A new isolation and purification method for staphylococcal protein A using membrane encapsulated rabbit IgG-agarose

A new isolation and purification method for bioproducts using membrane-encapsulated affinity adsorbents was investigated. The new method involves encapsulation of affinity adsorbents, batch adsorption of the bioproduct from whole fermentation broth and rapid batch desorption after dissolution of the capsule membranes. Recovery of protein A from Staphylococcus aureus was used as the model experimental system. Affinity adsorbents such as rabbit IgG-agarose were successfully encapsulated within calcium alginate membranes and used directly to recover protein A from whole cell homogenate containing a number of macromolecular contaminants as well as suspended solids. Both high yield and high purity of protein A were recovered by this method in comparison with various previously reported methods.     

Bilirubin removal from human plasma by Cibacron Blue F3GA using immobilized microporous affinity membranous capillary method

A novel affinity sorbent system for direct bilirubin removal from human plasma was developed. These new adsorbents comprise Cibacron Blue F3GA as the specific ligand, and microporous membranous poly(tetrafluoroethylene) capillary (modified by coating with a hydrophilic layer of poly(vinyl alcohol) after activation) as the carrier matrix. The affinity adsorbents carrying 126.5 micromol Cibacron Blue F3GA/g polymer was then used to remove bilirubin in a flow-injection system. Non-specific adsorption on the poly(vinyl alcohol) coated capillary remains low, and higher affinity adsorption capacity, of up to 76.2 mg/g polymer was obtained after dye immobilization. The bilirubin adsorption capacity of the affinity capillary decreased with increase in the recirculation rate of plasma. The adsorption capacity increased with increase the temperature while decreased with increase the ionic strength. The maximum adsorption was only observed in neutral solution (pH 6-7). The adsorption isotherm fitted the Langmuir model well. These new adsorbents have higher velocity of mass transfer, better adsorption capacity, less fouling, longer service life and good reusability. The results of blood tests suggested the dye affinity capillary has good blood compatibility.     

Plasmid adsorption to anion-exchange matrices: comments on plasmid recovery

A number of anion-exchange adsorbents were constructed, employing nonporous silica fibers, and examined with the aim of describing factors that influence desorption and recovery of plasmid DNA (pDNA). The fibers were provided with ligands via adsorption of the polymeric amines poly(ethyleneimine) or chitosan, or via graft-polymerization of primary, tertiary, or quaternary amine monomers to vinyl-silanized fibers. Several adsorbents showed an almost irreversible plasmid binding. It was suggested that important factors affecting the DNA releasing ability are (i) type of amine ligand used (primary amines bind plasmids the strongest), (ii) the structure of the nucleic acid (supercoiled pDNA may bind stronger than linear genomic DNA), (iii) shift of ligand pK(a) (due to the proximity of highly charged pDNA), and (iv) the solid support itself (steric factors may lead to kinetically stable complexes). The last factor was derived from several comparisons between support-bound ligand and free soluble ligand. It was thus observed that polyelectrolyte complexes associated with a surface were much more difficult to dissociate than the equivalent soluble complexes.     

Evaluation of two different Concanavalin A affinity adsorbents for the adsorption of glucose oxidase

Concanavalin A (Con A) was selected as ligand and thus immobilized onto two different supports, namely the polymeric Toyopearl and the inorganic silica, with the protection of its binding sites provided during the coupling procedure. The prepared Con A affinity adsorbents were then employed to evaluate their adsorption behaviour for the enzyme glucose oxidase (GOD). The immobilization kinetics showed that the immobilization of Con A on silica supports was much faster than that on Toyopearl supports, which could highly reduce the possibility of the denaturation of Con A. The optimal adsorption conditions for binding of GOD onto the ligand were determined in terms of the pH value and the ionic strength of the adsorption medium. The adsorption isotherms for binding GOD onto two Con A affinity adsorbents fitted well with the Langmuir equation. The maximum adsorption capacity q(m) of Toyopearl Con A and silica Con A were 7.9 mg/ml and 4.9 mg/ml, with a dissociation constant K(d) of 4.8 x 10(-7)M and 2.6 x 10(-6)M, respectively. Due to the less diffusive resistance, silica Con A showed both higher adsorption and desorption rates for GOD when compared with Toyopearl Con A. The nonspecific adsorption of GOD was less than 8% for both end-capped Toyopearl and silica supports. The dynamic adsorption of GOD for five times repeated processes showed a high stability for both prepared adsorbents. All the results indicate a good suitability of both Con A adsorbents for affinity adsorption of GOD.     

Binding isotherms for soluble immobilized affinity ligands from spectral titration

The method of spectral titration has been applied to binding equilibria between proteins and soluble immobilized ligands and evaluated using the interaction between Cibacron blue-dextran conjugates and lysozyme. The method is both simple and rapid and provides a convenient screening technique for characterization of soluble adsorbents designed for use in aqueous two-phase affinity extraction or as liquid-phase models for affinity chromatography systems. The results indicate that regardless of ligand density a constant 28% of the total coupled dye is available for high-affinity protein binding at saturation. The dissociation constant for the dye-protein interaction, however, decreases with dye loading. The potential for kinetic investigations has been demonstrated using a stopped-flow apparatus. The results indicate that a simple rate equation is inadequate to describe the data for lysozyme binding to dye-dextran conjugates. A modified model, which better describes the data, was developed by including a second rate limiting process, the transition from stacked to unstacked dye ligands on the dextran backbone. This effect could have practical significance for protein binding kinetics in affinity chromatography, especially in high-performance liquid affinity chromatography applications where mass transfer is rapid. (c) 1992 John Wiley & Sons, Inc.     

A novel process of cyclodextrin production by the use of specific adsorbents: Part I. Screening of specific adsorbents

Novel adsorbents that are composed of ligand, spacer and support were chemically synthesized and the two consecutive screenings of them made it possible to determine the adsorbents that were most recommendable for α- and β-cyclodextrin (CD) production. First, ligands of high adsorption selectivity for each CD were screened from among the candidates (carboxylic acids) that are tied in ionic bonds to two types of strongly basic anion exchange resins as support. Secondly, ligand derivatives (as ligand and spacer) were bound in covalent bonds onto chilosan beads as support and then, the most suitable spacer length for the CDs' adsorption selectivity and capacity were investigated. An optimal mol ratio of ligand to amino group of the beads was also examined. Stearic acid was the most effective ligand for α-CD, whereas cyclohexanepropanamide-n-caproic acid was the best for β-CD. Adsorption selectivity of adsorbents derived from carboxylic acids (stearic and/or palmitic) and chitosan beads was almost 100%, while their adsorption capacities were large enough to meet the demand for economic production and purification of CDs on an industrial scale.     

Operational intensification by direct product sequestration from cell disruptates: application of a pellicular adsorbent in a mechanically integrated disruption-fluidised bed adsorption process

A novel prototype adsorbent, designed for intensified fluidised bed adsorption processes, was assembled by the emulsification coating of 4% (w/v) porous agarose upon a zirconia-silica solid core. The adsorbent, designated ZSA (particle density 1.75 g/ml, maximum pellicle depth 40 microm), was subjected to physical and biochemical comparison with the performance of two commercial adsorbents (Streamline and Macrosorb K4AX). Bed expansion qualities and hydrodynamic characteristics (N, D(axl) and B(o)) of ZSA demonstrated a marked robustness in the face of elevated velocities (up to 550 cm/h) and biomass loading (up to 30% (ww/v)) disrupted yeast cells. Cibracron Blue derivatives of the pellicular prototype (ZSA-CB), evaluated in the batch and fluidised bed recovery of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from unclarified yeast disruptates, exhibited superior capacities and adsorption/desorption performance to the commercial derivatives. These advanced physical and biochemical properties facilitated a demonstration of the direct, mechanical coupling of bead-milling and fluidised bed adsorption in a fully integrated process for the accelerated recovery of G3PDH from yeast. The generic application of such pellicular adsorbents and integrated processes to the recovery of labile, intracellular products is discussed.     

乳腺生物反应器表达的重组人乳铁蛋白的分离纯化及生物活性鉴定

以国产高交联度的快流速琼脂糖为基质,合成了不同配基密度的SP(Sulfopropyl,磺酸基)离子交换介质,建立了乳腺生物反应器表达重组人乳铁蛋白(Recombinant Human Lactoferrin,rHLF)的纯化方法。以溶菌酶为模型蛋白考察了不同配基密度离子交换介质的静态和动态吸附行为,结果表明介质具有良好的吸附性能。不同配基密度离子交换介质均可纯化得到rHLF,其中,高配基密度(0.24mol/L)的离子交换介质每毫升可以处理50mL rHLF牛乳,rHLF收率为86.5%,纯度为98.5%。圆二色谱的测定结果表明纯化的rHLF二级结构与天然人乳铁蛋白一致。生物学功能实验结果表明,rHLF的铁结合与释放活性与天然人乳铁蛋白相似,浓度为5g/L的rHLF对大肠杆菌的生长具有明显的抑制作用。     

Fabrication and characterisation of a novel pellicular adsorbent customised for the effective fluidised bed adsorption of protein products

A dense pellicular solid matrix has been fabricated by coating 4% agarose gel on to dense zirconia-silica (ZS) spheres by water-in-oil emulsification. The agarose evenly laminated the ZS bead to a depth of 30 μm, and the resulting pellicular assembly was characterised by densities up to 2.39 g/mL and a mean particle diameter of 136 μm. In comparative fluidisation tests, the pellicular solid phase exhibited a two-fold greater flow velocity than commercial benchmark adsorbents necessary to achieve common values of bed expansion. Furthermore, the pellicular particles were characterised by improved qualities of chromatographic behaviour, particularly with respect to a three-fold increase in the apparent effective diffusivity of lysozyme within a pellicular assembly modified with Cibacron Blue 3GA. The properties of rapid protein adsorption/desorption were attributed to the physical design and pellicular deployment of the reactive surfaces in the solid phase. When combined with enhanced feedstock throughput, such practical advantages recommend the pellicular assembly as a base matrix for the selective recovery of protein products from complex, particulate feedstocks (whole fermentation broths, cell disruptates and biological extracts).     

In situ and Ex situ adsorption and recovery of betalains from hairy root cultures of Beta vulgaris

Various adsorbents were screened for in situ recovery of betalain pigments effluxed from hairy root cultures of red beet, Beta vulgaris. Alumina/silica (1:1) appeared ideal, showing in situ adsorption of 97% in a unit time of 30 min accounting for in situ recovery of 71.39% of the total betalaine effluxed. Other adsorbents such as Amberlite series (XAD-2 and -4), cyclodextrin, maltodextrin, dextrin white, and starches such as wheat starch and corn starch exhibited very poor in situ adsorption properties. Pretreatment of adsorbents with methanol significantly improved the adsorption capacities of some of the adsorbents, with a highest adsorption of 97.2% for alumina followed by alumina/silica (1:1) and higher adsorption by XAD-2 and -4. Complete in situ adsorption equilibrium was reached in 20 min for a solution containing 2.5 mg mL(-)(1) of betalain in adsorbents alumina, silica, and a mixture of alumina and silica. In situ betalain adsorption parameters for alumina/silica were determined using the Langmuir isotherm model where the adsorption capacity was found to be 0.174 mg g(-)(1) and the adsorption energy was 0.9 at pH 5.5 and 25 degrees C. Desorption of pigments from the adsorbents was invariably highest in poor adsorbents, indicating their poor adsorption energy for betalaines. Similarly, recovery by desorption was low in those adsorbents having high adsorption capacity, indicating that adsorbents such as activated ones with highest adsorption capacity with zero desorption property were unsuitable for the recovery of effluxed pigments. Ex situ recovery of betalain done using various combinations of alumina/silica and processed sand and different column geometries indicated that alumina with processed sand at a 2:1 ratio (w/w) and a minimum column material of 2 cm height and 2 cm diameter was good enough to cause 97% pigment adsorption from a solution containing 1.6 mg mL(-)(1). Desorption and recovery of pigments ex situ from columns were affected by various elution mixtures, where a gradient elution with ascending levels of HCl/ethanol in water resulted in 100% recovery of adsorbed pigments in a significantly lesser volume of eluent in a short period of 1 h. Different pigment flow rates of 0.2, 0.3, and 3.1 mL s(-)(1) through a column of alumina/processed sand indicated that a pigment equilibrium concentration of 0.18 mg mL(-)(1) at flow rates of 0.02 and 0.3 mL s(-)(1) resulted in a breakthrough at 110 and 14 min adsorbing 16.9 and 16.91 mg g(-)(1) betalain, respectively. From the breakthrough curves, the column capacities for respective flow rates were calculated as 8.86 and 9.6 mg g(-)(1), and the higher flow rates resulted in earlier breakthrough with lower capacity. Observations made in the present study are useful to develop a process for the on-line recovery of betalains effluxed from hairy roots.     

Use of affinity chromatography for the purification of specific endonuclease Eco RI and Bg1 II

The highly active preparations of specific endonucleases Eco RI and Bgl II were purified by affinity chromatography from E. coli and Bacillus globiggii cells, respectively. The isolation and purification procedures included cell disruption by ultrasonication, ultracentrifugation and chromatography. Blue dextrane-Sepharose, folate-Sepharose and phenyl-Sepharose were used as affinity adsorbents. The optimal conditions for the adsorption and elution of the endonucleases excluding intermediate steps of dialysis and concentration were selected. A high degree of purification was achieved by a consecutive use of adsorbents with different ligands. The purified enzyme does not contain non-specific nucleases or phosphatases, is sufficiently concentrated and can be used for specific hydrolysis of DNA.     

Derivatization of epoxy-activated agarose with various carbohydrates for the preparation of stable and high-capacity affinity adsorbents: Their use for affinity chromatography of carbohydrate-binding proteins

Two types of affinity adsorbents for lectins were prepared by new simple procedures. Both types of adsorbents had high ligand concentration and chemically stable linkage between ligand and Sepharose 4B. Oligosaccharide ligands were coupled by reductive amination with sodium cyanoborohydride to amino-Sepharose 4B prepared by amination of epoxy-activated Sepharose 4B. The glycamyl-Sepharose 4B thus obtained had particularly high adsorption capacities for lectins; lactamyl-Sepharose 4B, 58 mg/l ml of gel for peanut lectin; maltamyl-Sepharose 4B, 146 mg/ml for concanavalin A; and tetra-N-acetylchitotetraamyl-Sepharose 4B, 36 mg/ml for wheat germ agglutinin. Hexosamine was coupled by the aid of carbodiimide to carboxyl-Sepharose 4B prepared by succinylation of amino-Sepharose 4B. Galactosamine-Sepharose 4B adsorbed 145 mg soybean agglutinin/l ml gel. The columns turned from a semitransparent white to a milky white as they were saturated with lectins.     

Biomimetic-dye affinity adsorbents for enzyme purification: application to the one-step purification of Candida boidinii formate dehydrogenase

Formate dehydrogenase (FDH, EC 1.2.1.2) was purified from Candida boidinii cells in a single step by biomimetic-dye affinity chromatography. For this purpose, seven' biomimetic analogues of the monochlorotriazine dye, Cibacron(R) Blue 3GA (CB3GA), and parent dichloro-triazine dye, Vilmafix((R)) Blue A-R (VBAR), bearing a car-boxylated structure as their terminal biomimetic moiety, were immobilized on crosslinked agarose gel, Ultrogel((R)) A6R. The corresponding new biomimetic-dye adsorbents, along with nonbiomimetic adsorbents bearing CB3GA and VBAR, were evaluated for their ability to purify FDH from extracts obtained after press-disintegration of C. boidinii cells. Optimal conditions for maximizing specific activity of FDH in starting extracts (1.8 U/mg) were realized when cell growth was performed on 4% methanol, and press disintegration proceeded in four consecutive passages before the homogenate was left to stand for 1 h (4 degrees C). When compared to nonbiomimetic adsorbents, biomimetic adsorbents exhibited higher purifying ability. Furthermore, one immobilized biomimetic dye, bearing as its terminal biomimetic moiety mercap-topyruvic acid linked on the chlorotriazine ring (BM6), displayed the highest purifying ability. Adsorption equilibrium data which were obtained for the BM6 adsorbent in a batch system corresponded well to the Langmuir isotherm and, in addition, breakthrough curves were taken for protein and FDH adsorption in a fixed bed of BM6 adsorbent. The dissociation constant ( K(D)) of the complex between immobilized BM6 and FDH was found to equal 0.05 muM. Adsorbent BM6 was employed in the purification of FDH from a 18-L culture of C. boidinii in a single step (60% overall yield of FDH). The purified FDH afforded a single-band on sodium dodecyl sulphate poly-acrylamide gel electrophoresis, and a specific activity of 7,0 U/mg (30 degrees C). (c) 1995 John Wiley & Sons, Inc.     

Rate-limiting mass transfer in immunosorbents: characterisation of the adsorption of paraquat-protein conjugates to anti-paraquat Sepharose 4B

A series of paraquat-protein conjugates of different molecular size has been prepared by the coupling of paraquat hexanoate to the proteins lysozyme, ovalbumin, bovine serum albumin. The characteristics of the adsorption of these conjugates to an immunosorbent consisting of monoclonal anti-paraquat antibodies covalently immobilised to Sepharose 4B have been determined. Equilibrium adsorption isotherms were found to obey the Langmuir equation and indicated that 80% or more of the antibody binding sites were accessible to the conjugates. The rates of mass transfer of the conjugates to their adsorption sites on the immobilised antibodies was well described by a model in which mass transfer is controlled by transfer across the external film and diffusion within the porous adsorbent bead. The effective diffusivities of the conjugates within the immunosorbent were measured and has allowed the effect of the size of the adsorbing molecule on the rate of adsorption to be considered. The amount of paraquat that could be adsorbed and the rates of adsorption decreased as the size of the protein to which it is conjugated increased. The diffusivity of the conjugates within the pores of the adsorbent is reduced between two and five times compared to their diffusivities in free solution. The reduction is greater for the larger proteins and the variations of the effective diffusivities and the pore diffusivities with the molecular weight of the conjugate can be well described with simple correlations.     

Human serum albumin chromatography by Cibacron Blue F3GA-derived microporous polyamide hollow-fiber affinity membranes

An affinity dye ligand, Cibacron Blue F3GA was covalently attached onto commercially available microporous polyamide hollow-fibre membranes for human serum albumin (HSA) adsorption from both aqueous solutions and human plasma. Different amounts of Cibacron Blue F3GA were incorporated on the polyamide hollow-fibres by changing the dye attachment conditions, i.e. initial dye concentration, addition of sodium carbonate and sodium chloride. The maximum amount of Cibacron Blue F3GA attachment was obtained at 42.5 μmol g⁻¹ when the hollow-fibres were treated with 3 M HCl for 30 min before performing the dye attachment. HSA adsorption onto unmodified and Cibacron Blue F3GA-derived polyamide hollow-fibre membranes was investigated batchwise. The non-specific adsorption of HSA was very low (6.0 mg g⁻¹ hollow-fibre). Cibacron Blue F3GA attachment onto the hollow-fibres significantly increased the HSA adsorption (147 mg g⁻¹ hollow-fibre). The maximum HSA adsorption was observed at pH 5.0. Higher HSA adsorption was observed from human plasma (230 mg HSA g⁻¹ hollow-fibre). Desorption of HSA from Cibacron Blue F3GA derived hollow-fibres was obtained using 0.1 M Tris–HCl buffer containing 0.5 M NaSCN or 1.0 M NaCl. High desorption ratios (up to 98% of the adsorbed HSA) were observed. It was possible to reuse Cibacron Blue F3GA derived polyamide hollow-fibre without significant decreases in the adsorption capacities.     

Affinity adsorption of lysozyme on a macroligand prepared with Cibacron Blue 3GA attached to yeast cells

The objective of this study was the development of affinity adsorbent particles with the appropriate characteristics to be applied in protein purification using the affinity ultrafiltration method. To prepare affinity macroligands Cibacron Blue 3GA, as a ligand molecule, was immobilized by covalent bonding onto yeast cell walls, the support material or matrix. The maximum attachment of the ligand to the matrix was 212 μmol/g (ligand dry weight/yeast dry weight). Lysozyme was selected as the protein model for the adsorption studies. Its adsorption onto the matrix without ligand and matrix with attached ligand were investigated batch-wise. The adsorption equilibrium isotherms appeared to follow a typical Langmuir isotherm. The maximum adsorption capacity (q(m)) of the Cell-Cibacron macroligand for lysozyme was 110 mg/ml of wet macroligand. The adsorbent was also employed for the separation of lysozyme from hen egg white. High purity lysozyme was obtained.     

Preparation and evaluation of Ricinus communis agglutinin affinity adsorbents using polymeric supports

A practicable and efficient procedure for preparation of Ricinus communis agglutinin (RCA) affinity adsorbents has been developed. For immobilization of RCA two different polymer-based supports, Toyopearl and TSKgel (TosoHaas), were used. RCA has been successfully immobilized onto these supports with amounts of coupled ligand between 15 and 23 mg/g dry support and corresponding coupling yields of 69-93% (w/w). The prepared affinity adsorbents were characterized concerning their binding capacity for the glycoprotein asialofetuin (ASF) and accessibility of the ligand binding sites. The high accessibility of 80% showed that steric hindrance was negligible at the present ligand density. RCA-Toyopearl was successfully applied in affinity chromatography of glycoproteins indicating its high specificity. A long-term stability test proved no change in capacity for a period of at least 12 months. High-performance affinity chromatography (HPLAC) was carried out using RCA-TSKgel. Experimental results showed that the prepared adsorbents are suitable for selective separation of glycoproteins and oligosaccharides and therefore can be used for investigations of adsorption characteristics of glycoconjugates and for laboratory-scale preparations.     

Nisin adsorption on silica adsorbents

It has been shown that silica adsorbents can be used for adsorption of nizin from solutions obtained after centrifugation of the fermentation broth. The optimal structure of the adsorbent pores has been determined. Silica with pores 50 to 75 nm in size provided the highest adsorption rates. The value of silica adsorption of nizin depended on the medium pH. The maximum adsorption rates were observed at pH 6.5--7. At pH 3.5 the level of nizin adsorption was low.     

Performance of hydrophobic chromatography in purification of alpha-amylase

Hydrophobic ligands were introduced onto agarose beads, and the adsorption capacity of the beads was measured. The adsorption capacity increased with increase in the carbon number of the ligand, ionic strength of the buffer solution, and temperature. Crude alpha-amylase was purified with these hydrophobic adsorbents and the breakthrough and elution curves were estimated based on the mass transfer theory. Under strongly hydrophobic conditions, impurities contained in crude feeds and the lack of uniformity of packing caused by aggregation of beads affected adsorption and elution behaviors.     

Compatibility of column inlet and adsorbent designs for processing of corn endosperm extract by expanded bed adsorption

Corn has emerged as a viable host for expression of recombinant proteins; targeted expression to the endosperm has received particular attention. The protein extracts from corn endosperm differ from those of traditional hosts in regard to the nature of residual solids and extracted matrix contaminants. Each of these differences presents reasons for considering expanded bed adsorption for product capture and new considerations for limitations of the method. In this work three inlet-flow distribution devices (mesh, glass ballotini, and localized mixing) and six adsorbents with different physical (size and density), chemical (ligand), and base matrix properties were evaluated to determine conditions compatible with processing of crude corn endosperm extract by expanded bed adsorption. Of the inlet devices evaluated, the design with localized mixing at the inlet (as produced commercially by UpFront Chromatography A/S, Copenhagen, DK) allowed solids up to 550 microm into the column without clogging for all flow rates evaluated. A mesh at the inlet with size restriction of either 50 microm or 80 microm became clogged with very small corn particles (< 44 microm). When glass ballotini was used, large particles (550 microm) passed through for high flow rates (570 cm/h), but even small (< 44 microm) particles became trapped at a lower flow rate (180 cm/h). The physical and chemical properties of the resin determined whether solids could be eluted. The denser UpFront adsorbents allowed for complete elution of larger and more concentrated corn solids than the currently available Amersham Streamline adsorbents (Amersham Biosciences, Piscataway, NJ) as a result of the former's higher flow rate for the desired 2x expansion (570 cm/h for UpFront vs. 180 cm/h for Streamline). All corn solids < 162 microm eluted through nonderivatized UpFront resin. Larger corn solids began to accumulate due to their elevated sedimentation velocities. Feeds of < 44 microm solids at 0.45% and 2.0% dry weight successfully eluted through ion exchange adsorbents (DEAE and SP) from UpFront. However, significant accumulation occurred when the solids size increased to a feed of < 96 microm solids, thus indicating a weak interaction between corn solids and both forms of ion exchange ligands. Expanded beds operated with Streamline ion exchange adsorbents (DEAE and SP) did not allow full elution of corn solids of < 44 microm. A hyperdiffuse style EBA resin produced by Biosepra (Ciphergen Biosystems, Fremont, CA) with CM functionality showed a severe interaction with corn solids that collapsed the expanded bed and could not be eliminated with elevated flow rates or higher salt concentration.