Studies on Box-Behnken design experiments: cellulose acetate-polyurethane ultrafiltration membranes for BSA separation

Ultrafiltration membranes were prepared from mixtures of cellulose acetate-polyurethane blend membranes. During the last 1 or 2 decades, the concentration purification and separation of Albumin by ultrafiltration through semipermeable membranes have been put into practice and hence membrane separation is considered as the unit operation. The blend solution was prepared from cellulose acetate and polyurethane in polar solvent in presence of polyvinylpyrrolidone as additive. The performance of modified blend membranes applied for Bovine Serum Albumin (BSA) separation by ultrafiltration technique using Box-Behnken design with three variables: additive, time and pressure. Three different levels was studied to identify a significant correlation between the effect of these variables on the amount of separation of BSA. The methodology identifies the principal experimental variables, which have the greatest effect on the separation process. The experimental values are in good agreement with predicted values, the correlation coefficient was found to be 0.9871.     

Effects of chemical sanitization using NaOH on the properties of polysulfone and polyethersulfone ultrafiltration membranes

Membranes used in bioprocessing applications are typically sanitized before use to insure aseptic operation. However, there is almost no information in the literature on the effects of this preuse sanitization step on the properties of the membrane. Experiments were performed with commercially available hollow fiber polysulfone (PSf) and polyethersulfone (PES) membranes with different nominal molecular weight cutoffs. Data were obtained for the membrane hydraulic permeability, dextran retention coefficients, zeta potential (surface charge), and extent of protein adsorption both before and after sanitization with 0.5 N NaOH at 45°C for 30 min. Changes in chemical composition were examined using ATR‐FT‐IR and XPS. Sanitization caused a large increase in the net negative charge for all membranes. There was a small reduction in hydraulic permeability and a significant increase in dextran retention for the polyethersulfone membranes, consistent with a reduction in the effective pore size. Spectroscopic analyses suggest that this change is likely due to the base‐catalyzed hydrolysis of the lactam ring in polyvinylpyrrolidone (PVP) that is typically is used as a wetting/pore‐forming agent in PSf and PES membranes. Preuse sanitization also appeared to have a small effect on protein adsorption, although the extent of adsorption was quite low for both the virgin and sanitized membranes. The observed changes in membrane properties could have a significant impact on the ultrafiltration performance, demonstrating the importance of standardizing the sanitization procedures even in process development and scale‐down validation studies. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:90–96, 2015     

Performance characteristics of nanoporous carbon membranes for protein ultrafiltration

Nanoporous carbon membranes could be very attractive for applications of ultrafiltration in the biotechnology industry because of their greater mechanical strength and longer membrane life. The objective of this study was to obtain quantitative data on the performance characteristics of nanoporous carbon membranes formed within a stainless steel support that was first modified by deposition of silica particles within the macroporous support. The nanoporous carbon membrane effectively removed small solutes from a protein solution using diafiltration, with performance comparable to that of commercial polymeric membranes. Protein fouling was evident, although the nanoporous carbon membranes were easily regenerated; cleaning with 0.5 N NaOH at 50 degrees C completely restored the water permeability for multiple cycles. The nanoporous carbon membranes were also compatible with steam sterilization. Significant increases in process flux could be obtained using periodic back-pulsing, with no evidence of any structural alterations in the membrane. These results clearly demonstrate the potential benefits and opportunities for using nanoporous carbon membranes for protein ultrafiltration.     

Rapid separation of liposomes using ultrafiltration

Summary In order to make effective use of liposomes in studying biological systems, rapid and efficient methods are needed to separate liposomes from their unencapsulated contents. The method of partition ultrafiltration was used to monitor the release of a radioactive marker, tritium-labelled cytosine arabinoside (³H-Ara-C), from temperature-sensitive liposomes. Other physical techniques, dialysis, chromatography and ultracentrifugation, were also employed, and their effectiveness for separating ³H-Ara-C from the liposomes was studied. The results show that ultrafiltration is superior to the other physical techniques examined.     

Self-cleaning membranes for ultrafiltration

In order to reduce the severe flux losses encountered during ultrafiltration of protein solutions, proteases were immobilized on Ultrafiltration membranes to hydrolyze the deposited solute molecules. Over a standard 22 hr run 25 to 78% improvement in cumulative permeate yield was obtained when processing 0.5% albumin or hemoglobin. It was also demonstrated that the flux enhancements were due to the biochemical action of the absorbed protease and not to its physical effect as a prefilter coat. with the aid of a model retardation of gel formation mechanism was demonstrated. Economics of the system were shown to be favorable, improving the rate of return on capital investment up to 50% by reduction of the total membrane area of the plant.     

Characteristics of collodion membranes for ultrafiltration

An apparatus for the production of graded collodion membranes is described. The theoretical considerations of calibration are discussed in relation to the demonstrable structure and statistical characteristics of the membranes. A new definition of an "end-point" is suggested.     

Selective separation of tryptic beta-casein peptides through ultrafiltration membranes: Influence of ionic interactions

Peptide separation by selective membrane filtration has numerous potential applications such as production of peptides with biological activities or spectific enrichment in compounds acting as flavoring agents or as growth factors required by the fermentation industry. The retention of peptides arising from tryptic hdroysis of beta-casein using an M5 Carbosep membrane (molecular wieght cutoll = 10,000 D) has been studied. The peptides with known sequences were characterized by their molecular weight, isoelectric point, and hydrophobicity. Our experiments highlighted that their transmission involves mechanisms other than size exclusion as developed elsewhere. The effect of ionic interactions between peptides and membrance has been investgated by vrying pH, ionic strength of bulk, and electric potential of filtering material. The charge of both peptides and membrane plays an important role in the transmission, particularly with small size and high or lkow isoelectric point. Then, peptides with the same sign as the membrane have lower transmission than expected from the size xclusion model, whereas peptides with opposite sign have higher trnsmission than expected, and even higher than 1 with some of them. (c) 1995 John Wiley & Sons, Inc.     

Rational optimization of culture conditions for the most efficient ethanol production in Scheffersomyces stipitis using design of experiments

Optimization of culture parameters for achieving the most efficient ethanol fermentation is challenging due to multiple variables involved. Here we presented a rationalized methodology for multi‐variables optimization through the design of experiments DoE approach. Three critical parameters, pH, temperature, and agitation speed, affecting ethanol fermentation in S. stipitis was investigated. A predictive model showed that agitation speed significantly affected ethanol synthesis. Reducing pH and temperature also improved ethanol production. The model identified the optimum culture conditions for the most efficient ethanol production with the yield and productivity of 0.46 g/g and 0.28 g/l h, respectively, which is consistent with experimental observation. The results also indicated the scalability of the model from shake flask to bioreactor. Thus, DoE is a promising tool permitting the rapid establishment of culture conditions for the most efficient ethanol fermentation in S. stipitis. The approach could be useful to reduce process development time in lignocellulosic ethanol industry. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Selective separation of homogeneous catalysts using silicalite membranes

The recovery of a homogeneous catalysts can be performed effectively by means of a zeolite membrane, which in many cases has a crystalline structure with pores smaller than transition metal catalysts but larger than the products. This concept has been successfully applied to the Diels-Alder reaction between acroleine and cyclopentadiene, catalyzed by a cationic dinuclear Pd(II) complex. A reaction mixture containing 5-norbornene-2-carboxyaldehyde and the catalyst employed for its synthesis [Pd(μ-Cl)(Ph₂P-(CH₂)₄-PPh₂)](CF₃SO₃)₂ shows close to 100% recovery of the metallic compound.     

Efficient optimization of time-varying inputs in a fed-batch cell culture process using design of dynamic experiments

In cell culture process development, we rely largely on an iterative, one-factor-at-a-time procedure based on experiments that explore a limited process space. Design of experiments (DoE) addresses this issue by allowing us to analyze the effects of process inputs on process responses systematically and efficiently. However, DoE cannot be applied directly to study time-varying process inputs unless an impractically large number of bioreactors is used. Here, we adopt the methodology of design of dynamic experiments (DoDE) and incorporate dynamic feeding profiles efficiently in late-stage process development of the manufacture of therapeutic monoclonal antibodies. We found that, for the specific cell line used in this article, (1) not only can we estimate the effect of nutrient feed amount on various product attributes, but we can also estimate the effect, develop a statistical model, and use the model to optimize the slope of time-trended feed rates; (2) in addition to the slope, higher-order dynamic characteristics of time-trended feed rates can be incorporated in the design but do not have any significant effect on the responses we measured. Based on the DoDE data, we developed a statistical model and used the model to optimize several process conditions. Our effort resulted in a tangible improvement in productivity—compared with the baseline process without dynamic feeding, this optimized process in a 200-L batch achieved a 27% increase in titer and > 92% viability. We anticipate our application of DoDE to be a starting point for more efficient workflows to optimize dynamic process conditions in process development.     

Biocatalytic membranes for ultrafiltration treatment of wastewater containing dyes

A possibility to prepare the biofunctional membranes showing the biocatalytic properties and use those in post-treatment of wastewater containing synthetic dyes have been established. Selected Pseudomonas mendocina and Bacillus subtilis cultures were used as biocatalysts for dye destruction. It has been established that cells in spore form are able to survive in N-methylpyrrolidone that allow to use method of polymer solution casting for membrane preparation. The optimal conditions for entrapping of whole cells of microorganisms into the polymer matrix have been determined. Membrane biocatalytic activity has been studied depending on method of casting solution preparation, biocatalyst loading and operating parameters. Dye destruction occurs both in membrane pores and on membrane surface. Membrane obtained provide discolouring of treated solutions (permeate). The dye concentration in retentate depends on the trans-membrane fluxes. The concentration in retentate need not be observed at relatively low fluxes (up to 20 l/m² h).     

Virus removal from bioproducts using ultrafiltration membranes modified with latex particle pretreatment

Ultrafiltration is an attractive process for virusremoval from bioproducts owing to its high throughputas well as the fact that the operation is carried outunder ambient conditions (damage to proteins is highlylimited). The principal concern regarding the adoptionof conventional ultrafiltration membranes for virusremoval is the possibility of the virus passingthrough abnormally large pores or surfaceimperfections on the membrane surface. The chiefprinciple behind the present work is to pretreat themembrane by blocking the abnormally large pores usinglatex particles. Experimental work was conducted tovalidate this pretreatment using the bacteriophagex174 as a model virus.The results attained were highly encouraging.Different sizes of latex particles were tested bytreating a 100 KD molecular weight cut-off membrane,and the transmission of phage (suspended in buffer)through this membrane assessed. In the absence of anyparticle pretreatment, a virus clearance of 4.78 logreduction value was observed for this membrane. Thetransmission of phage through the membrane could bereduced by an order of magnitude using 0.11 mlatex particles, or two orders of magnitude using acombination of 0.11 and 0.50 m particles.The application of latex particles did nothinder the transport of protein through the 100 KDmembrane. Protein sieving coefficients obtained usingthis membrane were 91%, 16% and 2%, for lysozyme,HSA and IgG, respectively.     

Immobilization of lipase by ultrafiltration and cross-linking onto the polysulfone membrane surface

In this study, a biphasic enzymatic membrane reactor was made by immobilizing Candida Rugosa lipase onto the dense surface of polysulfone ultrafiltration membrane by filtration and then cross-linking with glutaraldehyde solution. The reactor was further applied for the hydrolysis of olive oil, the performance of which was evaluated in respect of apparent reaction rate based on the amount of fatty acids extracted into the aqueous phase per minute and per membrane surface. It was found that the ultrafiltration and cross-linking process greatly improved the reaction rate per unit membrane area and the enzyme lifetime. The highest reaction rate reached 0.089 micromol FFA/min cm2 when the enzyme loading density was 0.098 mg/cm2. The results also indicated that the performance of lipase immobilized on the membrane surface was superior to that immobilized in the pores, and the apparent reaction rate and stability of immobilized lipases were improved greatly after cross-linking. It suggested that immobilization of enzymes by filtration and then cross-linking the enzymes onto the membrane surface is a simple and convenient way to prepare a high-activity immobilized enzyme membrane.     

Multifactorial screening design and analysis of SELDI-TOF ProteinChip array optimization experiments

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry is a powerful tool for rapidly generating protein expression data (peptide and protein profiles) from a large number of samples. However, as with any technology, it must be optimized and reproducible for one to have confidence in the results. Using a classical statistical method called the fractional factorial design of experiments, we assessed the effects of 11 different experimental factors. We also developed several metrics that reflect trace quality and reproducibility. These were used to measure the effect of each individual factor, and the interactions between factors, to determine optimal factor settings and thus ultimately produce the best possible traces. Significant improvements to output traces were seen by simultaneously altering several parameters, either in the sample preparation procedure or during the matrix preparation and application procedure. This has led to the implementation of an improved method that gives a better quality, reproducible, and robust output.     

Influence of polysulfone and hemophan hemodialysis membranes on phagocytes

The aim of the study was to compare the effect of hemophane and polysulfone membranes on the phagocyte-derived production of reactive oxygen species (ROS) as well as on neutrophil CD11b and CD62L expression in patients undergoing regular hemodialysis. The effects of hemodialysis membranes were also studied in in vitro conditions after coincubating them with differentiated HL-60 cells. ROS production was measured using chemiluminometric and flow cytometric methods. Expression of CD11b, CD62L and mitochondrial membrane potential were detected by monoclonal antibodies and by the JC-1 fluorescent probe, respectively. Depressed ROS production was observed in patients already before dialysis. Further decrease in ROS production and an increase in CD11b expression were observed especially in patients after hemophan hemodialysis. Decreased ROS production and increased CD11b expression were observed also after incubation of HL-60 cells with hemophan membranes. Mitochondrial membrane potential dropped only after incubating cells with hemophan membranes proving its more serious adverse effects in comparison with the polysulfone membrane. In conclusion, deleterious effects of hemodialysis on the metabolic activity of phagocytes were proved. Combining chemiluminescent and flow cytometric methods for the detection of ROS production and determining mitochondrial membrane potential can be useful tools for the analysis of material biocompatibility.     

Evaluation of ultrafiltration membranes with biological macromolecules

Eighteen ultrafiltration membranes ranging in molecular weight cutoff ratings from 500 to 300,000 were tested with water, 0.5M NaCl solution, and, in some cases, with macromolecules and urea in a 3-in. stirred filter cell. Approximately half of the membranes showed a significant decrease in filtration rate during the first 24-hr period. The steady-state rates were less than the manufacturers' rating for about two thirds of the membranes, the discrepancy being greater for the membranes with high molecular weight cutoffs. The filtration rates were linearly dependent on applied pressure over the range at least as great as 15 to 55 psig. The rate decreased as the concentration of macromolecules such as transfer ribonucleic acid (tRNA) increased; the rate for a concentration of 3 mg tRNA/per ml was one-fourth of that observed when no tRNA was present. Some increase in rate (～33 to 50%) was obtained by increasing the stirring speed from 100 rpm to 1000 rpm. The membranes were effective for desalting and concentration of macromolecules but not for separation of large molecules from each other, such as tRNA from bovine serum albumin. Easily denatured molecules such as catalase were not deactivated by filtration at 4°C.     

Application of statistical design for the optimization of amino acid separation by reverse-phase HPLC

Modified resolution and overall separation factors used to quantify the separation of complex chromatography systems are described. These factors were proven to be applicable to the optimization of amino acid resolution in reverse-phase (RP) HPLC chromatograms. To optimize precolumn derivatization with phenylisothiocyanate, a 2^5-1 fractional factorial design in triplicate was employed. The five independent variables for optimizing the overall separation factor were triethylamine content of the aqueous buffer, pH of the aqueous buffer, separation temperature, methanol/acetonitrile concentration ratio in the organic eluant, and mobile phase flow rate. Of these, triethylamine concentration and methanol/acetonitrile concentration ratio were the most important. The methodology captured the interaction between variables. Temperature appeared in the interaction terms; consequently, it was included in the hierarchic model. The preliminary model based on the factorial experiments was not able to explain the response curvature in the design space; therefore, a central composite design was used to provide a quadratic model. Constrained nonlinear programming was used for optimization purposes. The quadratic model predicted the optimal levels of the variables. In this study, the best levels of the five independent variables that provide the maximum modified resolution for each pair of consecutive amino acids appearing in the chromatograph were determined. These results are of utmost importance for accurate analysis of a subset of amino acids.     

Strain design optimization using reinforcement learning

Engineered microbial cells present a sustainable alternative to fossil-based synthesis of chemicals and fuels. Cellular synthesis routes are readily assembled and introduced into microbial strains using state-of-the-art synthetic biology tools. However, the optimization of the strains required to reach industrially feasible production levels is far less efficient. It typically relies on trial-and-error leading into high uncertainty in total duration and cost. New techniques that can cope with the complexity and limited mechanistic knowledge of the cellular regulation are called for guiding the strain optimization.In this paper, we put forward a multi-agent reinforcement learning (MARL) approach that learns from experiments to tune the metabolic enzyme levels so that the production is improved. Our method is model-free and does not assume prior knowledge of the microbe’s metabolic network or its regulation. The multi-agent approach is well-suited to make use of parallel experiments such as multi-well plates commonly used for screening microbial strains.We demonstrate the method’s capabilities using the genome-scale kinetic model of Escherichia coli, k-ecoli457, as a surrogate for an in vivo cell behaviour in cultivation experiments. We investigate the method’s performance relevant for practical applicability in strain engineering i.e. the speed of convergence towards the optimum response, noise tolerance, and the statistical stability of the solutions found. We further evaluate the proposed MARL approach in improving L-tryptophan production by yeast Saccharomyces cerevisiae, using publicly available experimental data on the performance of a combinatorial strain library.Overall, our results show that multi-agent reinforcement learning is a promising approach for guiding the strain optimization beyond mechanistic knowledge, with the goal of faster and more reliably obtaining industrially attractive production levels.