for the rapid quantification of exopolysaccharides in continuous culture systems of acidophilic bioleaching bacteria

In this study, we investigate exopolysaccharide production by a bacterial consortium during the bioleaching of a cobaltiferrous pyrite. Whereas comparable studies have looked at exopolysaccharide production in batch systems, this study focuses on a continuous system comprising a series of four stirred bioreactors and reveals the difficulties in quantifying biomolecules in complex media such as bioleached samples. We also adapted the phenol/sulphuric acid method to take into account iron interference, thus establishing a new protocol for sugar quantification in bioleached samples characterised by low pH (1.4) and high iron concentration (2 g l⁻¹). This allows sugar analysis without any prior sample preparation step; only a small amount of sample is needed (0.5 ml) and sample preparation is limited to a single filtration step. We found that free exopolysaccharides represented more than 80% of the total sugars in the bioreactors, probably because stirring creates abrasive conditions and detaches sugars bound to pyrite or bacteria and that they were produced mainly in the first two reactors where bioleaching activity was greatest. However, we could not establish any direct link between the measured exopolysaccharide concentration and bioleaching activity. Exopolysaccharides could have another role (protection against stress) in addition to that in bacterial attachment.     

Engineering microenvironments for manufacturing therapeutic cells

There are a growing number of globally approved products and clinical trials utilizing autologous and allogeneic therapeutic cells for applications in regenerative medicine and immunotherapies. However, there is a need to develop rapid and cost-effective methods for manufacturing therapeutically effective cells. Furthermore, the resulting manufactured cells may exhibit heterogeneities that result in mixed therapeutic outcomes. Engineering approaches that can provide distinct microenvironmental cues to these cells may be able to enhance the growth and characterization of these cell products. This mini-review describes strategies to potentially enhance the expansion of therapeutic cells with biomaterials and bioreactors, as well as to characterize the cell products with microphysiological systems. These systems can provide distinct cues to maintain the quality attributes of the cells and evaluate their function in physiologically relevant conditions.     

Application of solid–liquid TPPBs to the production of L‐phenylacetylcarbinol from benzaldehyde using Candida utilis

The biotransformation of benzaldehyde and glucose to L ‐phenylacetylcarbinol (PAC) using Candida utilis was demonstrated in a solid–liquid two‐phase partitioning bioreactor (TPPB) with the aim of reducing substrate, product, and by‐product toxicity via sequestration. Previous work in the field had used octanol as the sequestering phase of liquid–liquid TPPBs but was limited by the toxic effects of octanol on C. utilis. To improve solvent selection in any future studies, the critical log P of C. utilis was determined in the current study to be 4.8 and can be used to predict biocompatible solvents. Bioavailability tests showed alkanes and alkenes to be non‐bioavailable. As polymers are biocompatible and non‐bioavailable, a wide range of commercially available polymers was screened and it was demonstrated that polymer softness plays a key role in absorptive capability. The polymer Hytrel G3548L was selected as the second phase to sequester benzaldehyde, PAC, and benzyl alcohol, with partition coefficients of 35, 7.5, and 10, respectively. With a 9% by volume partitioning phase, 13.6 g/L biomass of C. utilis achieved an overall PAC concentration of 11 g/L, a 1.9‐fold improvement over the single‐phase case. Benzyl alcohol concentration was 4.5 g/L, a 1.6‐fold reduction. The volumetric productivity was 0.85 g/L h, a 1.2‐fold improvement over the single‐phase system. These results demonstrate a promising starting point for solid–liquid TPPBs for PAC production. Biotechnol. Bioeng. 2010;107:633–641. © 2010 Wiley Periodicals, Inc.