Antibody capture from corn endosperm extracts by packed bed and expanded bed adsorption

Topical treatments of chronic infections with monoclonal antibodies will require large quantities of antibodies. Because plants have been proven capable of producing multisubunit antibodies and provide for large-scale production, they are likely hosts to enable such applications. Recovery costs must also be low because of the relatively high dosages required. Hence, we have examined the purification of a human secretory antibody from corn endosperm extracts by processing alternatives of packed bed and expanded bed adsorption (EBA). Because of the limited availability of the transgenic corn host, the system was modeled by adding the antibody to extracts of nontransgenic corn endosperm. Complete clarification of a crude extract followed by packed bed adsorption provided antibody product in 75% yield with 2.3-fold purification (with antibody accounting for 24% of total protein). The small size of the packed bed, cation-exchange resin SP-Sepharose FF and the absence of a dense core (present in EBA resins) allowed for more favorable breakthrough performance compared to EBA resins evaluated. Four adsorbents specifically designed for EBA operation, with different physical properties (size and density), chemical properties (ligand), and base matrices were tested: SP-steel core resin (UpFront Chromatography), Streamline SP and Streamline DEAE (Amersham Biosciences), and CM Hyper-Z (BioSepra/Ciphergen Biosystems). Of these, the small hyperdiffuse-style resin from BioSepra had the most favorable adsorption characteristics. However, it could not be utilized with crude feeds due to severe interactions with corn endosperm solids that led to bed collapse. UpFront SP-steel core resin, because of its relatively smaller size and hence lower internal mass transfer resistance, was superior to the Streamline resins and operated successfully with application of a crude corn extract filtered to remove all solids of >44 microm. However, the EBA performance with this adsorbent provided a yield of only 61% and purification factor of 2.1 (with antibody being 22% of total protein). Process simulation showed that capital costs were roughly equal between packed and expanded bed processes, but the EBA design required four times greater operating expenditures. The use of corn endosperm as the starting tissue proved advantageous as the amount of contaminating protein was reduced approximately 80 times compared to corn germ and approximately 600 times compared to canola. Finally, three different inlet designs (mesh, glass beads, and mechanical mixing) were evaluated on the basis of their ability to produce efficient flow distribution as measured by residence time distribution analysis. All three provided adequate distribution (axial mixing was not as limiting as mass transfer to the adsorption process), while resins with different physical properties did not influence flow distribution efficiency values (i.e., Peclet number and HETP) when operated with the same inlet design.     

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.     

Purification of filamentous bacteriophage M13 by expanded bed anion exchange chromatography

In this paper, we investigated the development of a simplified and rapid primary capture step for the recovery of M13 bacteriophage from particulate-containing feedstock. M13 bacteriophage, carrying an insert, was propagated and subsequently purified by the application of both conventional multiple steps and expanded bed anion exchange chromatography. In the conventional method, precipitation was conducted with PEG/NaCl, and centrifugation was also performed. In the single step expanded bed anion exchange adsorption, UpFront FastLine 20 (20 mm i.d.) from UpFront Chromatography was used as the contactor, while 54 ml (Ho = 15 cm) of STREAMLINE DEAE (rho = 1.2 g/cm3) from Amersham Pharmacia Biotechnology was used as the anion exchanger. The performance of the two methods were evaluated, analysed, and compared. It was demonstrated that the purification of the M13 bacteriophage, using expanded bed anion exchange adsorption, yielded the higher recovery percentage, at 82.86%. The conventional multiple step method yielded the lower recovery percentage, 36.07%. The generic application of this integrated technique has also been assessed.     

Capture of a recombinant protein from unclarified canola extract using streamline expanded bed anion exchange

The feasibility of applying expanded bed adsorption technology to recombinant protein recovery from extracts of transgenic canola (rapeseed) was assessed. The extraction step results in a suspension of high solids content that is difficult to clarify. The coarse portion of the solids can be removed easily, and our aim was to operate the expanded bed in the presence of the recalcitrant particulates. Recombinant beta-glucuronidase (rGUS) produced in transgenic canola seed was the model system. Diethylaminoethyl (DEAE) and Streamline DEAE resin exhibited similar binding and elution properties for both rGUS and native canola proteins. More than 95% of native canola proteins did not bind to DEAE resins at pH 7.5, whereas the bound proteins were fractionated by two-step salt elution into two groups with the first peak, containing 70% of total bound proteins, at 20 mS/cm, followed by elution of rGUS at 50 mS/cm. The adsorption isotherm was only slightly influenced by the presence of up to 14 mg solids/mL extract; C(m) and K(d) changed by -1% and +39%, respectively. Bed expansion was semiquantitatively predictable from physical properties of the fluid together with Stokes's law and the Richardson-Zaki correlation for both clarified and partially clarified extracts. The presence of 1.4% solids did not change rGUS breakthrough behavior of the expanded bed; however, a small difference between expanded bed and packed bed was observed early in the sample loading stage, during which bed expansion adjusts. Canola solids moved through the column in approximately plug flow with no detriment to bed stability. Seventy-two percent recovery of 34-fold purified rGUS was obtained after initial loading of 1.4% (w/w) solids extract to 25% breakthrough.     

Zeta potential as a diagnostic tool to evaluate the biomass electrostatic adhesion during ion-exchange expanded bed application

Expanded bed adsorption is an integrative technology in downstream processing allowing the direct capture of target proteins from biomass (cells or cell debris) containing feedstocks. Potential adhesion of biomass on the surface of adsorbent, however, may hamper the application of this technique. Since the electrostatic forces dominate the interactions between biomass and adsorbent, the concept of zeta potential was introduced to characterize the biomass/adsorbent electrostatic interactions during expanded bed application. The criterion of zeta potential evaluation proposed in the previous paper (Biotechnol Bioeng, 83(2):149-157, 2003) was verified further with the experimental validation. The zeta potential of intact cells and homogenates of four microorganisms (Escherichia coli, Bacillus subtilis, Pichia pastoris, and S. cerevisiae) were measured under varying pH and salt concentration, and two ion-exchange adsorbents (Streamline DEAE and Streamline QXL) were investigated. The biomass transmission index (BTI) from the biomass pulse response experiments was used as the indicator of biomass adhesion in expanded bed. Combining the influences from zeta potential of adsorbent (zeta(a)), zeta potential of biomass (zeta(b)) and biomass size (d), a good relationship was established between the zeta potential parameter (-zeta(a)zeta(b)d) and BTI for all experimental conditions. The threshold value of parameter (-zeta(a)zeta(b)d) can be defined as 120 mV2 microm for BTI above 0.9. This means that the systems with (-zeta(a)zeta(b)d) < 120 show neglectable electrostatic bio-adhesion, and would have a considerable probability of forming stable expanded beds in a biomass suspension under the particular experimental conditions.     

Evaluation of expanded bed adsorption chromatography for extraction of prothrombin complex from Cohn Supernatant I.

This study evaluated the feasibility of substituting expanded bed adsorption (EBA) chromatography for an existing chromatographic purification process for the isolation of prothrombin complex concentrate (PCC) from Cohn Supernatant I. The EBA chromatography (Streamline) resins were compared to the current DEAE-cellulose resin for the extraction of PCC from Cohn SNI. EBA chromatography resins efficiently bound PCC from Cohn SNI at a significantly higher flow rate of up to 300 cm/h compared to 30 cm/h for the current DEAE-cellulose process. Composition and yield of the recovered PCC reflected the elution conditions used. The results indicate that EBA chromatography could be used to efficiently produce PCC comparable to existing products.     

Impact of operating variables on the expanded bed adsorption of Saccharomyces cerevisiae cells using a concanavalin A derivatized perfluorocarbon

The use of fluidizable affinity adsorbents for the adsorption of cells in expanded mode is investigated. Affinity adsorbents have been synthesized by immobilizing the lectin Concanavalin A onto the surface of triazine-activated perfluorocarbon-solids. The adsorbents were found to adsorb Saccharomyces cerevisiae cells from solution with adsorption capacities of up to 6.8 x 10(9) cells mL(-1). Adsorption kinetics were rapid with a time constant of 相似文献   

Expanded bed protein A affinity chromatography of a recombinant humanized monoclonal antibody: process development, operation, and comparison with a packed bed method.

We show that expanded bed protein A affinity chromatography using Streamline rProtein A media is an efficient method for purifying a recombinant humanized monoclonal antibody from unclarified Chinese hamster ovary cell culture fluid and that it provides purification performance comparable to using a packed bed. We determined that the dynamic capacity of the expanded bed media is related to flow rate (measured in column volumes per hour) by a power function, which allows a high capacity at a low flow rate. At 250 cm h-1 with a 25 cm bed height (10 column volumes h-1), the dynamic capacity is 30 g l-1. The yield and purity (measured by the amount of host cell proteins, DNA, SDS-PAGE, and turbidity) of the antibody purified by expanded bed is comparable to the yield and purity obtained on a standard packed bed method using Prosep A media.     

Studies on protein adsorption in a vortex flow reactor

The performance of a vortex flow reactor (VFR) with suspended particles for protein adsorption was studied under varying operating conditions, and resin volume fractions. The VFR behaved as an expanded bed in the regimen of laminar vortices flow. Streamline DEAE was used for bovine serum albumin (BSA) adsorption. Expanded bed VFR experiments were performed with varying geometric aspect ratios (14.6, 28.6 and 40.0) and axial superficial velocity (100–300 cm h⁻¹) to investigate their influence on productivity and dynamic capacity. The results are compared with literature data on an expanded bed column (EBC). Adsorption breakthrough curves were fitting to a simple two-parameter model.     

Partial purification properties of human epidermal growth factor from recombinant Escherichia coli by expanded bed adsorption

Human epidermal growth factor is a polypeptide hormone having many diverse biological functions. This paper first presents the recovery results of human epidermal growth factor (hEGF) immediately from the fermentation broth of recombinant Escherichia coli by using an expanded bed system (a couple of STREAMLINE25 and ÄKTA explorer 100). The influences of operational conditions such as linear flow rate, gradient length of NaCl concentration, pH and sample concentration on the purification performances of hEGF in expanded and packed bed modes with STREAMLINE DEAE resin were systematically evaluated. After optimization, the practical recovery procedure in the expanded bed mode was carried out on a scaled-up system under the conditions of linear flow rates of 183 cm/h (upward) and 37 cm/h (downward), sample volume of 300 ml and column bed height of 13.8 cm which yielded a primary product of hEGF from the cell-free supernatant containing hEGF after centrifugation at 4000 rev/min for 15 min. As a result, the hEGF concentration in the product was higher than 20% (w/v), the concentration factor was greater than 4.3 and the total yield was higher than 80%, respectively. At the same time, the results of hEGF recovery by using expanded bed adsorption (EBA), packed bed chromatography (PBC) and salting out were compared. The results show that the procedure of hEGF recovery in expanded bed adsorption has some advantages over the other two procedures, because of its higher concentration factor, recovery yield, productivity, hEGF concentration in the primary product and shorter duration of purification run.     

Recovery of recombinant beta-glucosidase by expanded bed adsorption from Pichia pastoris high-cell-density culture broth

Methanol limited fed-batch cultivation was applied for production of a plant derived beta-glucosidase by Pichia pastoris. The beta-glucosidase was recovered by expanded bed adsorption chromatography applied to the whole culture broth. The new Streamline Direct HST1 adsorbent was compared with Streamline SP. Higher bead density made it possible to operate at two times higher feedstock concentration and at two times higher flow velocity. The higher binding capacity in the conductivity range 0-48 mS cm(-1) of Streamline Direct HST1 might be caused by the more complex interaction of multi-modal ligand in Streamline Direct HST1 compared to the single sulphonyl group in Streamline SP. Harsher elution condition had to be applied for dissociation of beta-glucosidase from Streamline Direct HST1 due to stronger binding interaction. The 5% dynamic binding capacity was 160 times higher for Streamline Direct HST1 compared to Streamline SP. The yield of beta-glucosidase on Streamline Direct HST1 (74%) was significantly higher than on Streamline SP (48%). Furthermore, beta-glucosidase was purified with a factor of 4.1 and concentrated with a factor of 17 on Streamline Direct HST1 while corresponding parameters were half of these values for Streamline SP. Thus, for all investigated parameters Streamline Direct HST1 was a more suitable adsorbent for recovery of recombinant beta-glucosidase from unclarified P. pastoris high-cell-density cultivation broth.     

The performance of anion exchange expanded bed adsorption chromatography on the recovery of G6PDH from unclarified feedstock with high biomass concentration

The bed stability of Streamline DEAE (p=1.2 g/mL) in a 20 mm (i.d.) glass expanded bed contactor, and its performance on the recovery of glucose 6-phosphate dehydrogenase (G6PDH) from unclarified yeast homogenate were investigated. A residence time distribution study showed that a stable expanded bed was achieved. The theoretical plate and Bodenstein numbers determined were 25 and 53, respectively. A recovery yield of 87% and purification factor of 4.1 were achieved in the operation using 5% (w/v) biomass concentration feedstock. The performance of the anion exchange EBAC was still considerable good at a biomass concentration as high as 15% (w/v).     

Expanded bed adsorption/desorption of proteins with Streamline Direct CST I adsorbent

Streamline Direct CST I is a new type of ion exchanger with multi-modal functional groups, specially designed for an expanded bed adsorption (EBA) process, which can capture directly the proteins from the high ionic strength feedstocks with a high binding capacity. In this study, an experimental study is carried out for two-component proteins (BSA and myoglobin) competitive adsorption and desorption in an expanded bed packed with Streamline Direct CST I. Based on the measurements of the single- and two-component bovine serum albumin (BSA)/myoglobin adsorption isotherm on Streamline Direct CST I, the binding and elution conditions for the whole EBA process are selected; and then frontal analysis for a longer timescale and column displacement experiments in a fixed bed (XK16/20 column) are carried out to evaluate the two-component proteins (BSA and myoglobin) competitive adsorption and displacement on Streamline Direct CST I. Finally, the feasibility of capturing both BSA and myoglobin by an expanded bed packed with Streamline Direct CST I is addressed in a Streamline 50 column packed with 300 mL Streamline Direct CST I.     

Bioprocess considerations for expanded-bed chromatography of crude canola extract: sample preparation and adsorbent reuse

Compared to the conventional microbial and mammalian systems, transgenic plants produce proteins in a different matrix. This provides opportunities and challenges for downstream processing. In the context of the plant host Brassica napus (canola), this work addresses the bioprocessing challenges of solid fractionation, resin fouling by native plant components (e.g., oil, phenolics, etc.), hydrodynamic stability, and resin reuse for expanded bed adsorption for product capture. Plant tissue processing and subsequent protein extraction typically result in an extract with a high content of solids containing a wide particle-size distribution. Without removal of larger particles, the column inlet distributor plugged. The larger particles (> 50 microm) were easily removed through centrifugal settling comparable to that attainable with a scroll decanter. The remaining solids did not affect the column performance. Less than 4% of the lipids and phenolics in the fed extract bound to STREAMLINE trade mark DEAE resin, and this small proportion could be satisfactorily removed using recommended clean-in-place (CIP) procedures. Hydrodynamic expansion and adsorption kinetics of the STREAMLINE trade mark DEAE resin were maintained throughout 10 cycles of reuse, as was the structural integrity of the resin beads. No significant accumulation of N-rich (e.g., proteins) and C/O-rich components (e.g., oil and phenolics) occurred over the same period.     

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.     

Comparison of two matrices for selective recovery of C595 diabody fragment (dbFv) from Escherichia coli lysates

A comparative study of the performance of two types of adsorbent (Streamline Quartz Base and Upfront Matrices), derivatized with the same affinity ligand (RPAP) to recover C595 diabody fragment (dbFv) from Escherichia coli lysates has been undertaken. Both streamline and Upfront Matrices are characterized by a particle size range of 100–300 μm. Streamline has a density of 1.20 g cm⁻³ and ligand concentration of 0.85 μmol ml⁻¹. Upfront has a density of 1.35 g cm⁻³ and ligand concentration of 0.83 μmol ml⁻¹. The release of C595 dbFv from E. coli cells was achieved by a chemical lysis method. The recovery performance of both adsorbents was evaluated in terms of operational productivity and elution yield of C595 dbFv in packed bed (clarified feedstock) and expanded bed (unclarified and clarified feedstock) chromatography systems. Streamline and Upfront adsorbents exhibited diabody operational productivities of 131 and 202 mg l⁻¹, respectively, with an elution yield of 92 and 94%, respectively, in packed bed operation. Streamline and Upfront adsorbents exhibited diabody operational productivities of 54.5 and 123.7 mg l⁻¹, respectively, with an elution yield of 89 and 92%, respectively, in expanded bed operation.     

Purification of recombinant nucleocapsid protein of Newcastle disease virus from unclarified feedstock using expanded bed adsorption chromatography

In the present work, a single-step purification of recombinant nucleocapsid protein (NP) of the Newcastle disease virus (NDV) directly from unclarified feedstock using an expanded bed adsorption chromatography (EBAC) was developed. Streamline 25 column (ID = 25 mm) was used as a contactor and Streamline chelating adsorbent immobilized with Ni2+ ion was used as affinity adsorbent. The dynamic binding capacity of Ni2+ -loaded Streamline chelating adsorbent for the NP protein in unclarified feedstock was found to be 2.94 mg ml(-1) adsorbent at a superficial velocity of 200 cm h(-1). The direct purification of NP protein from unclarified feedstock using expanded bed adsorption has resulted in a 31% adsorption and 9.6% recovery of NP protein. The purity of the NP protein recovered was about 70% and the volume of processing fluid was reduced by a factor of 10. The results of the present study show that the IMA-EBAC developed could be used to combine the clarification, concentration and initial purification steps into a single-step operation.     

Expanded and packed bed albumin adsorption on fluoride modified zirconia

The expanded bed characteristics of 75-103microm fluoride-modified zirconia (FmZr) particles synthesized by a fed batch oil emulsion process were investigated. These particles are distinguished from commercially available expanded-bed adsorbents by virtue of their high density (2.8 g/cc) and the mixed mode protein retention mechanism which allows for the retention of both cationic and anionic proteins. The linear velocity versus bed porosity data agree with the Richardson-Zaki relationship with the terminal velocity in infinite medium of 2858.4 cm/h and a bed expansion index of 5.1. Residence time distribution (RTD) studies and bovine serum albumin (BSA) adsorption studies were performed as a function of the height of the settled bed to the column diameter (H:D) ratio and degree of bed expansion with superficial velocities of 440 to 870 cm/h. The settled bed, a 2x expanded bed, and a 3x expanded bed were studied for the H:D ratios of 1:1, 2:1, and 3:1. The dynamic binding capacity (DBC) at 5% breakthrough was low (2-8 mg BSA/mL settled bed) and was independent of the H:D ratio or the degree of bed expansion. The saturation DBC was 32.3 +/- 7.0 mg BSA/mL settled bed. The adsorption-desorption kinetics and intraparticle diffusion for protein adsorption on FmZr (38-75 micrometer) were investigated by studying the packed bed RTD and BSA adsorption as a function of temperature and flow rate. The data show that the adsorption-desorption kinetics along with intraparticle diffusion significantly influence protein adsorption on FmZr. Low residence times ( approximately 0.8 min) of BSA result in a DBC at 5% breakthrough which is 3.5-fold lower compared to that at 6-fold higher protein residence time. At low linear velocity (45 cm/h) the breakthrough curve is nearly symmetrical and becomes asymmetrical and more dispersed at higher linear velocity (270 cm/h) due to the influence of slow adsorption-desorption kinetics and intraparticle diffusion.Copyright 1998 John Wiley & Sons, Inc. Bioeng 60: 333-340, 1998.     

Expanded bed adsorption: elution in expanded bed mode

Elution in expanded bed mode has been investigated in the expanded bed adsorption process. Elution was performed at different sample loads and at different liquid velocities using bovine serum albumin as a model. The effect on mixing in the liquid phase and on the volume of the eluted peak were determined. Mixing in the liquid phase was almost unaffected when elution was performed at 100 cm/h, regardless of sample load. However, mixing increased significantly when elution was carried out at high liquid velocities (300 cm/h) at high sample loads. The eluted peak volume increased with liquid velocity and increased sample load. It was approx. 80% higher in expanded bed mode than in packed bed from an adsorbent completely saturated with protein eluted at 300 cm/h.     

Protein adsorption and hydrodynamic stability of a dense, pellicular adsorbent in high-biomass expanded bed chromatography

A dense, pellicular UpFront adsorbent (ϱ=1.5 g/cm³, UpFront Chromatography, Cophenhagen, Denmark) was characterized in terms of hydrodynamic properties and protein adsorption performance in expanded bed chromatography. Cibacron Blue 3GA was immobilised into the adsorbent and protein adsorption of bovine serum albumin (BSA) was selected to test the setup. The Bodenstein number and axial dispersion coefficient estimated for this dense pellicular adsorbent was 54 and 1.63×10⁻⁵ m²/s, respectively, indicating a stable expanded bed. It could be shown that the BSA protein was captured by the adsorbent in the presence of 30% (w/v) of whole-yeast cells with an estimated dynamic binding capacity (C/C ₀=0.01) of approximately 6.5 mg/mL adsorbent.