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.     

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.     

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.     

Recovery and partial purification of penicillin G acylase from E. coli homogenate and B. megaterium culture medium using an expanded bed adsorption column

The adsorption of penicillin G acylase (PGA) from B. megaterium and from Escherichia coli on a cationic resin, Streamline SP XL, was studied using both packed and expanded beds. Stability assays showed that penicillin acylases from the two sources presented high irreversible deactivation at pH 4.0 and 4.5, but remained stable at pH 4.8. Adsorption experiments performed in a packed bed (PB), in the pH range 4.8–5.8, showed highest adsorption yields at pH 4.8, for both enzymes. Using small expanded bed adsorption (EBA) columns, PGA was directly recovered and partially purified from E. coli crude extracts, E. coli homogenates, and from B. megaterium centrifuged broth in a single unit operation. Global recovery yields of 91.0, 55.0 and 7.4% and purification factors of 4.5-, 7.5- and 12.7-fold were achieved, respectively. The elution yields of penicillin acylase obtained with these cationic EBA processes when working with E. coli homogenate and B. megaterium centrifuged medium were of 100 and 52%, respectively. The comparison of adsorption capacities of E. coli penicillin acylase from crude extracts onto Streamline SP XL showed similar results for packed-bed and for expanded-bed modes. However, PGA adsorption yields for E. coli (homogenate) and B. megaterium (centrifuged medium) were substantially lower than the values obtained for E. coli crude extract, due to the competition of cell debris and other components present in the B. megaterium medium.     

Comparative evaluation of three purification methods for the nucleocapsid protein of Newcastle disease virus from Escherichia coli homogenates

In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with Ni2+ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was 1.26% and 5.56%, respectively. It was demonstrated that EBA achieved the highest final protein yield of 9.6% with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.     

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.     

Minimising biomass/adsorbent interactions in expanded bed adsorption processes: a methodological design approach

Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from crude feedstock. Interactions between solid matter in the feed suspension and fluidised adsorbent particles influence bed stability and therefore have a significant impact on protein adsorption in expanded beds. In order to design efficient and reliable EBA processes a strategy is needed, which allows to find operating conditions, where these adverse events do not take place. In this paper a methodological approach is presented, which allows systematic characterisation and minimisation of cell/adsorbent interactions with as little experimental effort as possible. Adsorption of BSA to the anion exchanger Streamline Q XL from a suspension containing S. cerevisiae cells was chosen as a model system with a strong affinity of the biomass towards the stationary phase. Finite bath biomass adsorption experiments were developed as an initial screening method to estimate a potential interference. The adhesiveness of S. cerevisiae to the anion exchanger could be reduced significantly by increasing the conductivity of the feedstock. A biomass pulse response method was used to find optimal operation conditions showing no cell/adsorbent interactions. A good correlation was found between the finite bath test and the pulse experiment for a variety of suspensions (intact yeast cells, E. coli homogenate and hybridoma cells) and adsorbents (Streamline Q XL, DEAE and SP), which allows to predict cell/adsorbent interactions in expanded beds just from finite bath adsorption tests. Under the optimised operating conditions obtained using the prior methods, the stability of the expanded bed was investigated during fluidisation in biomass containing feedstock (up to 15% yeast on wet weight basis) employing residence time distribution analysis and evaluation by an advanced model. Based on these studies threshold values were defined for the individual experiments, which have to be achieved in order to obtain an efficient EBA process. Breakthrough experiments were conducted to characterise the efficiency of BSA adsorption from S. cerevisiae suspensions in EBA mode under varying operating conditions. This allowed to correlate the stability of the expanded bed with its sorption efficiency and therefore could be used to verify the threshold values defined. The approach presented in this work provides a fast and simple way to minimise cell/adsorbent interactions and to define a window of operation for protein purification using EBA.     

Evaluation of three expanded bed adsorption anion exchange matrices with the aid of recombinant enhanced green fluorescent protein overexpressed in Escherichia coli

Three anion exchanger expanded bed adsorption (EBA) matrices: Streamline DEAE, Streamline Q XL and Q Hyper Z were evaluated with the aid of EFGP from an ultrasonic homogenate of Escherichia coli. Two pH of buffer were tested. Capture was done in an expanded mode whereas elution was done in a packed mode. The same conditions were chosen for evaluation of the three matrices. We observed a loss of EGFP (8-15%) in the through flow fraction especially with the Streamline Q XL matrix, probably due to an aggregation of beads during sample application. The beads of this matrix possess tentacles which probably retain a lot of cellular and molecular debris. The two other matrices gave a good purification of the EGFP (7-15-fold) but the Q Hyper Z matrix appeared to give the best results. It is composed of little size and density beads which lead to a higher exchange surface and then a better mass transfer.     

Expanded bed adsorption in the purification of monoclonal antibodies: a comparison of process alternatives

Expanded bed adsorption (EBA) was examined as the initial capture/purification step in the purification of monoclonal antibodies from Chinese hamster ovary (CHO) cultures. Two process alternatives each using EBA were compared to a conventional Protein A process without EBA. One alternative used Protein A affinity EBA followed by packed-bed cation and anion-exchange steps. The other alternative used cation-exchange EBA as the capture step followed by packed-bed Protein A and anion-exchange steps. The process using Protein A EBA produced comparable purity (host cell protein, DNA, Protein A, antibody aggregate) to the conventional process. However, the Protein A EBA column showed a significant decrease in dynamic capacity with a limited number of cycles. The process using cation EBA achieved comparable levels of host cell proteins (HCP) and DNA but not antibody aggregate or leached Protein A compared to the conventional process.     

Isolation of a recombinant formate dehydrogenase by pseudo-affinity expanded bed adsorption.

Formate dehydrogenase (FDH) is an enzyme of industrial interest, which is recombinantly expressed as an intracellular protein in Escherichia coli. In order to establish an efficient and reliable purification protocol, an expanded bed adsorption (EBA) process was developed, starting from the crude bacterial homogenate. EBA process design was performed with the goal of finding operating conditions which, on one hand, allow efficient adsorption of the target protein and which, on the other hand, support the formation of a perfectly classified fluidised bed (expanded bed) in the crude feed solution. A pseudo-affinity ligand (Procion Red HE3B) was used to bind the FDH with high selectivity and reasonable capacity (maximum equilibrium capacity of 30 U/ml). Additionally, a simplified modelling approach, involving small packed beds for generation of process parameters, was employed for defining the operating conditions during sample application. In combination with extended elution studies, a process was set up, which could be scaled up to 7.5 l of adsorbent volume yielding a total amount of 100,000 U of 94% pure FDH per run. On this scale, 19 l of a benzonase-treated E. coli homogenate of 15% wet-weight (pH 7.5, 9 mS/cm conductivity) were loaded to the pseudo-affinity adsorbent (0.25 m sed. bed height, 5 x 10(-4) m/s fluid velocity). After a series of two wash steps, a particle-free eluate pool was obtained with 85% yield of FDH. This excellently demonstrates the suitability of expanded bed adsorption for efficient isolation of proteins by combining solid-liquid separation with adsorptive purification in a single unit operation.     

Purification by expanded bed adsorption and characterization of an alpha-amylases FORILASE NTL from A. niger

In this work the purification and biochemistry characterization of alpha-amylases from Aspergillus niger (FORILASE NTL) were studied. The effects of expansion degree of resin bed on enzyme purification by expanded bed adsorption (EBA) have also been studied. Residence time distributions (RTD) studies were done to achieve the optimal conditions of the amylases recovery on ion-exchange resin, and glucose solution was used as a new tracer. Results showed that height equivalent of the theoretical plates (HETP), axial dispersion and the Prandt number increased with bed height, bed voidage and linear velocity. The adsorption capacity of alpha-amylases, on the resin, increased with bed height and the best condition was at four-expansion degree. alpha-Amylase characterization showed that this enzyme has high affinity with soluble starch, good hydrolysis potential and molecular weight of 116 kDa.     

Clarification and capture of high‐concentration refold pools for E. coli‐based therapeutics using expanded bed adsorption chromatography

Expanded bed adsorption (EBA) chromatography was investigated for clarification and capture of high‐concentration refold pools of Escherichia coli‐based therapeutics. Refolding of denatured inclusion bodies (IBs) at high protein concentration significantly improved product throughput; however, direct filtration of the refold materials became very challenging because of high content of protein precipitates formed during refolding. In addition, irreversible protein precipitation caused by high local concentration was encountered in packed bed capture during cation exchange chromatography elution, which limited column loading capacity and capture step productivity. In this study, the two issues are addressed in one unit operation by using EBA. Specifically, EBA can handle feed streams with significant amount of particles and precipitates, which eliminated the need for refold pool clarification through filtration. The relatively broad EBA elution profile is particularly suitable for proteins of low solubility and can effectively avoid product loss previously associated with on‐column precipitation during capture. As the EBA resin (RHOBUST^® FastLine SP IEX) used here has unique properties, it can be operated at high linear velocity (800–1,600 cm/h), while achieving a selectivity and impurity clearance largely comparable to the packed bed resin of the same ligand chemistry (SP Sepharose FF). Furthermore, the filtration of the EBA elution pool is easily manageable within facility capability. Overall, this study demonstrates that the EBA process helps debottleneck the purification of high‐turbidity refold pools by removing precipitates and concurrently capturing the product, which can be applied to other E. coli‐based therapeutics that also requires refolding of IBs. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:113–123, 2014     

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.     

扩张柱床吸附层析回收纯化灌流培养生产的单克隆抗体

用扩张柱床吸附层析技术,一步回收纯化连续灌流培养的单克隆抗体。用Streamline SP阳离子交换介质在固定床柱XK16/20上进行条件摸索,扩张床柱Streamline25和50分别用于小规模条件优化和中试规模放大。培养液中的低浓度单抗经此步处理,浓缩10倍以上,纯度提高5～7倍,回收率＞90%,制备周期比固定柱床层析缩短一半以上。 根据培养液中单抗浓度的不同,一次处理量为18～50L,纯化规模由实验室水平（400mg）扩大至中试水平（2g）,生产成本和工艺复杂性大为降低。应用扩张柱床吸附层析技术,建立单克隆抗体回收纯化工艺,具有经济、简便、高效实用和良好的可放大性。     

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.     

Physicochemical parameters involved in the interaction of Saccharomyces cerevisiae cells with ion-exchange adsorbents in expanded bed chromatography

Expanded bed adsorption (EBA) is an interesting primary technology allowing the adsorption of target proteins from unclarified feedstock in order to combine separation, concentration, and purification steps. However, interactions between cells and adsorbent beads during the EBA process can strongly reduce the performance of the separation. So, to minimize these interactions, the mechanisms of cell adsorption on the support were investigated. Adsorption kinetics of the baker's yeast Saccharomyces cerevisiae on the anion exchanger Q Hyper Z were directly performed under real EBA operating conditions, in a lab-scale UpFront 10 column. The yeast was marketed either as rod-shaped pellets (type I yeast) or as spherical pellets (type II yeast). For both types, a complete series of experiments for determining the adsorption profile versus time was performed, varying the superficial velocity or the pH. In parallel, the surface physicochemical properties of the cells (surface charge and electron-donor and electron-acceptor components) and of the support were determined. First of all, whatever the yeast types, the relation between cell adsorption and bed expansion has been highlighted, demonstrating the important role of hydrodynamic. However, for the type II yeast cells, adsorption increased dramatically, compared to the type I, even though it was shown that both types exhibited the same surface charge. In fact, there were strong differences in the Lewis acidic and basic components of the two yeasts. These differences explain the variable affinity toward the support, which was characterized by a strong electron-donor and a weak electron-acceptor component. These observed behaviors agreed with the colloidal theory. This work demonstrates that all kinds of interaction between the cells and the support (electrostatic, Lifshitz-van der Waals, acid/base) have to be taken into account together with hydrodynamic characteristics inside the bed.     

Recovery of mouse endostatin produced by Pichia pastoris using expanded bed adsorption

Endostatin, a 20 KDa fragment of collagen XVIII, was shown to have an inhibitory effect on angiogenesis and can potentially be used as a tumor growth suppressor. To obtain the amount needed for testing, the protein was successfully cloned and expressed in Pichia pastoris. At the end of the fermentation process, the concentration of the endostatin in the culture was 50 mg per liter, accompanied by 400 gr per liter (wet weight) of biomass. Before the protein can be captured and purified on a packed bed of heparin-Sepharose, the biomass must be removed. Because of the high biomass concentration, conventional biomass removal techniques like centrifugation or filtration are inefficient and cumbersome. Therefore, the expanded-bed adsorption technique was chosen as an alternative approach. An efficient procedure for the initial recovery and purification of the endostatin was developed. The process utilized a cation- exchanger resin instead of a heparin-based affinity resin, because its dynamic capacity was higher, even though it was affected by the high linear flow on the expanded bed. After adjusting the conductivity, pH and biomass concentration, the complete broth was pumped directly on the expanded-bed matrix (Streamline SP XL). Though the yields of protein are similar, the expanded-bed approach is superior to the packed-bed method for several reasons. The expanded-bed process was shorter (only 8 hours compared to 16 hours for the packed bed), it is cheaper, and the product has higher specific activity (29% compared with 18%). Endostatin produced by the expanded-bed adsorption method showed the expected bioactivity and is currently being tested for its potential as a tumor suppressor.     

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.     

Separation of scutellarin from crude extracts of Erigeron breviscapus (vant.) Hand. Mazz. by macroporous resins

Scutellarin, a flavone glycoside, popularly used in the treatment of heart disease, has been efficiently separated using macroporous resins from crude extracts of Chinese medicinal plant Erigeron breviscapus (vant.) Hand. Mazz. HPD-800 resin offered the best adsorption and desorption capacity for scutellarin among the eight macroporous resins tested, and its adsorption data at 25 degrees C fit best to the Langmuir isotherm. The dynamic adsorption and desorption experiments have been carried out on a HPD-800 resin packed column to optimize the separation process of scutellarin from the crude extracts of E. breviscapus. After one run treatment with HPD-800 resin, the scutellarin content in the product was increased 15.69-fold from 2.61% to 40.96% with a recovery yield of 95.01%. The preparative separation process via adsorption-desorption method developed in this study provides a new approach for scale-up separation and purification of scutellarin for its wide pharmaceutical use.