Data and knowledge based experimental design for fermentation process optimization

A novel method for the sequential experimental design in order to optimize fed-batch fermentations was applied to a hyaluronidase fermentation by Streptococcus agalactiae. A Λ-optimal design was introduced to minimize the model parameter estimation error and to maximize the performance of the fermentation process. The method employs hybrid models that contain mechanistic, fuzzy and neural network components.     

Optimization of physicochemical parameters for gallic acid production by evolutionary operation-factorial design technique

The interaction effects of five variable parameters on gallic acid yield by the application of Evolutionary Operation (EVOP)-factorial design technique is presented in this paper. The methodology combines the advantage of factorial techniques for designing experiments with five parameters and that of EVOP methodology for the systematic analysis of experimental results. It facilitates the selection of optimum conditions or directs the changes desired for individual parameters for the designing of subsequent experiments. Decision were taken according to the decision-making procedure available in the literature. Standard deviation and error limits based on 95% confidence were also calculated according to the relationship given in literature. The best combinations of the physicochemical parameters at optimal levels of 32 °C, 93% relative humidity, 4.5 pH, 72 h incubation period, 20 g substrate weight and 0.4:1 solid–liquid ratio resulted in gallic acid yield of 90.9% under modified solid-state fermentation (MSSF).     

Optimization of biosurfactant production using waste from biodiesel industry in a new membrane assisted bioreactor

The main byproduct of biodiesel production is glycerol. Here, crude glycerol – byproduct of biodiesel industry – was evaluated as sole carbon source in rhamnolipids production by Pseudomonas aeruginosa. The optimal concentration of crude glycerol and sodium nitrate was assessed using response surface methodology, resulting in about 40–50 mg/L.h of rhamnolipids, which was about four times higher than previously reported in the literature. Fermentation parameters were similar to those observed with commercial glycerol as sole carbon source. The optimized medium was suitable for production using simple (22.9 mg/L.h) and fed-batch (32.4 mg/L.h) fermentation in oxygen-controlled bioreactor without foaming formation. Composition and relative abundance of rhamnolipid congeners showed that crude glycerol had little effect on metabolic pathways involved in their production. CMC values were approximately 130 mg/L and 230–260 mg/L for rhamnolipids from crude and commercial glycerol fermentation, respectively, which were about 2–6 times lower than CMC values of synthetic surfactants.     

Feasibility studies in spheronization and scale-up of ibuprofen microparticulates using the rotor disk fluid-bed technology

The aim of this study was to develop spheronized microparticulates as a drug delivery system using the 1-step closed rotor disk fluid-bed technology, and to scale up the batch spheronization process. Ibuprofen was used as the model drug and microcrystalline cellulose/sodium carboxymethyl cellulose hydrocolloid (Avicel(R) RC-581 or CL-611) was present as the diluent/binder. The mixture, in 1:1 ratio, was blended with and without 1% sodium lauryl sulfate (SLS) and spheronized with the rotor disk insert, using either water or hydroxypropylmethyl cellulose (HPMC) as binder. Fluid-bed machines (Vector/Freund Flo-Coater model) FLM-1 (with 9-inch rotor insert for 0.75 kg) and FLM-15 (with a 12-inch and 19-inch rotor inserts for 1 kg and 5, 10 kg, respectively) were used. The critical process parameters included inlet air temperature, rotor disk speed and configuration, air flow, and rate of binder application. The 1 kg batch containing SLS that was made with 12-inch smooth stainless steel or waffle teflon plates rotating at 500 rpm had desirable characteristics. The sphericity values were 0.88 and 0.91, with percent yield of 85.4 and 91.2 and drug content values of 94.47% and 91.44%, respectively. The spheroids showed good flow properties with respective rapid drug release (Q20 = 83.27 and 91.75). No difference was seen in the Avicel RC-581 and CL-611. Based on the 1 kg data, Avicel RC-581 and smooth stainless steel and waffle teflon plates (12 inch and 19 inch), the batch was scaled up to 5 and 10 kg. The scale-up parameters included rotor speed (124 -300 rpm) and spray rate (90-140 g/min). The scale-up batches had similar flow characteristics, release rate, and size distribution. The geometric mean diameter increased as batch size increased, and slightly bigger spheroids were obtained using the waffle teflon plate. Ibuprofen spheres with very good physical characteristics were developed using the rotor disk fluid-bed technology, a 1-step closed process that did not require additional unit processes.     

Optimization of ICP-MS internal standardization for 26 elements by factorial design experiment

IntroductionInternal standardization is a common tool used in inductively coupled plasma – mass spectrometry (ICP-MS) analysis in order to reduce matrix effects, and thus improve the reliability and the robustness of results. However, its efficacy relies on the choice of a proper internal standard (IS) which, ideally, undergoes the same signal variations as the analyte. Thus far, IS selection has mainly relied on the proximity of atomic mass between the analyte and the internal standard.However, while it may be a satisfactory rule of thumb, more recent works suggest that this criterium might not be suitable in several conditions, among which the presence of high amounts of carbon atoms in the sample. This may thus be of particular interest in the case of trace elements determination in biological samples.Materials and methodsIn this study, we propose an empirical and global approach to IS selection in ICP-MS through the use of a factorial design of experiments (DoE), with a focus on biological matrices of interest in clinical analysis: human blood and urine. The suitability of 13 potential IS was evaluated for 26 clinically-relevant analytes, including a polyatomic ion obtained through reaction with oxygen, across 324 experimental conditions.Results and discussionThe results underline several exceptions to the rule of IS selection based on mass proximity, notably when considering heavy or polyatomic analytes. As a consequence, measurements of said analytes in several extreme experimental conditions using IS selected by mass proximity could yield vastly erroneous results (up to 30 times the theoretical concentrations). By contrast, the use of empirically selected IS yielded much more acceptable results.     

Problems in physiological experimental animal models investigated with factorial design

In the present study we investigated four variables using factorial design to decide if any of these could explain the variations in the control measurements of interstitial fluid pressure (P_if) in rat trachea that were experienced. This approach requires only a fraction of the animals normally needed when studying each factor separately. P_if in tracheal tissue was measured with the servocontrolled counterpressure system using sharpened micropipettes. The measurements were performed over a period of 60 min and are presented as mean for every 15 min period. The factors investigated in the study were: three strains of female rats (Strain) two brands of diets (Food); two breeder companies (Source); and finally two batches of the same set of animals to repeat the experiment twice (Week), using a total of 48 animals. There was a highly significant effect within Strain the first week (p=0.007), but this response was not observed the second week. The interaction between Strain×Week was significant (p=0.007) while the main effects Strain or Week alone were not significant. The response pattern for Strain and Food was inconsistent for the two experimental weeks studied. These experiments made it possible for us to simultaneously test several factors and exclude these factors as the reason for the observed changes in our experiments since the experiments did not allow the conclusion that one or several of these factors could explain the variation in P_if.     

Optimization of a recombinant human growth hormone purification process using quality by design

This work describes a strategy to optimize a downstream processing of a recombinant human growth hormone (rhGH) by incorporating a quality by design approach toward meeting higher quality specifications. The optimized process minimized the presence of impurities and degradation by-products during manufacturing by the establishment of in-process controls. Capillary zone electrophoresis, reverse phase, and size-exclusion chromatographies were used as analytical techniques to establish new critical process parameters for the solubilization, capture, and intermediate purification steps aiming to maintain rhGH quality by complying with pharmacopeial specifications. The results indicated that the implemented improvements in the process allowed the optimization of the specific recovery and purification of rhGH without compromising its quality. In addition, this optimization facilitated the stringent removal of the remaining impurities in further polishing stages, as demonstrated by the analysis of the obtained active pharmaceutical ingredient.     

Determining conditions for nitric oxide synthesis in Caco-2 cells using Taguchi and factorial experimental designs

Intestinal inflammation correlates well with the increased synthesis of nitric oxide (NO), which is attributed mainly to the up-regulation of inducible nitric oxide synthase (iNOS). We optimized the use of interferon γ (IFN-γ), tumour necrosis factor α (TNF-α), interleukin 1β (IL-1β), lipopolysaccharide (LPS), and phorbol myristate acetate (PMA) as inducers to stimulate NO synthesis in Caco-2 cells using a Taguchi design. The results indicated that IFN-γ was the most important inducer of iNOS in Caco-2 cells. Treating Caco-2 cells with both IFN-γ and PMA using an optimal mixture of 8000 U/ml IFN-γ and 0.1 μg/ml of PMA resulted in a synergistic induction of NO synthesis. Further experiments using a 5-factor/2-level factorial design including Caco-2 growth conditions such as cell passage, culture medium composition, cell seeding time and density, and stimulation time were also performed. Cell seeding and stimulation times significantly (P < 0.05) affected NO synthesis, whereas culture medium and seeding density did not appreciably affect NO synthesis in Caco-2 cells. Western blotting and RT-PCR findings confirmed that the optimal mixture of IFN-γ and PMA effectively up-regulated iNOS mRNA and protein. The induced NO, iNOS mRNA, and protein were all inhibited by the iNOS selective inhibitor, aminoguanidine (AG).     

Optimization of non-detergent treatment for enveloped virus inactivation using the Taguchi design of experimental methodology (DOE)

Abstract

In mammalian cell culture technology, viral contamination is one of the main challenges; and, so far, various strategies have been taken to remove or inactivate viruses in the cell-line production process. The suitability and feasibility of each method are determined by different factors including effectiveness in target virus inactivation, maintaining recombinant protein stability, easiness—in terms of the process condition, cost-effectiveness, and eco-friendliness. In this research, Taguchi design-of-experiments (DOE) methodology was used to optimize a non-detergent viral inactivation method via considering four factors of temperature, time, pH, and alcohol concentration in an unbiased (orthogonal) fashion with low influence of nuisance factors. Herpes Simplex Virus-1 (HSV1) and Vero cell-line were used as models for enveloped viruses and cell-line, respectively. Examining the cytopathic effects (CPE) in different dilutions showed that pH (4), alcohol (15%), time (120?min), and temperature (25?°C) were the optimal points for viral inactivation. Evaluating the significance of each parameter in the HSV-1 inactivation using Taguchi and ANOVA analyses, the contributions of pH, alcohol, temperature and time were 56.5%, 19.2%, 12%, and 12%, respectively. Examining the impact of the optimal viral treatment condition on the stability of model recombinant protein-recombinant human erythropoietin, no destabilization was detected.     

Cyclodextrin glucosyltransferase production by <Emphasis Type="Italic">Bacillus megaterium</Emphasis> NCR: evaluation and optimization of culture conditions using factorial design

Statistically-based experimental designs were used to optimize the production of cyclodextrin glucosyltransferase (CGTase) from a local isolate of Bacillus megaterium using shack culture fermentation. Seven cultural conditions were examined for enzyme production and specific activity using Plackett-Burman factorial design. Fermentation time and K₂HPO₄ level were the crucial for factors improving enzyme production process. The steepest ascent design was adopted-based on the results recorded with Plackett-Burman design. Maximal enzyme estimates (activity 56.1 U/ml, and specific activity 62.7 U/mg protein) were achieved. A verification experiment was carried out to examine model validation of this optimization.     

Optimization of thermostable lipase production from a thermophilic Geobacillus sp. using Box-Behnken experimental design

Thermostable lipase production by Geobacillus thermoleovorans was optimized in shake-flask cultures using Box-Behnken experimental design. An empirical model was developed through response surface methodology to describe the relationship between tested variables (Tween 80, olive oil, temperature and pH) and enzyme activity. Maximum enzyme activity (495 U l^–1) was attained with Tween 80 at 5 g l^–1; olive oil at 60 g l^–1; 70 °C and pH 9. Experimental verification of the model showed a validation of 95%, which is more than 4-fold increase compared to the basal medium.     

Joint effect of nitrogen and phosphorous on glucose oxidase production by Aspergillus niger: Discussion of an experimental design with a risk of co-linearity

The combined effects of nitrogen and phosphorous on the production of glucose oxidase and gluconic acid by Aspergillus niger cannot be adequately described with Monod-type model, neither do they fit well to linear equations with interactions N × P, nor quadratic with N² and P² terms. On the other hand, the interactions of type N²P and NP², although common in real cases such as enzymatic kinetics in the presence of inhibitors, should be verified – if included in empiric models – by means of designs that can lead to artefactual results derived from the co-linearity. To avoid this risk we propose a procedure, based on the ‘bootstrap’ algorithm, which provided consistent results in the mentioned bioproductions. Applied together with methods of response surface and gradient, said procedure allowed to optimize the enzyme production as a function of the concentrations of N and P, to quintuple the initially obtained levels, and to explain other culture behaviours related with the sources of these nutrients.     

正交试验优化鼠疫活疫苗冻干稳定剂

目的优化皮上划痕用鼠疫活疫苗的稳定剂配方。方法采用正交试验,以鼠疫杆菌冻干存活率为检测指标,分别对乳糖、谷氨酸钠、硫脲和尿素4种成分组成的稳定剂配方,以及乳糖、蔗糖、谷氨酸钠、硫脲和尿素5种成分组成的稳定剂配方进行优化。结果 4种成分组成的稳定剂最佳组合为乳糖7.5%、谷氨酸钠0.5%、硫脲1.0%、尿素0.1%(均为质量分数),此配方可使菌体的冻干存活率达到(68.49±6.19)%;5种成分组成的冻干稳定剂最佳组合为乳糖7.5%、蔗糖7.5%、谷氨酸钠0.5%、硫脲0.5%、尿素0.1%(均为质量分数),此配方可使菌体的冻干存活率达到(74.71±6.34)%,均高于现有稳定剂配方。结论优化的两种稳定剂均对鼠疫杆菌具有较好的保护作用。     

Optimization of a packed bed bioreactor with immobilized cells using experimental design

A central composite design was employed for the optimization of heterogeneous enzymatic hydrolysis of sucrose. The reaction was catalyzed by whole yeast cells of Saccharomyces cerevisiae immobilized in Ca-pectate gel. Bioreactor volumetric productivity was chosen as an optimization criterion, while temperature and gel biomass concentration were optimization parameters. Sucrose inlet concentration of 700 kg m^–3 and outlet conversion of 65% were constant in all experiments. In the temperature range 51–73 °C and biomass concentration range 11–39 kg m^–3 (dry mass of cells), the dependence of bioreactor productivity on the two factors was described by a second order polynom regression equation. No simple optimum was revealed by the experimental design. The bioreactor productivity increased within the whole experimental range of biomass concentration, whereas a temperature optimum was found to be between 60 and 65 °C.List of Symbols b _j jth regression coefficient - c _Si kg m^–3 inlet sucrose concentration - F m³ min^–1 flow rate - F F distribution - f _LF degrees of freedom of lack of fit variance - f _P degrees of freedom of pure error variance - N total number of runs - n ₀ number of runs in the centre of design - P kg m^–3 min^–1 productivity - s _LF ² lack of fit variance - SS _LF lack of fit sum of squares - S _p ² pure error variance - SS _P pure error sum of squares - SS _R total residual sum of squares - V _b m³ bioreactor bed volume - X _O outlet conversion - x ₁ 1st factor - coded temperature - x ₂ 2nd factor - coded biomass concentration - y kgm^–3min^–1 measured response (productivity) - kg m^–3 min^–1 estimated response (productivity) - y _Oi kg m^–3 min^–1 measured response in the centre of design - ¯y ₀ kg m^–3 min^–1 average of response in the centre of design     

Optimizing the rotor design for controlled-shear affinity filtration using computational fluid dynamics

Controlled shear affinity filtration (CSAF) is a novel integrated processing technology that positions a rotor directly above an affinity membrane chromatography column to permit protein capture and purification directly from cell culture. The conical rotor is intended to provide a uniform and tunable shear stress at the membrane surface that inhibits membrane fouling and cell cake formation by providing a hydrodynamic force away from and a drag force parallel to the membrane surface. Computational fluid dynamics (CFD) simulations are used to show that the rotor in the original CSAF device (Vogel et al., 2002) does not provide uniform shear stress at the membrane surface. This results in the need to operate the system at unnecessarily high rotor speeds to reach a required shear stress of at least 0.17 Pa at every radial position of the membrane surface, compromising the scale-up of the technology. Results from CFD simulations are compared with particle image velocimetry (PIV) experiments and a numerical solution for low Reynolds number conditions to confirm that our CFD model accurately describes the hydrodynamics in the rotor chamber of the CSAF device over a range of rotor velocities, filtrate fluxes, and (both laminar and turbulent) retentate flows. CFD simulations were then carried out in combination with a root-finding method to optimize the shape of the CSAF rotor. The optimized rotor geometry produces a nearly constant shear stress of 0.17 Pa at a rotational velocity of 250 rpm, 60% lower than the original CSAF design. This permits the optimized CSAF device to be scaled up to a maximum rotor diameter 2.5 times larger than is permissible in the original device, thereby providing more than a sixfold increase in volumetric throughput.     

Optimization of furfural production from hemicellulose extracted from delignified palm pressed fiber using a two-stage process

This study aims to optimize the conditions for furfural production from hemicellulose extracted from delignified palm pressed fiber (dPPF) via two-stage process: acid hydrolysis followed by dehydration, using response surface methodology (RSM). The extracted hemicellulose contained 80.8% xylose. In order to convert hemicellulose to xylose in the acid hydrolysis step, there were four important parameters consisting of reaction temperature (100–150 °C), sulfuric acid concentration (1–10% v/v), ratio of sulfuric acid to hemicellulose (L/S ratio) (10, 9, and 8 v/w), and reaction time (30–120 min). The maximum xylose production (12.58 g/L) was achieved at 125 °C, 5.5% sulfuric acid, L/S ratio of 9 mL/g for 30 min with the determination coefficient (R²) value of 0.90. For the dehydration process, two parameters; reaction temperature (120–160 °C) and reaction time (30–150 min), were optimized. The maximum furfural production (8.67 g/L) was achieved at a reaction temperature of 140 °C for 90 min with the determination coefficient (R²) value of 0.93.     

Optimization of a mAb production process with regard to robustness and product quality using quality by design principles

Quality by Design principles are well described and widely used in biopharmaceutical industry. The characterization of a monoclonal antibody (mAb) production process is crucial for novel process development and control. Yet, the application throughout the entire upstream process was rarely demonstrated. Following previously published research, this study marks the second step toward a complete process characterization and is focused on the effect of critical process parameters on the antibody production efficiency and quality of the process. In order to conduct the complex Design of Experiments approach with optimal control and comparability, the ambr®15 micro bioreactor platform was used. Investigated parameters included the pH and dissolved oxygen set points, the initial viable cell density (iVCD) as well as the N‐1 duration. Various quality attributes (e.g., growth rate, viability, mAb titer, and peak proportion) were monitored and analyzed using multivariate data analysis to evaluate the parameter effects. The pH set point and the initial VCD were identified as key process parameters with strong influence on the cell growth as well as the mAb production and its proportion to the total protein concentration. For optimization and improvement in robustness of these quality attributes the pH must be increased to 7.2, while the iVCD must be lowered to 0.2 × 10⁶ cells/mL. Based on the defined design space, additional experiments verified the results and confirmed the intact bioactivity of the antibody. Thereby, process control strategies could be tuned toward high cell maintenance and mAb production, which enable optimal downstream processing.     

Optimization of waste stabilization pond design for developing nations using computational fluid dynamics

Waste stabilization ponds (WSPs) have been used extensively to provide wastewater treatment throughout the world. However, no rigorous assessment of WSPs that account for cost in addition to hydrodynamics and treatment efficiency has been performed. A study was conducted that utilized computational fluid dynamics (CFD) coupled with an optimization program to optimize the selection of the best WSP configuration based on cost and treatment efficiency. The results of monitoring the fecal coliform concentration at the reactor outlet showed that the conventional 70% pond-width baffle pond design is not consistently the best pond configuration as previously reported in the literature. The target effluent log reduction can be achieved by reducing the amount of construction material and tolerating some degree of fluid mixing within the pond. As expected, the multi-objective genetic algorithm optimization did produce a lower-cost WSP design compared to a SIMPLEX optimization algorithm, however, with only a marginal increase in the effluent microbial log reduction. Several other designs generated by the CFD/optimization model showed that both shorter and longer baffles, alternative depths, and reactor length to width ratios could improve the hydraulic efficiency of the ponds at a reduced overall construction cost.