A new coiled hollow-fiber module design for enhanced microfiltration performance in biotechnology.

The microfiltration performance of a novel membrane module design with helically wound hollow fibers is compared with that obtained with a standard commercial-type crossflow module containing linear hollow fibers. Cell suspensions (yeast, E. coli, and mammalian cell cultures) commonly clarified in the biotechnology industry are used for this comparison. The effect of variables such as transmembrane pressure, particle suspension concentration, and feed flow rate on membrane performance is evaluated. Normalized permeation fluxes versus flow rate or Dean number behave according to a heat transfer correlation obtained with centrifugal instabilities of the Taylor type. The microfiltration performance of this new module design, which uses secondary flows in helical tubes, is significantly better than an equivalent current commercial crossflow module when filtering suspensions relevant to the biotechnology industry. Flux and capacity improvements of up to 3.2-fold (constant transmembrane pressure operation) and 3.9-fold (constant flux operation), respectively, were obtained with the helical module over those for the linear module.     

Dean vortex membrane microfiltration and diafiltration of rBDNF E. coli inclusion bodies

Cross-flow microfiltration (CMF) and diafiltration were used to concentrate and purify recombinant Brain-Derived Neutrophic Factor (rBDNF) inclusion bodies from an E. coli cell suspension and a homogenized E. coli cell suspension (homogenate/lysate). Although these processes have been tested industrially in pilot scale with conventional linear membrane microfiltration modules, their performances were severely limited due to membrane fouling. The purpose of this work was to determine whether Dean vortex microfiltration with controlled centrifugal instabilities (Dean vortices produced in helical flow) could be used to improve filtration performance over that observed with conventional linear cross-flow microfiltration (CMF). For the microfiltration experiments with the feeds containing cell and homogenate suspensions, improvements in flux of about 50 and 70%, respectively, were obtained with the helical module as compared with that obtained with the linear module. For diafiltration with the homogenate suspension as feed, solute transport (as measured by mass) was from 100 to 40% higher after 40 and 100 min, respectively, with the helical module as compared with that obtained with the linear module. In the presence of the neutral surfactant, Tween 20, solute transport for diafiltration was at least 25 times higher during the first 10 min of operation and 100% higher after 300 min with the helical module as compared with that obtained with the linear module. Clearly, improved filtration performance, a purer and more concentrated product, and substantial savings can be expected with the new Dean vortex filters.     

A predictive aggregate transport model for microfiltration of combined macromolecular solutions and poly-disperse suspensions: testing model with transgenic goat milk

To meet the technical challenge of recovering human IgG fusion protein from transgenic whole goat milk at reasonable cost with high purity and yield, a predictive aggregate transport model for microfiltration has been developed (Baruah and Belfort, 2003). Here, to test the model's predictability of permeate flux and mass transport, a comprehensive series of experiments with varying wall shear rate, feed temperature, feed concentration, and module design are presented. A very good fit was obtained between the model predictions and measurements for a wide variety of experimental conditions. For microfiltration module design comparison, a linear hollow fiber module (representing current commercial technologies) gave lower permeation flux and higher yield than a helical hollow fiber module (representing the latest self-cleaning methodology). These results are easily explained with the model that is now being used to define operating conditions for maximizing performance. The procedure described by the model is generalizable and can be used to obtain optimal filtration performance for applications other than milk.     

Optimized recovery of monoclonal antibodies from transgenic goat milk by microfiltration

The Predictive Aggregate Transport Model for microfiltration is used in combination with optimum fluid mechanics and electrostatics to maximize recovery of a heterologous immunoglobulin (IgG) from transgenic goat milk. The optimization algorithm involved varying pH (6.8-9), transmembrane pressure (2-4.5 psi), milk feed concentration (1-2X), membrane module type (linear vs. helical design), and axial velocity (Reynolds number: 830-1170). Operation in the pressure-dependent regime at low uniform transmembrane pressures (approximately 2 psi) using permeate circulation in co-flow, at the pI of the protein (9 in this case) was used to increase IgG recovery from less than 1% to over 95%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and attenuated total reflection Fourier transform infrared spectroscopy of the microfiltration permeate samples confirmed that all the fat globules and most of the casein micelles were retained in the MF membrane whereas a large amount of the target IgG was transported through the membrane. Transmembrane pressure and hence permeation flux was kept low (approximately 15 lmh) to maximize IgG membrane transport and thus recovery, due to a sparse deposit on the membrane which facilitated high solute transport. Next, an analytical method was used to optimize the diafiltration process using the aggregate transport model, experimental target protein sieving coefficients and permeation flux (Baruah and Belfort, 2003). The methodology reported here should be generalizable to the recovery of target proteins found in other complex suspensions of biological origin using the microfiltration process.     

Membrane fouling by cell-protein mixtures: in situ characterisation using multi-photon microscopy

Fouling of the membrane by cell and protein mixtures can result in severe flux declines, leading to the eventual need to clean or replace the membrane. In this study multi-photon microscopy, a fluorescence-based technique is used to 3-D image in situ the fouling of microfiltration membranes by suspensions containing combinations of washed yeast, bovine serum albumin (BSA) and ovalbumin. Appropriate fluorescent labelling allows the three foulant species to be clearly identified. Images correlate well with filtration data and clearly show the cake of yeast cells capturing protein aggregates. The proteins exhibited very different filtration behaviour. When filtering washed yeast together with ovalbumin and/or a 50:50 mixture by mass of BSA and ovalbumin, the ovalbumin fouling dominates the system. Capture of aggregates by the cake did not reduce fouling of the membrane by the protein and increased the resistance of the cake. For mixtures of BSA and washed yeast, the presence of a cake of yeast cells did reduce fouling of the membrane by the protein, however, the extra resistance due to the cake resulted in a flux lower than that when filtering BSA alone.     

A new integrated membrane filtration and chromatographic device

To improve protein separation, a novel integrated device combining membrane filtration and chromatography has been developed. The device basically consists of a hollow fiber filtration module whose shell side is filled with chromatographic resin beads. However, there is an essentially impermeable coated zone near the hollow fiber module outlet. The integrated device enjoys the advantages of both membrane filtration and chromatography; it also allows one to load the chromatographic media directly from the fermentation broth or lysate and separate the adsorbed proteins through the subsequent elution step in a cyclic process. Interfacial polymerization was carried out to coat the bottom section of the hollow fiber membrane; the rest of the hollow fiber membrane remained unaffected. Myoglobin (Mb) and alpha-lactalbumin (alpha-LA) were primarily used as model proteins in a binary mixture; binary mixtures of Mb and bovine serum albumin (BSA) were also investigated. Separation behaviors of binary protein mixtures were studied in devices having either an ultrafiltration (UF) or a microfiltration (MF) membrane. Experimental results show that the breakthrough time and the protein loading capacities were dramatically improved after introducing the impermeable coating in both UF and MF modules. For a synthetic yeast fermentation broth feed, four loading-washing-elution-reequilibration-based cyclic runs for separation of Mb and alpha-LA were performed in the device using a MF membrane with a coated zone without cleaning in between. The Mb and alpha-LA elution profiles for the four consecutive runs were almost superimposable. Due to lower transmembrane flux in this device plus the periodical washing-elution during the chromatographic separation, fouling was not a problem, unlike in conventional microfiltration.     

Effects of colloidal fouling and gas sparging on microfiltration of yeast suspension

Cross-flow microfiltration is an important step in separating Baker’s yeast (Saccharomyces cerevisiae) from aqueous suspension in many processes. However the permeate flux often declines rapidly due to colloidal fouling of membranes and concentration polarisation. The present work explores the possibility of maintaining acceptable permeate flux by co-current sparging of gas along with the feed, which would scour away colloidal deposits and reduce concentration polarisation of membranes. In this work, both washed and unwashed yeast were used to study the effect of washing to reduce protein fouling of membranes. It was found that permeate flux increased by 45% for liquid throughput of 75 kg/h for a feed concentration of 2.0 kg/m³ of washed yeast as compared with unwashed yeast suspension without gas sparging. For washed yeast suspension, the increase in gas flow rate from 0.5 lpm to 1.5 lpm (30 l/h to 90 l/h) had beneficial effect on permeate flux. It is concluded that in the present case, the gas flow rate should be less than or equal to the liquid flow rate for enhancement of permeates flux.     

Protein fouling of virus filtration membranes: effects of membrane orientation and operating conditions

The capacity of virus filters used in the purification of therapeutic proteins is determined by the rate and extent of membrane fouling. Current virus filtration membranes have a complex multilayer structure that can be used with either the skin-side up or with the skin-side facing away from the feed, but there is currently no quantitative understanding of the effects of membrane orientation or operating conditions on the filtration performance. Experiments were performed using Millipore's Viresolve 180 membrane under both constant pressure and constant flux operation with sulfhydryl-modified BSA used as a model protein. The capacity with the skin-side up was greater during operation with constant flux and at low transmembrane pressures, with the flux decline or pressure rise due primarily to osmotic pressure effects. In contrast, data obtained with the skin-side down showed a slower, steady increase in total resistance with the cumulative filtrate volume, with minimal contribution from osmotic pressure. The capacity with the skin-side down was significantly greater than that with the skin-side up, reflecting the different fouling mechanisms in the different membrane orientations. These results provide important insights for the design and operation of virus filtration membranes.     

Factors affecting the fermentative production of a lysozyme-binding antibody fragment in Escherichia coli

Fed-batch fermentation for production of a single-chain Fv antibody fragment (scFv) expressed as a recombinant periplastic protein from Escherichia coli was investigated. A high cell density of 50 g dry cell weight per liter was routinely achieved in a 14-L vessel by controlled exponential feeding of glucose to impose a constant specific growth rate. Following biomass accumulation, induction of the tac promoter by addition of IPTG was accompaied by a linear feed of yeast extract. The concentration of yeast extract feed was found to be highly influential upon both concentration and location of active product. Although scFv fragments were specifically targeted to the periplasmic space, at yeast extract feed rates of 0.72 g/h the final location was largely extracellular (68% to 79%). Total concentrations (extracellular + periplasmic) were of the order of 5 to 8 mg/L. A ten-fold increase in yeast extract supply increased total scFv concentration to almost 200 mg/L and 78% of this yield was retained in the periplasm. Control of such leakage of the recombinant product is fundamental to process design of downstream operations for product recovery. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 611-622, 1997.     

Application of rotary microfiltration in debittering process of spent brewer's yeast

This study concerns the production of yeast extract from spent brewer's yeast using rotary microfiltration as a means to combine debittering and cell debris separation into a single step, without using a toxic alkali wash. The pH of yeast homogenate was found to affect protein yield and bitterness of the product. Rotary filtration of yeast homogenate at various pHs resulted in different percent protein transmissions. These were found to be 5.05%, 9.83%, and 30.83% for pH 5, 6, and 7.5, respectively. The bitterness concentration in the permeate was also found to be higher at higher pHs. Autolysis of the cell homogenate prior to filtration increased protein yield and decreased bitterness considerably. At pH 5.5, the protein transmission was increased to 60% and debittering efficiency was increased from 59% to 86%. The permeate flux and protein productivity could be further increased by increasing the rotational speed, but this resulted in a decrease in debittering efficiency. Thus, the rotational speed should be carefully selected to compromise between the yield and product quality. Furthermore, for the tested rotational speeds of 600 and 1000 rpm, the change in feed flow rate from 11 to 35 L h(-1) changes the flow behavior from turbulent vortex flow to laminar vortex flow, thus decreasing the flux and protein productivity.     

High performance tangential flow filtration

Conventional tangential flow filtration (TFF) has traditionally been limited to separation of solutes that differ by about ten-fold in size. Wide pore-size distributions, membrane fouling, and concentration polarization phenomena have commonly been cited as reasons for this limitation. The use of TFF in the biotechnology industry has therefore been restricted to cell-protein, virus-protein, and protein-buffer separations. A multi-disciplinary team with industrial and academic members was formed to overcome these limitations and enable protein-protein separations using High Performance TFF (HPTFF) systems. Pore-size distributions have been improved with the development of new membrane formulation and casting techniques. Membrane fouling has been controlled by operating in the transmembrane pressure-dependent regime of the filtrate flux curve and by carefully controlling fluid dynamic start-up conditions. Concentration polarization was exploited to enhance, rather than limit, the resolution of solutes. Concentration polarization has also been controlled by operating a co-current filtrate stream that maintains transmembrane pressure constant along the length of the TFF module. High yields and purification factors were obtained even with small differences in protein sieving. IgG-BSA and BSA monomer-oligomer mixtures have successfully been separated with these systems. HPTFF technology provides a competitive purification tool to complement chromatographic processing of proteins. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 71-82, 1997.     

Copper II complex of a tridentate ligand: an artificial metalloprotease for bovine serum albumin

A copper(II) complex of 2, 6-bis(benzimidazo-2-yl) pyridine was synthesized and its binding properties with bovine serum albumin (BSA) has been evaluated. The binding plot obtained from the absorption titration data gives a binding constant of 2.4 (+/-0.3) x10(3) M(-1). It was found that the charge transfer band of the metal complex was perturbed in the presence of BSA. The gel electrophoresis pattern of BSA incubated with copper(II) complex shows the metalloproteolytic activity of the metal complex. In the presence of oxygen, protein undergoes site-specific cleavage by binding to the histidine residues of domain III, with the resultant formation of four fragments of molecular weight 49, 45, 22 and 17 kDa. This indicates the presence of two specific binding sites in the protein molecule. In the absence of molecular oxygen, the metal complex was found unable to cleave the protein. The circular dichroism (CD) spectrum of the isolated fragments shows nearly 38% and 32% of alpha helical content in 49 and 45 kDa fragments, respectively, which shows that the cleavage leads to no changes in the secondary structure of the protein fragments.     

酵母饲料的营养价值及应用研究

本文报道了以热带假丝酵母ＺＡＵ－１为生产菌株,应用固体发酵技术生产的酵母饲料的营养价值,并通过对比饲养试验评价应用效果。试验结果表明,粗蛋白提高１１．９７％,粗脂肪提高２．４４倍;赖氨酸、蛋氨酸和苏氨酸分别提高８０．００％、３４．０７％和６９．４２％;维生素Ｂ２提高１１．４１倍,Ｂ５和Ｂ６分别提高１９．５７％和６８．８４％;维生素Ｅ提高６３．４３倍,还含有维生素Ａ和Ｄ３用多种微量元素。饲养试验结果     

Cellulose affinity purification of fusion proteins tagged with fungal family 1 cellulose-binding domain

N- or C-terminal fusions of red-fluorescent protein (RFP) with various fungal cellulose-binding domains (CBDs) belonging to carbohydrate binding module (CBM) family 1 were expressed in a Pichia pastoris expression system, and the resulting fusion proteins were used to examine the feasibility of large-scale affinity purification of CBD-tagged proteins on cellulose columns. We found that RFP fused with CBD from Trichoderma reesei CBHI (CBD(Tr)(CBHI)) was expressed at up to 1.2g/l in the culture filtrate, which could be directly injected into the cellulose column. The fusion protein was tightly adsorbed on the cellulose column in the presence of a sufficient amount of ammonium sulfate and was efficiently eluted with pure water. Bovine serum albumin (BSA) was not captured under these conditions, whereas both BSA and the fusion protein were adsorbed on a phenyl column, indicating that the cellulose column can be used for the purification of not only hydrophilic proteins but also for hydrophobic proteins. Recovery of various fusion proteins exceeded 80%. Our results indicate that protein purification by expression of a target protein as a fusion with a fungal family 1 CBD tag in a yeast expression system, followed by affinity purification on a cellulose column, is simple, effective and easily scalable.     

Crossflow filtration of yeast broth cultivated in molasses

A broth of yeast cells cultivated in molasses was crossfiltered with a thin-channel module. The permeation flux gradually decreased at a constant cell concentration. The flux was much lower than that obtained for yeast broth cultivated in yeast extract, polypeptone, and dextrose (YPD) medium during the filtration. The flux did not depend on the membrane pore size (0.45 to 5 mum). The steady-state flux was one-twentieth that calculated for a cake filtration mode from the amount of cake per unit filtration area and the specific resistance of the cake measured in a dead-end filtration apparatus. The lower flux was due to small particles (most of which were less than 1 mum in diameter) in the molasses. The mehanism of crossflow filtration of broths of yeast cells cultivated in molasses was clarified by analysis of the change in flux with time and observations with scanning electron microscopy. At the initial stage of crossflow filtration the yeast cells and particles from the molasses were deposited on the membrane to form the molasses were deposited on the membrane to form a cake in a similar way to dead-end filtration. After the deposition of cells onto the membrane ceased, the fine particles from molasses formed a thin layer, which had higher resistance than the cake formed next to the membrane. The backwashing method was effective to increase the flux. The flux increased low when the pore size was 0.45 to 0.08 mum, but using larger pores of 3 to 5 mum it returned almost to the bases line. (c) 1994 John Wiley & Sons, Inc.     

Copper(II) complex of a tridentate ligand: an artificial metalloprotease for bovine serum albumin

A copper(II) complex of 2, 6-bis(benzimidazo-2-yl) pyridine was synthesized and its binding properties with bovine serum albumin (BSA) has been evaluated. The binding plot obtained from the absorption titration data gives a binding constant of 2.4 (±0.3) ×10³ M⁻¹. It was found that the charge transfer band of the metal complex was perturbed in the presence of BSA. The gel electrophoresis pattern of BSA incubated with copper(II) complex shows the metalloproteolytic activity of the metal complex. In the presence of oxygen, protein undergoes site-specific cleavage by binding to the histidine residues of domain III, with the resultant formation of four fragments of molecular weight 49, 45, 22 and 17 kDa. This indicates the presence of two specific binding sites in the protein molecule. In the absence of molecular oxygen, the metal complex was found unable to cleave the protein. The circular dichroism (CD) spectrum of the isolated fragments shows nearly 38% and 32% of alpha helical content in 49 and 45 kDa fragments, respectively, which shows that the cleavage leads to no changes in the secondary structure of the protein fragments.     

A multitechnique approach to probe the interaction of a therapeutic tyrosine kinase inhibitor nintedanib and bovine serum albumin

Drug and protein interaction provides a structural guideline in the rational drug designing and in the synthesis of new and improved drugs with greater efficacy. We have examined here the interaction tendency and mechanism of nintedanib (NTB), an anticancer drug (tyrosine kinase inhibitor) with bovine serum albumin (BSA), by spectroscopic techniques. The decline in Stern–Volmer quenching constants and binding constant with the temperature rise suggests that BSA forms a complex with NTB. Binding constant obtained by modified Stern–Volmer equation at 3 temperatures was realized to be of the order of ~10⁴?M^?1. Negative ΔG (~?5.93?kcal?mol^?1), ΔH (?3.74?kcal?mol^?1), and ΔS (?1.50?kcal?mol^?1) values exhibited a spontaneous and exothermic reaction between BSA and NTB. NTB molecule interacts with BSA by forming hydrogen bonds, as elucidated by fluorescence results. Moreover, a minor increment in the helical conformation of BSA upon its binding to NTB was observed by circular dichroism spectroscopy. The modification in protein’s symmetry and a decline in hydrodynamic radii were observed in the presence of NTB (from ~3.6 to ~3?nm) as obtained by the dynamic light scattering measurement results.     

Effect of an unstirred layer on the membrane permeability of anandamide

To study the effect of an unstirred layer (UL), we have investigated the exchange efflux kinetics of anandamide at 0 degrees C, pH 7.3, from albumin-free as well as from albumin-filled human red blood cell ghosts to media of various BSA concentrations ([BSA](o)). The rate constant (k(m)) of unidirectional flux from the outer membrane leaflet to BSA in the medium increased with the square root of [BSA](o) in accordance with the existence of a UL, which is a water layer adjacent to the membrane that is not subject to the same gross mixing that takes place in the rest of the medium. From k(m), it is possible to calculate the rate constant of anandamide dissociation from BSA (k(1)) if we know the membrane binding of anandamide, the equilibrium dissociation constant of BSA-anandamide complexes, and the diffusion constant of anandamide. We estimated k(1) to be 3.33 +/- 0.27 s(-1). The net flux of [(3)H]anandamide is balanced by an equal and opposite movement of nonradioactive anandamide in exchange efflux experiments. This means that our results are also valid for uptake. We show that for anandamide with rapid membrane translocation, UL causes a significant resistance to cellular uptake. Depicting the rate of anandamide uptake as a function of equilibrium water phase concentrations results in a parabolic uptake dependence. Such apparent "saturation kinetics" is often interpreted as indicating the involvement of transport proteins. The validity of such an interpretation is discussed.     

流加发酵提高产胡萝卜素红酵母产量的研究

本文对产胡萝卜素的红酵母进行了间歇、恒速及变速流加发酵实验,建立了简单的数学模型控制流加。实验结果表明,变速流加可显著地提高红酵母的产量,当流加控制因子K为88×10－5,变速流水发酵比间歇发酵红酵母产量提高了56．2％。     

Activity coefficients of salts in highly concentrated protein solutions: I. Alkali chlorides in isoionic bovine serum albumin solutions

In order to understand the thermodynamic state of simple salts in living cells, the mean activity coefficients of LiCl, NaCl, KC1, RbCl, CsCl were determined in concentrated isoionic bovine serum albumin (BSA) solutions by use of the EMF method with ion exchange membrane electrodes. The protein concentration range extended up to 22 wt %, whereas the salt concentration was kept constant at 0.1 mole per kilogram water. These solutions may be regarded as crude but appropriate model systems for the cytoplasm of cells as far as type and magnitude of the macromolecular component influence on the chemical potential of the salts is concerned. The mean stoichiometric activity coefficients of the alkali chlorides in the isoionic BSA solutions decreased linearly with the protein molality; this decrease, however, did not exceed ca. 10% compared with the pure 0.1 molal salt solutions. Only very small differences in the behaviour of the different alkali chlorides were observed. The results may be interpreted by the superposition of the effects of specific Cl^? ion binding to BSA and BSA bound “non-solvent” water with probably electrostatic long range interactions of the BSA(Cl^?)_v polyions with the salt ions in solution. The resulting mean activity coefficients, corrected for ion binding and non-solvent water, showed a very slight linear dependence on the protein concentration. The departure from the value in the pure 0.1 molal salt solutions did not exceed ± 2%.