Application of magnetic agarose support in liquid magnetically stabilized fluidized bed for protein adsorption

A novel magnetic agarose support (MAS) was fabricated for application in a liquid magnetically stabilized fluidized bed (MSFB). It was produced by water-in-oil emulsification method using a mixture of agarose solution and nanometer-sized superparamagnetic Fe(3)O(4) particles as the aqueous phase. The MAS showed good superparamagnetic responsiveness in a magnetic field. A reactive triazine dye, Cibacron blue 3GA (CB), was coupled to the gel to prepare a CB-modified magnetic agarose support (CB-MAS) for protein adsorption. Lysozyme was used as a model protein to test the adsorption equilibrium and kinetic behavior of the CB-MAS. The dependence of bed expansion in the MSFB with a transverse magnetic field on liquid velocity and magnetic field intensity was investigated. Liquid-phase dispersion behavior in the MSFB was examined by measurements of residence time distributions and compared with that obtained in packed and expanded beds. Dynamic lysozyme adsorption in the MSFB was also compared with those in packed and expanded beds. The dynamic binding capacity at 10% breakthrough was estimated at 55.8 mg/mL in the MSFB, higher than that in the expanded bed (31.1 mg/mL) at a liquid velocity of 45 cm/h. The results indicate that the CB-MAS is promising for use in liquid MSFB for protein adsorption.     

The effects of magnetic stabilization on the structure and performance of liquid fluidized beds.

Liquid fluidized beds containing porous magnetic ion-exchange particles with densities ca. 1.03-1.16 g mL-1 were examined. The effect of magnetic stabilization was studied, both in terms of bed physical characteristics and sorptive behavior. Maximum applied magnetic field strength was approximately 200 oersted. Breakthrough and pulse analyses were carried out with protein and acetone solutions, respectively, with liquid flow rates ranging from approximately 1 to 3 cm min-1. Acetone pulses in columns containing 7 mL of particles had plate numbers ranging from 2.5 to 18 for magnetically stabilized beds and from 7.8 to 20 for non-stabilized fluidized beds. Under any particular set of conditions, magnetic stabilization always resulted in poorer efficiency, both in pulse analyses and in protein breakthrough experiments.     

A novel magnetic affinity support for protein adsorption and purification

A novel magnetic support was prepared by an oxidization-precipitation method with poly(vinyl alcohol) (PVA) as the entrapment material. Transmission electron microscopy indicated that the magnetic particles had a core-shell structure, containing many nanometer-sized magnetic cores stabilized by the cross-linked PVA. The particles showed a high magnetic responsiveness in magnetic field, and no aggregation of the particles was observed after the particles had been treated in the magnetic field. These facts indicated that the particles were superparamagnetic. Cibacron blue 3GA (CB) was coupled to the particles to prepare a magnetic affinity support (MAS) for protein adsorption. Lysozyme was used as a model protein to test the adsorption properties of the MAS. The adsorption equilibrium of lysozyme to the MAS was described by the Langmuir-type isotherm. The capacity for lysozyme adsorption was more than 70 mg/g MAS (wet weight) at a relatively low CB coupling density (3-5 micromol/g). In addition, 1.0 M NaCl solution could be used to dissociate the adsorbed lysozyme. Finally, the MAS was recycled for the purification of alcohol dehydrogenase (ADH) from clarified yeast homogenates. Under proper conditions, the magnetic separation yielded over 5-fold purification of the enzyme with 60% recovery of the enzyme activity.     

Performance of dye-affinity beads for aluminium removal in magnetically stabilized fluidized bed

BACKGROUND: Aluminum has recently been recognized as a causative agent in dialysis encephalopathy, osteodystrophy, and microcytic anemia occurring in patients with chronic renal failure who undergo long-term hemodialysis. Only a small amount of Al(III) in dialysis solutions may give rise to these disorders. METHODS: Magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads in the size range of 80-120 microm were produced by free radical co-polymerization of HEMA and ethylene dimethacrylate (EDMA) in the presence of magnetite particles (Fe3O4). Then, metal complexing ligand alizarin yellow was covalently attached onto mPHEMA beads. Alizarin yellow loading was 208 micromol/g. These beads were used for the removal of Al(III) ions from tap and dialysis water in a magnetically stabilized fluidized bed. RESULTS: Al(III) adsorption capacity of the beads decreased with an increase in the flow-rate. The maximum Al(III) adsorption was observed at pH 5.0. Comparison of batch and magnetically stabilized fluidized bed (MSFB) maximum capacities determined using Langmuir isotherms showed that dynamic capacity (17.5 mg/g) was somewhat higher than the batch capacity (11.8 mg/g). The dissociation constants for Al(III) were determined using the Langmuir isotherm equation to be 27.3 mM (MSFB) and 6.7 mM (batch system), indicating medium affinity, which was typical for pseudospecific affinity ligands. Al(III) ions could be repeatedly adsorbed and desorbed with these beads without noticeable loss in their Al(III) adsorption capacity. CONCLUSIONS: Adsorption of Al(III) demonstrate the affinity of magnetic dye-affinity beads. The MSFB experiments allowed us to conclude that this inexpensive sorbent system may be an important alternative to the existing adsorbents in the removal of aluminium.     

Separation of yeast cells from aqueous solutions using magnetically stabilized fluidized beds

AIMS: To separate Saccharomyces cerevisiae cells from aqueous solutions using magnetically stabilized fluidized beds (MSFB) that utilize a horizontal magnetic field, and to study the effect of some parameters, such as bed porosity and height, liquid flow rate and inlet concentration on cell removal efficiency and breakthrough curves. METHODS AND RESULTS: The separation process was conducted in an MSFB under the effect of horizontal magnetic field. The magnetic particles used consist of a ferromagnetic core of magnetite (Fe3O4) covered by a stable layer of activated carbon to adsorb the yeast cells from the suspension. The yeast cell concentration in the effluent was determined periodically by measuring the absorbance at 610 nm. The effect of the magnetic field intensity on the bed porosity and consequently the exit-normalized cell concentration from the bed was studied. It was found that bed porosity increased by 75%, and the normalized cell concentration in the bed effluent decreased by 30%, when the magnetic field intensity was increased from 0 to 110 mT. In addition, increasing the magnetic field intensity and bed height delayed the breakthrough point, and allowed efficient cell removal. These results demonstrate an improved method to separate cells of low concentration from cell suspension. CONCLUSIONS: This study allows the continuous separation of yeast cells from aqueous solutions in an MSFB. The removal efficiency is affected by different parameters including the bed height, flow rate and initial concentration. The removal efficiency reaches 82%, and could be improved by varying the operational parameters. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained in this investigation show that the MSFB using horizontal fields represents a potential tool for the continuous separation of cell suspension from aqueous solution. This study will contribute to a better understanding of the hydrodynamic parameters on the separation efficiencies of the cell.     

Modeling of product removal during enzymatic conversions by using affinity molecules

The feasibility of using magnetic particles for in-line product isolation during enzymatic conversion was studied. A comparison was made between a process based on magnetic particles and a conventional adsorption column. The enzymatic reaction was described by two consecutive first-order reactions (synthesis and subsequent hydrolysis), while the adsorption of substrate and product was described by multicomponent Langmuir isotherms. The yield as well as synthesis/hydrolysis ratio were calculated for various system characteristics. The results show that magnetic particles are very effective when the affinity with the particles is specific and for enzymatic conversions involving low ratios of the rate of synthesis versus the rate of hydrolysis. For slow conversions and for low specific affinity molecules column separations are more appropriate.     

A novel magnetic adsorbent for immunoglobulin-g purification in a magnetically stabilized fluidized bed

A novel magnetic poly(ethylene glycol dimethacrylate-N-methacryloly-L-histidinemethylester) [m-poly(EGDMA-(MAH)] support was prepared for purification of immunoglobulin G (IgG) in a magnetically stabilized fluidized bed by suspension polymerization. Elemental analysis of the magnetic beads for nitrogen was estimated as 70 micromol MAH/g polymer. Magnetic poly(EGDMA-MAH) beads were used in the separation of immunoglobulin-G (IgG) from aqueous solutions and/or human plasma in a magnetically stabilized fluidized bed system. IgG adsorption capacity of the beads decreased with an increase in the flow rate. The maximum IgG adsorption was observed at pH 6.0 for MES buffer. IgG adsorption onto the m-poly(EGDMA) was negligible. Higher adsorption values (up to 262 mg/g) were obtained in which the m-poly(EGDMA-MAH) sorbents were used from aqueous solutions. Higher amounts of IgG were adsorbed from human plasma (up to 320 mg/g) with a purity of 87%. IgG molecules could be repeatedly adsorbed and desorbed with these sorbents without noticeable loss in their IgG adsorption capacity.     

Protein purification using magnetic adsorbent particles

The application of functionalised magnetic adsorbent particles in combination with magnetic separation techniques has received considerable attention in recent years. The magnetically responsive nature of such adsorbent particles permits their selective manipulation and separation in the presence of other suspended solids. Thus, it becomes possible to magnetically separate selected target species directly out of crude biological process liquors (e.g. fermentation broths, cell disruptates, plasma, milk, whey and plant extracts) simply by binding them on magnetic adsorbents before application of a magnetic field. By using magnetic separation in this way, the several stages of sample pretreatment (especially centrifugation, filtration and membrane separation) that are normally necessary to condition an extract before its application on packed bed chromatography columns, may be eliminated. Magnetic separations are fast, gentle, scaleable, easily automated, can achieve separations that would be impossible or impractical to achieve by other techniques, and have demonstrated credibility in a wide range of disciplines, including minerals processing, wastewater treatment, molecular biology, cell sorting and clinical diagnostics. However, despite the highly attractive qualities of magnetic methods on a process scale, with the exception of wastewater treatment, few attempts to scale up magnetic operations in biotechnology have been reported thus far. The purpose of this review is to summarise the current state of development of protein separation using magnetic adsorbent particles and identify the obstacles that must be overcome if protein purification with magnetic adsorbent particles is to find its way into industrial practice.     

Examination of protein adsorption in fluidized bed and packed bed columns at different temperatures using frontal chromatographic methods

The influences of various experimental parameters on the dynamic adsorption capacity (DAC) and the dynamic adsorption rate (DAR) of a biomimetic affinity silica-based adsorbent in fluidized and packed bed columns operated under plug flow conditions and at different temperatures have been investigated with different inlet concentrations of hen egg white lysozyme (HEWL) and human serum albumin (HSA). The DACs as well as the DARs of both the fluidized and packed beds were examined at 10% saturation (i.e., at the QB value) and the experimental data compared with the corresponding data obtained from batch equilibrium adsorption procedures. Parameters examined included the fluid superficial velocity and protein concentration and their effect on the binding capacity and column efficiency. Consistent with various results reported from this and other laboratories on the behavior of biospecific affinity adsorbents derived from porous silica and zirconia particles, adsorbents prepared from Fractosil 1000 were found to exhibit appropriate rheological characteristics in fluidized bed systems under the experimental conditions. Moreover, changes in temperature resulted in a more significant effect on the breakthrough profiles of HSA compared to HEWL with the immobilized Cibacron Blue F3G-A with Fractosil 1000 adsorbent. This result suggests that temperature effects can possibly be employed profitably in some processes as part of a strategy to enhance column performance with fluidized bed systems for selective recovery of target proteins. At relatively low superficial velocities of the feed, the DARs with HEWL and HSA were similar for both the fluidized and packed bed column systems, whereas, at high superficial velocities, the DARs for these proteins were larger with the packed bed columns.     

Lysozyme purification with dye-affinity beads under magnetic field

Magnetic poly(2-hydroxyethyl methacrylate) mPHEMA beads carrying Cibacron Blue F3GA were prepared by suspension polymerization of HEMA in the presence of Fe3O4 nano-powder. Average size of spherical beads was 80-120 microm. The beads had a specific surface area of 56.0m(2)/g. The characteristic functional groups of dye-attached mPHEMA beads were analyzed by Fourier transform infrared spectrometer (FTIR) and Raman spectrometer. mPHEMA with a swelling ratio of 68% and carrying 28.5 micromol CibacronBlueF3GA/g were used for the purification of lysozyme. Adsorption studies were performed under different conditions in a magnetically stabilized fluidized bed (i.e., pH, protein concentration, flow-rate, temperature, and ionic strength). Lysozyme adsorption capacity of mPHEMA and mPHEMA/Cibacron Blue F3GA beads were 0.8 mg/g and 342 mg/g, respectively. It was observed that after 20 adsorption-desorption cycle, mPHEMA beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 87.4% with recovery about 79.6%. The specific activity of the desorbed lysozyme was high as 41.586 U/mg.     

Use of magnetic poly(glycidyl methacrylate) monosize beads for the purification of lysozyme in batch system

The hydrophobic affinity ligand L-tryptophan immobilized magnetic poly(glycidyl methacrylate) [m-poly(GMA)] beads in monosize form (1.6 microm in diameter) were used for the affinity purification of lysozyme from chicken egg white. The m-poly(GMA) beads were prepared by dispersion polymerization in the presence of Fe3O4 nano-powder. The epoxy groups of the m-poly(GMA) beads were converted into amino groups with 1,6 diaminohexane (i.e., spacer arm). l-tryptophan was then covalently immobilized on spacer arm attached m-poly(GMA) beads. Elemental analysis of immobilised L-tryptophan for nitrogen was estimated as 42.5 micromol/g polymer. Adsorption studies were performed under different conditions in a batch system (i.e., medium pH, protein concentration and temperature). Maximum lysozyme adsorption amount of m-poly(GMA) and m-poly(GMA)-L-tryptophan beads were 1.78 and 259.6 mg/g, respectively. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium adsorption capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. It was observed that after 10 adsorption-elution cycle, m-poly(GMA)-L-tryptophan beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. It was found to be successful in achieving purification of lysozyme in a high yield of 76% with a purification fold of 71 in a single step. The specific activity of the eluted lysozyme (62,580 U/mg) was higher than that obtained with a commercially available pure lysozyme (Sigma (60,000 U/mg).     

Interaction of mammalian cell culture broth with adsorbents in expanded bed adsorption of monoclonal antibodies

The interaction of a mammalian cell culture broth with two commercially available adsorbents for the use in expanded bed adsorption (EBA) has been studied. A cation exchange resin (Streamline SP) and an affinity adsorbent (Streamline rProtein A) were compared with regard to adsorption of hybridoma cells during sample application as well as potential cell damage. The results showed that hybridoma cells interact significantly with an expanded bed of cation exchange adsorbents but not with the Protein A adsorbent. After application of 17–20 sedimented bed volumes a saturation of the Streamline SP resin with cells was noted. With both adsorbents no measurable cell damage was found and IgG₁ was recovered in approximately 95% yield. The capacity for IgG₁ adsorption at 3% breakthrough was 2.7 mg IgG₁/ml Streamline rProtein A at a constant fluid velocity of 380 cm/h and 1.0 mg IgG_l/ml Streamline SP at 215–240 cm/h fluid velocity.     

A novel magnetic silica support for use in chromatographic and enzymatic bioprocessing

A limited number of support matrices have so far been developed for use in magnetically stabilized fluidized bed (MSFB) applications. We have developed a versatile magnetic silica support which can be derivatized readily for both adsorption chromatography and enzyme immobilization by well-known techniques. A magnetic pellicular bead is prepared by electrostatically depositing alternating layers of colloidal silica and cationic polymer onto macroscopic nickel core particles. The polymer is then burned out and the silica partially sintered to yield a porous shell with 5-80 m(2)/g of surface area. This magnetic composite was tested as a support for immobilizing invertase. Sucrose was continuously converted to its component monosaccharides with nearly constant activity over the first 8 days and retention of 50% of initial activity after 25 days.     

Cell separation by expanded bed adsorption: use of ion exchange chromatography for the separation of <Emphasis Type="Italic">E. coli</Emphasis> and <Emphasis Type="Italic">S. cerevisiae</Emphasis>

In a wide variety of biotechnological and medical applications it is necessary to separate different cell populations from one another. A promising approach to cell separations is demonstrated to be the adoption of chromatographic techniques conducted in expanded beds. The high voidage between the adsorbent beads in an expanded bed allows for the efficient capture of particulate entities such as cells together with washing and subsequent elution without entrapment and loss. In addition, the combination of a gentle hydrodynamic environment, a high surface area and low mixing within the expanded bed make this technique highly favourable. A model system for the separation of two types of microbial cells using STREAMLINE DEAE adsorbent in expanded bed procedures has been investigated. The use of a less selective ligand such as an ion exchange group, which is often characterised by gentle elution procedures, has been investigated as an alternative to affinity ligands whose strong binding characteristics can result in harsh elution procedures with consequent loss of yield and cell viability. Expanded bed experiments have demonstrated selective and high capacity capture of cells from feedstocks containing either a single type of cell or as a mixture of cells of Saccharomyces cerevisiae and Eschericia coli. The capture, washing and elution phases of the separation have been studied with respect to capacity, selectivity and yield of released cells. In these procedures, separation of cell types is achieved by the presence of multiple equilibrium stages within the expanded bed. The results show the potential for carrying out cell separations in expanded beds as an alternative to immunomagnetic cell separations. The combination of these recently developed technologies promises to be a powerful, but economic technique for cell separations involving simple equipment that can readily be scaled up.     

Silane-modified magnetic beads: application to immunoglobulin G separation

The magnetic poly(2-hydroxyethyl methacrylate ethylene glycol dimethacrylate) [m-poly(HEMA-EGDMA)] beads (150-250-microm diameter in spherical form) were prepared by a radical suspension polymerization technique. The pseudo-specific ligand, reactive imidazole containing 3-(2-imidazoline-1-yl)propyl (triethoxysilane) (IMEO) was selected as a silanization agent. IMEO was covalently immobilized onto the magnetic beads. IMEO-immobilized m-poly(HEMA-EGDMA) beads were used for the affinity adsorption of immunoglobulin-G (IgG) from aqueous solutions and human plasma. To evaluate the degree of IMEO attachment, the m-poly(HEMA-EGDMA) beads were subjected to Si analysis by using flame atomizer atomic absorption spectrometer, and it was estimated as 36.6 mg IMEO/g of polymer. The nonspecific IgG adsorption onto the plain m-poly(HEMA-EGDMA) beads was very low (about 0.4 mg/g). Higher adsorption values (up to 55 mg/g) were obtained when the m-poly(HEMA-EGDMA)/IMEO beads were used from both aqueous solutions and human plasma. The maximum IgG adsorption on the m-poly(HEMA-EGDMA)-IMEO beads was observed at pH 7.0. The IgG molecules could be repeatedly adsorbed and desorbed with m-poly(HEMA-EGDMA)-IMEO beads without noticeable loss in the IgG adsorption capacity. The adsorption capacity from human plasma in magnetically stabilized fluidized bed decreased drastically from 78.9 to 19.6 mg/g with the increase of the flow rate from 0.2 to 3.5 mL/min.     

Overloaded hollow-fiber liquid chromatography.

Liquid chromatography in hollow fibers can separate solutes like flavors and proteins by using a stationary phase of organic solvent, sometimes containing reversed micelles. Such separations, which have a much smaller pressure drop than equivalent separations in packed beds, show dispersion consistent with chromatographic theories at low flows and dilute feeds. These separations behave less predictably at high flows and concentrated feeds, which overload the hollow fibers. The results for flavors correlate well with the Graetz number, consistent with available theories of chromatography and adsorption. The results for proteins correlate poorly with the Graetz number but better with a dimensionless flux based on facilitated diffusion in the stationary phase.     

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.     

Development of Magnetic Beads for Rapid and Efficient Metal-Chelate Affinity Purifications

Metal-chelate affinity chromatography offers multiple advantages for protein purification, yet existing resins make its applications to sparse, hydrophobic, or particularly labile proteins and peptides quite difficult. In this work, we have developed a simple method to covalently modify commercially available superparamagnetic beads with a six-carbon spacer and nitrilotriacetic acid to provide a novel resin for extremely rapid and efficient metal chelate affinity purifications. Further, the small size and surface chemistry of these beads provide clear improvement in applicability to small scale purifications with reduced nonspecific adsorption. These advantages have been demonstrated relative to a commercially available nickel resin.     

Pathogenic antibody removal using magnetically stabilized fluidized bed

Magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads were used in the removal of anti-dsDNA antibodies from systemic lupus erythematosus (SLE) patient plasma in a magnetically stabilized fluidized bed. mPHEMA beads, in the size range of 80-120 microm, were produced by suspension technique. Then, DNA was immobilized onto mPHEMA beads by carbodiimide activation. Magnetic beads were contacted with blood in in vitro systems. Loss of blood cells and clotting times were followed. mPHEMA beads were characterized by scanning electron microscopy (SEM). Important results obtained in this study are as follows: the mPHEMA beads have a spherical shape and porous structure. Loss of cells in the blood contacting with mPHEMA/DNA was negligible. The anti-dsDNA adsorption capacity decreased significantly with the increase of the flow-rate. With increasing anti-dsDNA antibody concentration, the amount of antibody adsorbed per unit mass increased, then reached saturation. Maximum anti-dsDNA antibody adsorption capacity was found to be 97.8 mg/g. Pathogenic antibody molecules could be repeatedly adsorbed and desorbed with these magnetic beads without noticeable loss in their antibody adsorption capacity. Because of the good blood-compatibility, mPHEMA is hopeful for the treatment of SLE by magnetically stabilized fluidized bed systems in the future.     

On-line monitoring of breakthrough curves within an expanded bed adsorber

By abstracting samples of the liquid phase from various positions along the height of an expanded bed, it has been possible to monitor the breakthrough profiles of adsorbing components during the application of feedstock. Similarly, the concentration profiles of the subsequent washing and elution procedures were also followed. The procedure involves the abstraction of liquid samples from the voids of the expanded bed using a specially modified column and assaying the levels of proteins in the withdrawn stream by on-line rapid chromatographic monitoring. Studies of the residence time distribution showed that the modifications to the expanded bed did not cause additional mixing and dispersion. Breakthrough profiles have been measured in a simple single component system and in a complex feedstock in which the adsorption of lysozyme from skimmed cows' milk was monitored. The system shows promise for the on-line control and monitoring of expanded bed adsorption separations, together with providing additional insight into the hydrodynamic and adsorption/desorption processes that occur during bioseparations using expanded bed adsorption.