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Suspension culture of mammalian cells.

Mammalian cell suspension culture systems are being used increasingly in the biotechnology industry. This is due to their many advantages including simplicity and homogeneity of culture. Suspension systems are very adaptable (e.g., for microcarrier, microencapsulation, or other methods of culture). Their engineering is thoroughly understood and standardized at large scale, and automation and cleaning procedures are well established. Suspension systems offer the possibility of quick implementation of production protocols due to their ability to be scaled easily once the basic culture parameters are understood. The only main disadvantage of the suspension culture systems to date is their inapplicability for the production of human vaccines from either primary cell lines or from normal human diploid cell lines (Hayflick et al., 1987 and references therein). One of the great advantages of suspension culture is the opportunity it provides to study interactions of metabolic and production phenomena in chemostat or turbidostat steady-state systems. Furthermore, in suspension culture systems from which cell number and cell mass measurements are easy to obtain, rigorous and quantitative estimations of the effects of growth conditions or perturbations of metabolic homeostasis can be made. Such studies can speed up the development of optimal processes. With our increasing understanding of factors influencing expression in mammalian cells (Cohen and Levinson, 1988; Santoro et al., 1988) and the direct application of new methods in suspension culture (Rhodes and Birch, 1988), its usefulness and importance is likely to increase in the future. In this chapter, we have described some of the potential uses of the various suspension culture systems and have covered most of the established technology and literature. Due to the rapid developments and needs in the biotechnology industry and the versatility of suspension culture systems, it is probable that many more variations on this theme will evolve in the near future at both the pilot and production scales.

Animals

[Thermoanalytic techniques for the study of pharmaceutical products].

During the past years, there have been frequent demonstrations that thermoanalytical methods have found wide-spread use in pharmacy. Analysts have developed these techniques in pharmaceutical field in the estimation of impurities, in quality control procedures, in preformulation studies, in the active principle identification, in accelerated stability and in production process optimization. Wide-spread use of thermal methods is devoted to the identification of solid state of drugs (polymorphism, solvate, inclusion compound,...) due to its possible implication on bioavailability or on the stability of a final dosage form. Some examples of thermoanalytical applications are reported.

Chemistry, Pharmaceutical

Effects of extracellular matrix on cytoskeletal and myofibrillar organization in vitro.

The distribution and three-dimensional relationship of myofibrillar and cytoskeletal components during myofibrillogenesis were examined in preparations of neonatal rat cardiac myocytes processed in parallel for scanning electron microscopy (SEM), intermediate voltage transmission electron microscopy (IVEM) and immunofluorescence (IF). Of the various methods used for processing, optimal results were achieved by pre-extraction with Triton X-100 in an actin-stabilizing buffer. This procedure effectively removed the surface membrane, as viewed by SEM images, while preserving myofibrillar and cytoskeletal structure, as evidenced by IF for actin, alpha-actinin and vinculin. Cytoskeletons in SEM images consisted of a cortex of anastomosing filaments through which ran parallel filament bundles oriented in the long axis of the cell and attached along their length to the substrate by numerous fine filaments. In IVEM images, myofibrils were laterally connected at the level of the Z bands. Myocytes grown on different extracellular matrices showed different patterns and distributions of both striated myofibrils and focal adhesions, as determined by IF for alpha-actinin and vinculin, respectively. Cells on collagen I and III contained striated myofibrils which extended to the cell perimeters where focal adhesions were predominately located. Cells on laminin and fibronectin matrices exhibited myofibrils and focal adhesions more centrally located. In addition, cells on laminin contained circumferential arcs of filaments near the cell periphery.

Animals

Optimal background estimation in EELS.

In quantitative electron energy loss spectrometry, it is desirable to estimate the background law below core edge energy in a way that provides the maximum signal-to-noise ratio. Assuming an inverse power background model and independently Poisson distributed measurements, it is shown how to achieve this goal by using a maximum likelihood (ML) estimation technique which provides unbiased and minimum mean square error estimates of all parameters of interest. An efficient and computationally stable implementation of this procedure is proposed. Standard logarithmic least squares estimations are then compared with the ML approach and the gain in performance due to optimal processing is quantified.

Electrons

Microscale synthesis of nitrogen-13-labeled cisplatin.

A microscale synthesis of [13N]cisplatin (cis-dichlorodiammineplatinum(II), cis-DDP) from cyclotron-produced [13N]ammonia is presented. Temperature, reaction time, ratios, and concentration of reactants have been optimized for each step of the synthesis. Purification is performed by ion exchange chromatography. Radiochemical purity and optimization processes are controlled by high performance liquid chromatography and high performance thin layer chromatography--22 mCi [13N]cisplatin in 10 ml of solution is produced. The entire procedure takes approximately 15 min and the specific activity is approximately 300 mCi/mumole at EOB.

Ammonia

[Optimization of immobilization conditions for acid proteinase from Aspergillus awamori].

To optimize the immobilization conditions for acid proteinase from Aspergillus awamori by covalent binding through glutaraldehyde, experiments were carried out using the Box-Wilson method. The optimization process was assessed on the basis of absolute activity A, coefficient of activity retention gamma and their product A gamma. The following conditions can be recommended: glutaraldehyde concentration 50--60 mg/g, enzyme concentration not less than 40 mg/g, time of glutaraldehyde treatment 2--2.5 hrs, immobilization time 2 hrs, pH about 4.0, and temperature 35--40 degrees C. Under these conditions A=220--230 U/g, gamma = 23--24% Agamma = 5,000--6,000.

Aspergillus

Optimal temperature control policy for a two-stage recombinant fermentation process.

The optimal temperature control policy to be followed in the operation of a two-stage fermentation system in which gene expression is induced by a temperature-sensitive gene switching system was studied. A genetically structured model was used to describe product formation, and kinetic equations based on experimental data were used to quantify the specific gene expression rate and parameters that affect plasmid instability. A constant temperature control policy and temperature profiling control policy including temperature cycling were studied and compared. Maximum average production rate was obtained from a temperature control policy in which the second stage was operated initially at about 40.5 degrees C and the temperature decreased slightly to a constant value at 40.0 degrees C. The maximum average production rate, which corresponds to the optimal temperature control policy, for an operation of 180 h was 29.7 units of protein (mg of cells)-1 h-1.

DNA, Recombinant

Effects of optimization and image processing in digital chest radiography: an ROC study with an anthropomorphic phantom.

A digital system for chest radiography based on a large image intensifier was compared to a conventional film-screen system. The digital system was optimized with regard to spatial and contrast resolution and dose. The images were digitally processed for contrast and edge enhancement. A simulated pneumothorax and two simulated nodules were positioned over the lungs and the mediastinum of an anthropomorphic phantom. Observer performance was evaluated with ROC analysis. Five observers assessed the processed digital images and the conventional full-size radiographs. The time spent viewing the full-size radiographs and the digital images was recorded. For the simulated pneumothorax, the results showed perfect performance for the full-size radiographs and detectability was high also for the processed digital images. No significant difference in the detectability of the simulated nodules was seen between the two imaging systems. The results for the digital images showed a significantly improved detectability for the nodules in the mediastinum as compared to a previous ROC study where no optimization and image processing was available. No significant difference in detectability was seen between the former and the present ROC study for small nodules in the lung. No difference was seen in the time spent assessing the conventional full-size radiographs and the digital images. The study indicates that processed digital images produced by a large image intensifier are equal in image quality to conventional full-size radiographs for low-contrast objects such as nodules.

Evaluation Studies as Topic

[Principles for optimizing and guaging several processes in the technology of vaccine production. I. Several problems in guaging and optimizing thermal sterilization of fluids].

Thermal sterilization of fluids--one of the widespread operations in microbiological laboratories and in vaccine production--solves two tasks: provides sterility and maximal preservation of biological value of the sterilized fluid. A possibility and expediency of using some indices for the quantitative assessment of the efficacy of the sterilization regimens, including measurement of the sterilization processes is considered in this work. A possible way of the regimen optimization on the basis of known values of the activation energies of the processes of decomposition of thermolabile components and inactivation of foreign microbial flora is assessed.

Hot Temperature

Optimization of a process for the production of (R)-2-hydroxy-4-phenylbutyric acid--an intermediate for inhibitors of angiotensin converting enzyme.

(R)-2-Hydroxy-4-phenylbutyric acid, an intermediate in the manufacture of inhibitors of angiotensin converting enzyme, can be produced continuously in an enzyme membrane reactor by enzymatic reduction of its corresponding alpha-keto acid. D-Lactate dehydrogenase (D-LDH) from Staphylococcus epidermidis was chosen as the most appropriate enzyme to carry out the NADH-dependent reduction. Formate dehydrogenase (FDH) was used for NADH regeneration. Detailed kinetic measurements and a mathematical model for the coupled enzyme reactions were applied to calculate the optimal conditions for continuous production of the alpha-hydroxy acid. A mass of 1 kg [corrected] (R)-2-hydroxy-4-phenylbutyric acid was synthesized in a 220 ml enzyme membrane reactor over a period of 4 weeks. A mean space-time-yield of 165 g l-1 d-1 was achieved at low enzyme consumptions of 150 U kg-1 alpha-hydroxy acid for FDH and D-LDH.

Angiotensin-Converting Enzyme Inhibitors

The role of glucocorticoid hormones as biological amplifiers.

Recent research in hormone action has been aimed at studying single effects in well-defined systems. As exemplified in several chapters of this book, it has been possible to deduce a general mechanism of action of the glucocorticoids using this approach. Most hormones, and the glucocorticoids in particular, do not act as independent agents in the intact animal. Although the best known example of how glucocorticoids interact with other hormones is the amplification of the effect of those whose action is mediated by cAMP, these steroids also augment the effects of a variety of other hormones and effectors. Such interactions are of interest in clinical medicine as well, since glucocorticoid hormones are used in combination with other drugs in a number of conditions, including the treatment of asthma, allergies, and certain kinds of shock and cancer. Neither the biochemical nor the pharmacologic basis for the effects of the glucocorticoids is known. In some cases the actions of other hormones are not observed unless the tissue has first been exposed to glucocorticoids. In these instances the glucocorticoids are said to exert a "permissive effect," since they allow a process to proceed at a maximal rate even though the steroid itself has no effect on this process. There is no doubt that such examples exist, as documented above: thus the concept of a "permissive effect" does have utility. The term fails to describe the more general role the glucocorticoids play, since in many instances the steroid also has a direct effect on the process itself, or optimizes a process in which the primary effector is not as yet known. Because of these cases, and because the historically more general usage first proposed by INGLE [1] seems to have been forgotten, use of the term "permissive effect" has been avoided in this chapter. An ultimate goal in glucocorticoid hormone research is to identify the mechanisms involved in the amplification effect these hormones exert. Now that the actions of these hormones and of the hormones they interact with are being defined, such work is within the realm of feasibility.

Animals

Standardization of process parameters for salbutamol sulphate microcapsules coated with cellulose acetate phthalate and computation of in-vitro release kinetics.

Salbutamol sulphate, a bronchodilatatory drug for asthma, is encapsulated by Emulsion-Solvent-Evaporation technique using cellulose acetatephthalate as coating polymeric material. Different process parameters like Stirring Speed, Drug-Polymer Ratio, Solvent-Polymer Ratio, Internal Phase-External Phase Ratio, Effects of Temperature, and of Surfactants have been studied to standardise the process. At optimal condition of process parameters maximum encapsulation efficiency is obtained and microcapsules produced are free flowing and spherical. The dissolution of the microcapsules is carried out and the data obtained from in-vitro dissolution profile are computed in the light of different kinetic models. Diffusion coefficient (Da) and diffusivity rate constant (KBL) are evaluated with the help of Baker-Lonsdale model.

Albuterol

[Ways to optimize the technological process of oleandomycin reextraction].

Dependence of the oleandomycin distribution coefficient on pH of the acqueous phase and temperature in the system of butylacetate extract-water acidified with orthophosphoric acid was studied. With a purpose of intensification of the process of oleandomycin reextraction, decreasing the antibiotic inactivation and evaporation of the organic solvent it was proposed to perfom oleandomycin extraction at pH 4.0--5.0 accompanied by simultaneous decreasing of the temperature.

Acetates

[Optimizing the learning process in short-term autogenic training by practice protocols].

The results of two studies are presented, in which the question was investigated, whether the learning process of autogenic training (AT) in short group programs can be improved by the use of a self-control technique (records for the AT-exercises realized without the group at home). Study I refers to 24 university students, who learned AT within their psychology education; Study II refers to 28 adults, who learned AT within a psychosocial community service for adults. Within control-group designs evaluative and follow-up data were gathered before, during, at the end, and 4 months after the AT-treatment. The data refer to psychosomatic complaints (SSP, ATSYM), depression (BDI), relaxation capacity and wellbeing (VFE) as well as subjective valuations of AT and the frequency of AT-exercising. The results of both studies show that the basal AT-exercises can be learned in short group programs (6 meetings within 6 weeks). The self-control technique promotes the learning process at short as well as at longer sight. Implications of the results for the realization of introductions to AT in out- and in-patient group treatment are discussed.

Adult