Fractionation of transfer RNA on Sepharose 4B. Effects of Sepharose batch differences.
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Chromatography, Gel↗
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Adaptive, model-based control by the Open-Loop-Feedback-Optimal (OLFO) controller for the effective fed-batch cultivation of hybridoma cells.
Although fed-batch suspension culture of animal cells continues to be of industrial importance for the large scale production of pharmaceutical products, existing control concepts are still insufficient. Changes in cell metabolism during cultivation and between similar cultivations, the complexity of the cell metabolism, and the lack of on-line state variables restrict the transfer of available control strategies established in bioprocess engineering. A process control strategy designed to achieve optimized process control must account for all these difficulties and fit sophisticated requirements toward adaptability and flexibility. The combination of a fed-batch process and an Open-Loop-Feedback-Optimal (OLFO) control provides a new approach for cell culture process control that couples an efficient cultivation concept to a capable process control strategy. The application of an adaptive, model-based OLFO controller to a hybridoma cultivation and experimental results are presented.
Algorithms↗
Evaluation of daptomycin susceptibility testing by Etest and the effect of different batches of media.
One hundred and ninety-five Gram-positive bacteria representing 17 species were tested for susceptibility to daptomycin by broth microdilution and Etest methods. The geometric mean daptomycin MIC was 0.46 mg/L by broth microdilution tests and 0.73 mg/L by Etest. The concentration of calcium in 12 different batches of agar varied from 4 to 36 mg/L. Daptomycin Etest MICs varied inversely with the calcium concentration. Etest daptomycin MICs for quality control strains were within proposed quality control range on media with >20 mg/L of calcium. Monitoring the calcium levels of agar media by testing appropriate quality control strains is important for daptomycin Etests.
Anti-Bacterial Agents↗
Effect of batch-process solar disinfection on survival of Cryptosporidium parvum oocysts in drinking water.
The results of batch-process solar disinfection (SODIS) of Cryptosporidium parvum oocysts in water are reported. Oocyst suspensions were exposed to simulated sunlight (830 W m(-2)) at 40 degrees C. Viability assays (4',6'-diamidino-2-phenylindole [DAPI]/propidium iodide and excystation) and infectivity tests (Swiss CD-1 suckling mice) were performed. SODIS exposures of 6 and 12 h reduced oocyst infectivity from 100% to 7.5% (standard deviation = 2.3) and 0% (standard deviation = 0.0), respectively.
Animals↗
The oral contraceptive pill effectively permits cycle batching for an intermittent in vitro fertilization programme in Trinidad and Tobago.
Assisted reproductive technology (ART) in small island states like Trinidad and Tobago is usually provided in batches so as to minimize the cost of providing the service. As a result, patients 'cycles have to be synchronized in order to coincide with the arrival of a visiting embryologist. This is a retrospective study which evaluates the experience of pre-treatment with an oral contraceptive pill (OCP) as a means of batching cycles for an intermittent ART programme. Seventy-four in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles in which OCP usage was employed (Group A), were compared with 121 cycles which did not require pharmaceutical manipulation (Group B). In both groups more than 50% of women were older than 36-years. Two cycles were cancelled in Group A and seven in Group B, because of poor ovarian response. Although the pregnancy rate per treatment cycle was higher in Group A than in Group B (26.3% vs 17.3%), this difference was not significant. More spontaneous miscarriages occurred in the non-OCP women and ovarian cyst formation was more common in these women. The authors experience indicates that the OCP is a simple, cheap and efficient means of batching patients for an intermittent ART programme and can be utilized in other small ART centres.
Adult↗
[Effect of reagent batch on activated partial thromboplastin time in canine plasma and use of the ratio system for standardization].
In order to test the variability of the results of the activated partial thromboplastin time (APTT) in different reagent batches, 40 samples (20 from healthy dogs, 15 from patients with prolonged APTT as a result of different congenital or acquired haemostasis disorders, 5 from healthy dogs after in vitro addition of heparin) were used to compare 6 different lot Nos. of two commercial APTT-reagents (Pathromtin, PTT Reagent). Although the Friedman test showed a reagent batch dependency (p < 0.0001) for both reagents, only minor quantitative differences were observed with a variation coefficient of 2.7% (Pathromtin) and 2.4% (PTT Reagent), respectively. A second experiment was based on 105 samples measured with two batches of a third reagent (APTT-FS) with remarkable differences of results. Convergence of the results was achieved by converting into ratio values (quotient measurement value/control). However, statistical comparison still showed a significant difference. The study shows the good reproducibility of the APTT measured with different batches of the reagents Pathromtin and PTT Reagent in canine plasma, indicating that standardization is unnecessary. A standardization based on the ratio system can be used for reagents with a low batch consistency, requiring a high-quality control.
Animals↗
Sialidase activity in culture fluid of Chinese hamster ovary cells during batch culture and its effects on recombinant human antithrombin III integrity.
Sialidase activity in cell-free supernatant of batch-cultivated Chinese hamster ovary (CHO) cells producing human recombinant antithrombin III (rhAT III) was monitored during cultivation using 4-methylumbelliferyl substrate and HPLC for free sialic acid determination. Supernatant sialidase as well as lactate dehydrogenase activity increased significantly during batch growth. The enhanced number of dead cells correlated with increasing sialidase activity which seemed to be principally due to cell lysis, resulting in release of cytosolic sialidase. Loss of terminally alpha (2-->3) bound sialic acids of the oligosaccharides of rhAT III was analyzed in lectin-based Western blot and enzyme-linked lectin assays, using Maackia amurensis and Datura stramonium agglutinins for specific determination of Neu5Ac alpha (2-->3)Gal- and Gal beta (1-->4)-GlcNAc-terminated glycoproteins, respectively. Results show a remarkable loss of terminal sialic acids of rhAT III along with decrease in CHO cell viability and concomitant increase of dead cells throughout long-term batch cultivation. To avoid this degradation effect, process parameters forcing high viability are essential and harvesting of culture at an early time even at suboptimal recombinant protein concentration is highly recommended to avoid product desialylation.
Animals↗
Effects of extracellular polymeric substances on aerobic granulation in sequencing batch reactors.
The effects of extracellular polymeric substances on aerobic granulation in sequencing batch reactors were investigated by evaluating the content and compositions of extracellular polymeric substances, and the relationship between extracellular polymeric substances composition and surface properties of glucose-fed aerobic granules. The results showed that extracellular polymeric substances could affect surface properties of cells, such as surface charge and hydrophobicity, enhance polymeric interaction and promote aerobic granulation. Moreover, extracellular polymeric substances were produced mainly in the exponential phase, and served as carbon and energy source in starvation phase during granulation process, thus regulating the growth of bacteria in the interior and exterior of granules, and maintaining the integrality of granules.
Aerobiosis↗
Effects of fed-batch fermentation and pH profiles on nisin production in suspended-cell and biofilm reactors.
A biofilm reactor not only shortens the lag phase of nisin production, but also enhances nisin production when combined with an appropriate pH profile. Due to the substrate inhibition that takes place at high levels of carbon source, fed-batch fermentation was proposed as a better alternative for nisin production. In this study, the combined effects of fed-batch fermentation and various pH profiles on nisin production in a biofilm reactor were evaluated. The tested pH profiles include 1) a constant pH profile at 6.8 (profile 1), 2) a constant pH profile with an autoacidification after 4 h (profile 2), and 3) a step-wise pH profile with pH adjustment every 2 h (profile 3). When profile 1 was applied, fed-batch fermentation enhanced nisin production for both suspended-cell (4,188 IU ml(-1)) and biofilm (4,314 IU ml(-1)) reactors, yielded 1.8- and 2.3-fold higher nisin titer than their respective batch fermentation. On the other hand, pH profiles that include periods of autoacidification (profiles 2 and 3) resulted in a significantly lower nisin production in fed-batch fermentation (2,494 and 1,861 IU ml(-1) for biofilm reactor using profile 2 and 3, respectively) due to toxicity of excess lactic acid produced during the fermentation. Overall, this study suggested that fed-batch fermentation can be successfully used to enhance nisin production for both suspended-cell and biofilm reactors.
Anti-Bacterial Agents↗
The inhibitory effects of lindane in batch and upflow anaerobic sludge blanket reactors.
In this study, the inhibitory effects of lindane (LIN) on originally unacclimated mixed anaerobic cultures were investigated by anaerobic toxicity assay (ATA) experiments. ATA experiments revealed that 10 mg/l LIN exerted inhibitory effects on anaerobic cultures, which was recoverable. Continuous reactor experiments conducted to determine the inhibitory effects of LIN and the maximum LIN loading rate achievable in two-stage upflow anaerobic sludge blanket (UASB) reactors indicated that anaerobic granular cultures were successfully acclimated to 30 mg/l LIN. The maximum LIN loading rate and minimum hydraulic retention time (HRT) possible for the UASB system were 10 mg/l day and 18 h, respectively, which resulted in the overall chemical oxygen demand (COD) removal efficiency of 89%.
Adaptation, Physiological↗
Further studies related to the scale-up of high cell density Escherichia coli fed-batch fermentations: the additional effect of a changing microenvironment when using aqueous ammonia to control pH.
In this work, we report on the further development of the scale-down, two-compartment (STR + PFR) experimental simulation model. For the first time, the effect on high cell density Escherichia coli fed-batch fermentations of a changing microenvironment with respect to all three of the major spatial heterogeneities that may be associated with large-scale processing (pH, glucose, and dissolved oxygen concentration) were studied simultaneously. To achieve this, we used traditional microbiological analyses as well as multiparameter flow cytometry to monitor cell physiological response at the individual cell level. It was demonstrated that for E. coli W3110 under such conditions in a 20 m(3) industrial fed-batch fermentation, the biomass yield is lower and final cell viability is higher than those found in the equivalent well-mixed, 5L laboratory scale case. However, by using a combination of the well-mixed 5L stirred tank reactor (STR) with a suitable plug flow reactor (PFR) to mimic the changing microenvironment at the large scale, very similar results to those in the 20 m(3) reactor may be obtained. The similarity is greatest when the PFR is operated with a mean residence time of 50 sec with a low level of dO(2) and a high glucose concentration with either a pH of 7 throughout the two reactors or with pH controlled at 7 in the STR by addition into the PFR where the pH is > 7.
Ammonia↗
Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 2. Effects of serum concentration, dissolved oxygen concentration, and medium pH in a batch reactor.
The effects of serum, dissolved oxygen (DO) concentration, and medium pH on hybridoma cell physiology were examined in a controlled batch bioreactor using a murine hybridoma cell line (167.4G5.3). The effect of serum was also studied for a second murine hybridoma cell line (S3H5/gamma 2bA). Cell growth, viability, cell density, carbohydrate and amino acid metabolism, respiration and energy production rates, and antibody production rates were studied. Cell growth was enhanced and cell death was decreased by increasing the serum level. The growth rates followed a Monod-type model with serum being the limiting component. Specific glucose, glutamine, and oxygen uptake rates and specific lactate and ammonia production rates did not change with serum concentrations. Amino acid metabolism was slightly influenced by the serum level. Cell growth rates were not influenced by DO between 20% and 80% air saturation, while the specific death rates were lowest at 20-50% air saturation. Glucose and glutamine uptake rates increased at DO above 10% and below 5% air saturation. Cell growth rate was optimal at pH 7.2. Glucose and glutamine uptake rates, as well as lactate and ammonia production rates, increased above pH 7.2. Metabolic rates for glutamine and ammonia were also higher below pH 7.2. The consumption or production rates of amino acids followed the glutamine consumption very closely. Cell-specific oxygen uptake rate was insensitive to the levels of serum, DO, and pH. Theoretical calculations based on experimentally determined uptake rates indicated that the ATP production rates did not change significantly with serum and DO while it increased continually with increasing pH. The oxidative phosphorylation accounted for about 60% of total energy production. This contribution, however, increased at low pH values to 76%. The specific antibody production rate was not growth associated and was independent of serum and DO concentrations and medium pH above 7.20. A 2-fold increase in specific antibody production rates was observed at pH values below 7.2. Higher concentrations of antibody were obtained at high serum levels, between 20% and 40% DO, and at pH 7.20 due to higher viable cell numbers obtained.
Amino Acids↗
Bench-scale production of acrylamide using the resting cells of Brevibacterium sp. CH2 in a fed-batch reactor.
Effects of various organic acids and salts on the stabilization of nitrile hydratase were investigated. The stability of the nitrile hydratase of Brevibacterium CH2 during storage was greatly enhanced by the addition of n-butyric acid. Effects of temperature, pH, and concentrations of acrylonitrile and n-butyric acid on acrylamide production by the resting cells were also investigated. Acrylamide production per unit dry weight of the cells increased 1.33 times by the addition of 0.05% n-butyric acid. A 20% acrylamide solution was successfully produced in a bench-scale reactor (12 l) with only a trace amount of salts after 10 h of hydration reaction under optimum reaction conditions without using an isotonic substrate. The conversion yield was nearly 100%, and acrylic acid as a by-product was not produced. Final acrylamide production of 400 g g-1 cells and productivity of 20 g/(g cells l-1 x h-1) were obtained.
Acrylamide↗
Logistic equations effectively model Mammalian cell batch and fed-batch kinetics by logically constraining the fit.
A four-parameter logistic equation was used to fit batch and fed-batch time profiles of viable cell density in order to estimate net growth rates from the inoculation through the cell death phase. Reduced three-parameter forms were used for nutrient uptake and metabolite/product formation rate calculations. These logistic equations constrained the fits to expected general concentration trends, either increasing followed by decreasing (four-parameter) or monotonic (three-parameter). The applicability of this approach was first verified for Chinese hamster ovary (CHO) cells cultivated in 15-L batch bioreactors. Cell density, metabolite, and nutrient concentrations were monitored over time and used to estimate the logistic parameters by nonlinear least squares. The logistic models fit the experimental data well, supporting the validity of this approach. Further evidence to this effect was obtained by applying the technique to three previously published batch studies for baby hamster kidney (BHK) and hybridoma cells in bioreactors ranging from 100 mL to 300 L. In 27 of the 30 batch data sets examined, the logistic models provided a statistically superior description of the experimental data than polynomial fitting. Two fed-batch experiments with hybridoma and CHO cells in benchtop bioreactors were also examined, and the logistic fits provided good representations of the experimental data in all 25 data sets. From a computational standpoint, this approach was simpler than classical approaches involving Monod-type kinetics. Since the logistic equations were analytically differentiable, specific rates could be readily estimated. Overall, the advantages of the logistic modeling approach should make it an attractive option for effectively estimating specific rates from batch and fed-batch cultures.
Animals↗
The snowball effect in fed-batch bioreactions.
The bioreactor will play an important role in future biological manufacturing. For economic profit, important profiles of the feed rate in fed-batch cultures have been discussed. Unfortunately, the optimal feed rate is less robust. In these studies there exists the snowball effect in a substrate-inhibited bioprocess, in which substrate is accumulated due to uncertain parameters in the model or feed-rate error. The snowball effect also exists in multi-substrate-limited processes. In further studies, the interaction between the substrates has been higher in essential substrates than in growth-enhancing substrates. In a typical fed-batch bioreactor, the amount of the product can be reduced to 1% or less when the snowball effect arises. A new control structure, i.e., an off-line optimized feedforward controller added to a gain-scheduling PI(2)D feedback controller, is proposed to eliminate the troublesome snowball effect. The proposed control strategy recovers the yield up to 95%. Moreover, the robustness of the proposed control structure is demonstrated by simulation.
Algorithms↗
Effect of temperature on batch elastase production by Bacillus sp. EL31410.
The production of elastase by Bacillus sp. EL31410 at various temperatures was investigated. In order to study the effect of temperature on elastase fermentation, different cultivation temperatures, ranging from 39 degrees C to 28 degrees C, were evaluated in shake flask. The result indicated that 37 degrees C was best for cell growth at earlier stage; while maximum elastase activity was obtained when the cells were cultivated at 30 degrees C. This result was verified by batch fermentation in 5-L bioreactor under 37 degrees C and 30 degrees C temperature, respectively. The specific cell growth rate at 37 degrees C was higher than that at 30 degrees C during earlier stage of cultivation. The maximum value [5.5 U/(h x g DCW)] of elastase formation rate occurred at 24 h at 30 degrees C compared to 4.6 U/(h x g DCW) at 30 h at 37 degrees C. Based on these results, two-stage temperature shift strategy and oscillatory temperature cultivation mode were evaluated in the next study. When compared to single temperature of 37 degrees C or 30 degrees C, both two-stage temperature shift strategy and oscillatory temperature strategy improved biomass but did not yield the same result as expected for elastase production. The maximum biomass (both 8.6 g/L) was achieved at 30 h at 37 degrees C, but at 42 h using two-stage temperature cultivation strategy. The highest elastase production (652 U/ml) was observed at 30 degrees C in batch process. It was concluded that cultivation at constant temperature of 30 degrees C was appropriate for elastase production by Bacillus sp. EL31410.
Bacillus↗
Batch uptake of lysozyme: effect of solution viscosity and mass transfer on adsorption.
In this study, solid-phase adsorption by macroporous and hyper-diffusive resins was investigated in a batch uptake adsorption system to quantify solid-phase diffusion rates as a function of bulk phase viscosity. The performance of chromatographic resins used for adsorption of proteins is dependent on several factors including solid and liquid-phase diffusivity, boundary layer mass transfer, and intraparticle mass transfer effects. Understanding these effects is critical to process development and optimization of both packed and fluidized bed adsorption systems. The macroporous resin used here was Streamline SP, and the hyper-diffusive resin was S-HyperD LS. Both have been frequently used in fluidized bed adsorption of proteins; however, factors that affect uptake rates of these media are not well quantified. Adsorption isotherms were well represented by an empirical fit of a Langmuir isotherm. Solid-phase diffusion coefficients obtained from simulations were in agreement with other models for macroporous and hyper-diffusive particles. S-HyperD LS in the buffer system had the highest uptake rate, but increased bulk phase viscosity decreased the rate by approximately 50%. Increases in bulk phase viscosity increased film mass transfer effects, and uptake was observed to be a strong function of the film mass transfer coefficient. Uptake by Streamline SP particles was slower than S-HyperD in buffer, due to a greater degree of intraparticle mass transfer resistance. The effect of increased film mass transfer resistance coupled with intraparticle mass transfer resistances at an increased bulk phase viscosity resulted in a decrease of 80% in the uptake rate by Streamline SP relative to S-HyperD.
Adsorption↗
Effect of asynchronous nitrogen and energy supply on growth of ruminal bacteria in batch culture.
The effects of an asynchronous supply of fixed amounts of N and carbohydrate on bacterial growth were measured in two batch culture experiments. In Exp. 1, aqueous glucose and urea solutions were added at hourly intervals to culture flasks containing strained ruminal liquor and phosphate/bicarbonate buffer. The ratio of urea N to glucose was either constant (Synchrony, 26 mg of N/g of glucose) or increased exponentially over time (Asynchrony, .013 to 48,900 mg of N/g of glucose). After 12 h, identical quantities of glucose and urea had been added in both treatments. Bacterial population size (estimated from optical density) was greater (P less than .001) from 5 to 8 h of incubation for Synchrony than for Asynchrony, but after 12 h there was no difference (P greater than .1) between treatments. In Exp. 2, large (1 to 1.5 mm) and small (less than .5 mm) corn particles were used as slowly and rapidly degraded energy sources, with soybean meal (1 to 1.5 mm) and a papaic digest of soybean meal as sources of slowly and rapidly degraded N. At incubation times, when the ratio between total starch and N degraded was equal between treatments, bacterial population size was unaffected by the relative rate of N and OM supply. In both experiments, bacterial growth recovered quickly from transient restriction caused by deficits of N.
Ammonia↗