On-line process analysis and control in biotechnology

Measuring techniques can be developed and adapted for the characterization of bioreactors, for on-line process analysis and for the determination of biological parameters of cells during fermentation. Mathematical models can be formulated for bioreactors and cell regulation and a combined model for these can be reduced for use in process control. Microprocessors and minicomputers give further scope for data acquisition, model implementation and process control.     

Optimization of a trypsin-bioreactor coupled with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry for quality control of biotechnological drugs

The optimization of a silica-based trypsin bioreactor and its use in the quality control of biotechnological drugs like peptides and proteins is described. Five bioreactors based on monolithic material have been prepared, with different amount of bound trypsin. The performances of these bioreactors were compared to the proteolytic activity of a bioreactor based on silica material. The trypsin-based chromatographic columns were coupled on-line with an LC/ESI/MS/MS system for digestion and identification of proteins. First, human serum albumin has been used as test protein to compare the ability of the bioreactors to hydrolyse high-molecular-weight proteins. The best chromatographic material (epoxy monolithic silica) and the optimum amount of enzyme bound (7.13 mg) have been identified to obtain the highest protein recovery and an analytical reproducibility of the whole digestion, separation and identification process. The optimized enzyme-reactor has been used for the on-line digestion of some biotechnological drugs such as somatotropin. Somatotropin for parentheral use has been analyzed, without sample pre-treatment, with both an on-line procedure and the traditional off-line procedure described in the European Pharmacopoeia. It was found that the cleavage efficiency (aminoacidic recovery, %AA) achieved within minutes by the developed protocol is at least comparable or even better than the conventional 4h consuming method.     

Development of flow injection potentiometric methods for the off-line and on-line determination of fluoride to monitor the biodegradation of a monofluorophenol in two bioreactors

Water treatment has become a source of concern as new pollutants and higher volumes of waste water must be treated. Emerging biological approaches, namely the use of bioreactors, for cleaning processes have been introduced. The use of bioreactors requires the development of efficient monitoring tools, preferably with real-time measurements. In this work, a couple of flow injection systems were developed and optimized for the potentiometric determination of fluoride to monitor a rotating biological contactor (RBC) bioreactor and a sequencing batch reactor (SBR) with off-line and on-line sampling. Both the RBC and the SBR bioreactors were set up for the biodegradation of the halogenated organic compound 2-fluorophenol and, as fluoride was a degradation byproduct, the process was monitored by following up its concentration.The described flow injection potentiometric methods enabled the fluoride determination within the required quantification range 0.10-100 mM. The possible interferences from the growth medium were minimized in-line. The determination rate was 78 h⁻¹ for the off-line monitoring of RBC and 50⁻¹ h for the on-line monitoring of the SBR, with a sample consumption of 0.500 mL and 0.133 mL per determination, respectively. Furthermore, the overall reagent consumption was quite low. The accuracy of the system was evaluated by comparison with a batch procedure. The SBR efficiency was monitored both on-line by the flow system and off-line by HPLC, for comparison purposes.     

Multiplexing fibre optic near infrared (NIR) spectroscopy as an emerging technology to monitor industrial bioprocesses

The application of near infrared spectroscopy in bioprocessing has been limited by its dependence on calibrations derived from single bioreactor at a given time. Here, we propose a multiplexed calibration technique which allows calibrations to be built from multiple bioreactors run in parallel. This gives the flexibility to monitor multiple vessels and facilitates calibration model transfer between bioreactors. Models have been developed for the two key analytes: glucose and lactate using Chinese hamster ovary (CHO) cell lines and using analyte specific information obtained from the feasibility studies. We observe slight model degradation for the multiplexed models in comparison to the conventional (single probe) models, decrease in r² values from 89.4% to 88% for glucose whereas for lactate from 92% to 91.8% and a simultaneous increase in the number of factors as the model incorporates the inter-probe variability, nevertheless the models were fit for purpose. The results of this particular application of implementing multiplexed-NIRS to monitor multiple bioreactor vessels are very encouraging, as successful models have been built on-line and validated externally, which proffers the prospect of reducing timelines in monitoring the vessels considerably, and in turn, providing improved control.     

On-line analysis and control of production of antibiotics

The prerequisites for the utility of on-line techniques for monitoring and control of biotechnological production processes are discussed. Aseptic filters, air-segmented continuous-flow automatic analyzers, flow-injection analyzers and on-line HPLC systems are discussed. These were used for the process analysis and control of penicillin V and cephalosporin C production in stirred-tank and airlift-tower loop reactors. The results of on-line and off-line analyses are compared, as are different analytical methods. The suitability of these techniques for process control is demonstrated in several examples.     

Optimization of Multiparameters for Increased Yields of Cytochrome B5 in Bioreactors

The production of recombinant proteins is gaining increasing importance as the market requests high quality proteins for several applications. However, several process parameters affect both the growth of cells and product yields. This study uses high throughput systems and statistical methods to assess the influence of fermentation conditions in lab-scale bioreactors. Using this methodology, it was possible to find the best conditions to produce cytochrome b5 with recombinant cells of Escherichia coli. Using partial least squares, the height-to-diameter ratio of the bioreactor, aeration rate, and PID controller parameters were found to contribute significantly to the final biomass and cytochrome concentrations. Hence, we could use this information to fine-tune the process parameters, which increased cytochrome production and yield several-fold. Using aeration of 1 vvm, a bioreactor with a height-to-ratio of 2.4 and tuned PID parameters, a production of 72.72 mg/L of cytochrome b5 in the culture media, and a maximum of product to biomass yield of 24.97 mg/g could be achieved.     

On-line application of ion chromatography in a thermal power plant

Complete monitoring of the “condensate-feedwater” cycle in power plants requires reliable automatic methods suitable for very low concentrations of various chemical species. A complete on-line ion-chromatography monitoring system is under development. It comprises an automatic on-line sampling system two Dionex Model QUIC (process instrument) ion chromatographs, an on-line calibration systems, and a data acquisition/processing system. The present model serves for the determination of sodium, chloride and sulfate ions. Detection limits are < 1 μg l^?1 (P = 95%) with liner ranges up to 10 μ l^?1 or about 200 μ l^?1 depending on settings. The importance of statistical evaluation of data is emphasized. The instrumentation was tested for sequential samples from crucial points of a 320-MW thermal power station with satisfactory results. Problems of reliability are discussed.     

On-line measurement of extracellular enzymes during fermentation by using membrane techniques

The measurement of enzymes during fermentation is of general interest for process control in industrial biotechnology. Studies are presented to develop an on-line measurement for extracellular hydrolases by using membrane separations as the main unit operation, first for achieving continuous sampling from the bioreactor prior to analysis, and secondly for measuring hydrolytic activities, by separating the cleavage products during reaction of enzymes with high-molecular-weight substrates by an ultrafiltration membrane. The evaluation of batchwise and continuous flow analysis for an alkaline protease and pullulanase are described, and the feasibility of on-line measurement during fermentation of Bacillus licheniformis and Klebsiella pneumoniae on a 30- and 70-l pilot scale is demonstrated, including aseptic continuous sampling.     

On-line coupling of counter-current chromatography and macroporous resin chromatography for continuous isolation of arctiin from the fruit of Arctium lappa L.

In this work, we have developed a novel hybrid two-dimensional counter-current chromatography and liquid chromatography (2D CCC × LC) system for the continuous purification of arctiin from crude extract of Arctium lappa. The first dimensional CCC column has been designed to fractionalize crude complex extract into pure arctiin effluent using a one-component organic/salt-containing system, and the second dimensional LC column has been packed with macroporous resin for on-line adsorption, desalination and desorption of arctiin which was effluent purified from the first CCC dimension. Thus, the crude arctiin mixture has been purified efficiently and conveniently by on-line CCC × LC in spite of the use of a salt-containing solvent system in CCC separation. As a result, high purity (more than 97%) of arctiin has been isolated by repeated injections both using the ethyl acetate–8% sodium chloride aqueous solution and butanol–1% sodium chloride aqueous solution. By contrast with the traditional CCC processes using multi-component organic/aqueous solvent systems, the present on-line CCC × LC process only used a one-component organic solvent and thus the solvent is easier to recover and regenerate. All of used solvents such as ethyl acetate, n-butanol and NaCl aqueous solution are low toxicity and environment-friendly. Moreover, the lower phase of salt-containing aqueous solution used as mobile phase, only contained minor organic solvent, which will save much organic solvent in continuous separation. In summary, our results indicated that the on-line hybrid 2D CCC × LC system using one-component organic/salt-containing aqueous solution is very promising and powerful tool for high-throughput purification of arctiin from fruits of A. lappa.     

Ultrasonic nebulization extraction coupled with on-line gas chromatography for determination of trans-anethole in spices

Ultrasonic nebulization extraction (UNE) coupled with on-line gas chromatography (GC) was proposed for the determination of trans-anethole in fruits of Illicium verum Hook. f. and Foeniculum vulgare Mill. The extraction was performed in a common self-made extraction system. In the UNE the analyte was transferred and enriched from the solid sample to gas phase. The sample gas containing analyte obtained by UNE was introduced into the sampling loop with the purging gas (N₂). And then the sample gas in the sampling loop was introduced into the GC column. Several experimental parameters of on-line UNE-GC, including sampling time, flow rate of purging gas, standstill time and temperature of tubing, were optimized. The calibration curve ranging from 0.05 to 1.5 mg g⁻¹ for determining the trans-anethole was obtained. The recoveries for determining trans-anethole are between 99.2% and 111.2% and RSDs are less than 8.3% when the UNE was applied. The analytes can rapidly be extracted and transferred from the solid sample to gas phase. The analytes in the gas phase are easier to be introduced into GC system than those in the solid and liquid phase. Compared with off-line systems, the proposed on-line system is more suitable to detect volatile compounds.     

Lipase production by solid-state fermentation

The production of lipase by Penicillium simplicissimum in solid-state fermentation was studied using babassu cake as the basal medium. Tray-type and packed-bed bioreactors were employed. In the former, the influence of temperature; content of the medium, and medium supplementation with olive oil, sugarcane molasses, corn steep liquor, and yeast hydrolysate was studied. For all combinations of supplements, a temperature of 30°C, a moisture content of 70%, and a concentration of carbon source of 6.25% (m/m, dry basis) provided optimum conditions for lipase production. When used as single supplements olive oil and molasses also were able to provide high lipase activities (20 U/g). Using packed-bed bioreactors and molasses-supplemented medium, optimum conditions for enzyme production were air superficial velocities above 55 cm/min and temperatures below 28°C. The lower temperature optimum found for these reactors is probably related to radial heat gradient formation inside the packed bed. Maximum lipase activities obtained in these bioreactors (26.4 U/g) were 30% higher than in tray-type reactors.     

On-line simultaneous monitoring of glucose and acetate with FIA during high cell density fermentation of recombinant E. coli

A two-channel flow injection analysis (FIA) system was developed for the simultaneous on-line monitoring of acetate and glucose during high cell density fed-batch fermentations of recombinant Escherichia coli. Acetate measurement was performed with a modified and optimised version of an existing method, based on acetate diffusion through a gas-diffusion chamber into a stream containing an acid-base indicator. The subsequent decrease in the absorbance was detected with an incorporated photometer. After method optimisation, it was possible to achieve linearity until 10 g/kg with no dilution step and with a detection level of 0.05 g/kg. Although some interferences were found, the performance of the method proved to be sufficiently reliable for on-line control purposes Commercially packed glucose oxidase (GOD) was used for the amperometric measurement of glucose. The method was linear up to 5 g/kg and it was possible to detect concentrations lower than 0.06 g/kg. For these measurements, no significant interferences were detected when the results were compared with other reference methods. The application of a simultaneous parallel configuration of the methods to a high cell density fed-batch E. coli fermentation was tested and reliable results were obtained within a 3 min delay. This information was made available to a supervisory computer running a developed LabVIEW™ programme via an Ethernet network, allowing the immediate implementation of control actions, improving the process performance.     

Four-channel enzyme thermistor system for process monitoring and control in biotechnology

A newly developed four-channel enzyme thermistor system is presented and its application to biotechnological process monitoring is discussed. Different sugars were detected on-line during the cultivation of Cephalosporium acremonium and Bacillus licheniformis on technical media and during a starch hydrolysis process with immobilized thermostable enzymes. Immobilized enzymes and entrapped microorganisms were used as biological compounds in this biosensor.     

Electrokinetically driven micro flow cytometers with integrated fiber optics for on-line cell/particle detection

This paper presents an innovative micro flow cytometer which is capable of counting and sorting cells or particles. This compact device employs electrokinetic forces rather than the more conventional hydrodynamic forces technique for flow focusing and sample switching, and incorporates buried optical fibers for the on-line detection of cells or particles. This design approach results in a compact microfluidic system and an easier integration process. The proposed cytometer integrates several critical modules, namely electrokinetic-focusing devices, built-in control electrodes, buried optical fibers for on-line detection, and electrokinetic flow switches for bio-particle collection. A linear relationship exists between the focused stream width (d) and the focusing ratio (F/φ), which is estimated to be D≈134.5−53.8F/φ. The relationship between the particle velocity (U) and the applied voltage (V) is also investigated. Numerical and experimental data confirm the effectiveness of the device when applied to the counting and sorting of 10 μm diameter particles and red blood cells.     

Ex Vitro Expansion of Human Placenta-Derived Mesenchymal Stem Cells in Stirred Bioreactor

The high demand of human placenta-derived mesenchymal stem cells (hPDMSCs) for therapeutic applications requires reproducible production of large numbers of well-characterized cells under well-controlled conditions. However, no method for fast hPDMSCs proliferation has yet been reported. In the present study, the feasibility of using a stirred bioreactor system to expand hPDMSCs was examined. hPDMSCs were cultured either in stirred bioreactors or in tissue culture flasks (T-flasks) for 5 days. Total cell density and several parameters of physical microenvironments were monitored in the two culture systems every 24 h. The maintenance of the antigenic phenotype of hPDMSCs before and after culturing in the stirred bioreactor system was cytometrically assessed. Data suggested that the physical microenvironment in the stirred bioreactors was much more favorable than that of the T-flasks. At the end of 144 h culturing, the total cell number was increased 1.73 times from the T-flasks to the stirred bioreactors. In addition, hPDMSCs could maintain their antigenic phenotype when cultured in stirred bioreactors. These results provide the initial assessment for large-scale hPDMSCs production using suspension culture bioreactors.     

Semiconductor gas sensors in bioreactor control

A simple semiconductor gas sensor (TGS 812) is used for the on-line measurement and control of indole during the production of l-tryptophan from indole and l-serine with immobilized E. coli cells. Indole is estimated in the reactor gas space. In combination with an automatic indole supply system, a feed-batch process became possible. The indole concentration was monitored and kept within the optimal range (300–600 mg l^?1). A simple gas-sensing electrode dipped in the reaction medium provides direct measurement of organic solvents and gases in the liquid. Such a system is suitable for on-line determination of ethanol (10–70 g l^?1) during continuous production of ethanol with immobilized yeast cells.     

Bioreactor monitoring with spectroscopy and chemometrics: a review

Biotechnological processes are crucial to the development of any economy striving to ensure a relevant position in future markets. The cultivation of microorganisms in bioreactors is one of the most important unit operations of biotechnological processes, and real-time monitoring of bioreactors is essential for effective bioprocess control. In this review, published material on the potential application of different spectroscopic techniques for bioreactor monitoring is critically discussed, with particular emphasis on optical fiber technology, reported for in situ bioprocess monitoring. Application examples are presented by spectroscopy type, specifically focusing on ultraviolet-visible, near-infrared, mid-infrared, Raman, and fluorescence spectroscopy. The spectra acquisition devices available and the major advantages and disadvantages of each spectroscopy are discussed. The type of information contained in the spectra and the available chemometric methods for extracting that information are also addressed, including wavelength selection, spectra pre-processing, principal component analysis, and partial least-squares. Sample handling techniques (flow and sequential injection analysis) that include transport to spectroscopic sensors for ex-situ on-line monitoring are not covered in this review.     

Trypsin immobilization in ordered porous polymer membranes for effective protein digestion

Fast and effective protein digestion is a vital process for mass spectrometry (MS) based protein analysis. This study introduces a porous polymer membrane enzyme reactor (PPMER) coupled to nanoflow liquid chromatography-tandem MS (nLC-ESI-MS/MS) for on-line digestion and analysis of proteins. Poly (styrene-co-maleic anhydride) (PS-co-MAn) was fabricated by the breath figure method to make a porous polymer membrane in which the MAn group was covalently bound to enzyme. Based on this strategy, microscale PPMER (μPPMER) was constructed for on-line connection with the nLC-ESI-MS/MS system. Its capability for enzymatic digestion with bovine serum albumin (BSA) was evaluated with varied digestion periods. The on-line proteolysis of BSA and subsequent analysis with μPPMER-nLC-ESI-MS/MS revealed that peptide sequence coverage increased from 10.3% (digestion time 10 min) to 89.1% (digestion time 30 min). μPPMER can efficiently digest proteins due to the microscopic confinement effect, showing its potential application in fast protein identification and protease immobilization. Applications of on-line digestion using μPPMER with human plasma and urinary proteome samples showed that the developed on-line method yielded equivalent or better performance in protein coverage and identified more membrane proteins than the in-solution method. This may be due to easy accommodation of hydrophobic membrane proteins within membrane pores.     

On-line coupling of macroporous resin column chromatography with direct analysis in real time mass spectrometry utilizing a surface flowing mode sample holder

A surface flowing mode sample holder was designed as an alternative sampling strategy for direct analysis in real time mass spectrometry (DART-MS). With the sample holder, the on-line coupling of macroporous resin column chromatography with DART-MS was explored and the new system was employed to monitor the column chromatography elution process of Panax notoginseng. The effluent from macroporous resin column was first diluted and mixed with a derivatization reagent on-line, and the mixture was then directly transferred into the ionization region of DART-MS by the sample holder. Notoginsenosides were methylated and ionized in a metastable helium gas stream, and was introduced into MS for detection. The on-line system showed reasonable repeatability with a relative standard deviation of 12.3% for the peak area. Three notoginsenosides, i.e. notoginsenoside R₁, ginsenoside Rb₁ and ginsenoside Rg₁, were simultaneously determined during the eluting process. The alteration of the chemical composition in the effluent was accurately identified in 9 min, agreeing well with the off-line analysis. The presented technique is more convenient compared to the traditional UPLC method. These results suggest that the surface flowing mode DART-MS has a good potential for the on-line process monitoring in the pharmaceutical industry.     

Achievement of optimum laser cleaning in the restoration of artworks: expected improvements by on-line optical diagnostics

In view of the optimization of the laser cleaning techniques in the conservation of artworks, non-standard laser systems and specific intervention methodologies were devised on the basis of shadowgraphic and spectroscopic diagnostics analyses. Optimized cleaning procedures were achieved through on-line diagnostics that had the role to monitor and control the interaction process. The potential improvements of the laser cleaning treatment by integration of time-resolved shadowgraphy and plasma spectroscopy as on-line or off-line diagnostic techniques are discussed.