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.     

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).     

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.     

Expanded bed adsorption at production scale: Scale-up verification, process example and sanitization of column and adsorbent

Expanded bed adsorption is a technique for recovery of biomolecules directly from unclarified feedstocks. The work described here demonstrates that expanded bed adsorption is a scaleable technique. The methods used to test scaleability were “determination of degree of bed expansion”, “determination of axial dispersion” and “determination of protein breakthrough capacity”. The performance of a production scale expanded bed column with 600?mm diameter was tested using these methods and the results were found to be consistent with the results obtained from lab scale and pilot scale expanded bed columns. The scaleability and function of the expanded bed technique was also tested by performing a “process example”: a purification mimicking a real process using a yeast culture spiked with bovine serum albumin as feedstock. The results show that the 600?mm diameter production scale column was as efficient as a 25?mm diameter lab scale column in recovering bovine serum albumin from the unclarified yeast culture. The production scale runs were fully automated using a software controlled system containing an adaptor position sensor and an adsorbent sensor. A cleaning study was performed which showed that after use of a proper cleaning protocol, no surviving microorganisms could be detected in the column or in the adsorbent.     

A study of the influence of yeast cell debris on protein and alpha-glucosidase adsorption at various zones within the expanded bed using in-bed sampling

Expanded bed adsorption chromatography is used to capture products directly from unclarified feedstocks, thus combining solid-liquid separation, product concentration and preliminary purification into a single step. However, when non-specific ion-exchangers are used as the adsorbent in the expanded bed, there is the possibility that electrostatic interactions of cells or cell debris with the adsorbent may interfere with the adsorption of soluble products. These interactions depend on the particle size of the cell debris and its surface charge, which in turn depend on the extent of disruption used to release the intracellular products. The interactions occurring during expanded bed adsorption between the anionic ion-exchanger STREAMLINE DEAE and particulate yeast homogenates obtained by high pressure homogenisation at different intensities of disruption achieved by operating at different pressures were studied, while maintaining all other parameters constant. In-bed sampling from the expanded bed using ports fitted up the height of expanded bed was used to study the retention of yeast cells and cell debris within the bed and its influence on the adsorption of total soluble protein and alpha-glucosidase within various zones of the expanded bed. The retention of the biomass present in the homogenate obtained at a lower intensity of disruption was found to be high at the lower end of the column (17% from 13.8 MPa sample compared to 1% from 41.4 MPa sample). This interaction of the particulate material with the adsorbent was found to reduce the dynamic binding capacity of the adsorbent for total soluble protein from 3.6 mg/mL adsorbent for 41.4 MPa sample to 3.0 mg/mL adsorbent for 13.8 MPa sample. The adsorption of alpha-glucosidase was found to increase with an increase in the concentration of the enzyme in the feed, which increased with the intensity of disruption. Selective adsorption of 6,732 U alpha-glucosidase per mg of total protein bound, was noticed for the feedstock prepared at a higher disruption intensity at 41.4 MPa compared to adsorption of 1,262 U/mg of total protein bound for that prepared at 13.8 MPa. The selective adsorption of alpha-glucosidase due to its high concentration together with simultaneous high specific activity of the enzyme in the feed indicated the significance of selective release of enzymes during microbial cell disruption for efficient expanded bed adsorption processes.     

Mechanistic analysis on the effects of salt concentration and pH on protein adsorption onto a mixed-mode adsorbent with cation ligand

Streamline Direct HST is a new kind of mixed-mode adsorbent with cation exchange ligand, especially developed for the expanded bed adsorption process, which can capture target protein directly from the moderate ionic strength feedstock without the need of dilution or other additives. In this study, the isotherm adsorption behaviors and the isocratic retention factors of bovine serum albumin (BSA) on Streamline Direct HST were measured, and the corresponding adsorption mechanisms were also described. The results indicated that Streamline Direct HST shows the typical property of salt-independent adsorption and the maximum binding capacity of BSA occurs near the isoelectric point of BSA. When there are some amounts of electrostatic repulsion protein-adsorbent interactions, the multilayer adsorption could be found, and high salt concentration does not favor the adsorption of protein. A patch-controlled adsorption process and an oriented adsorption model are proposed for describing the adsorption behaviors under electrostatic repulsion condition.     

Ion exchange purification of G6PDH from unclarified yeast cell homogenates using expanded bed adsorption

The use of expanded beds of STREAMLINE ion exchange adsorbents for the direct extraction of an intracellular enzyme glucose-6-phosphate dehydrogenase (G6PDH) from unclarified yeast cell homogenates has been investigated. It has been demonstrated that such crude feedstocks can be applied to the bed without prior clarification steps. The purification of G6PDH from an unclarified yeast homogenate was chosen as a model system containing the typical features of a direct extraction technique. Optimal conditions for the purification were determined in small scale, packed bed experiments conducted with clarified homogenates. Results from these experiments were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 EBA apparatus. The use of an on-line rotameter for measuring and controlling the height of the expanded bed when operated in highly turbid feedstocks was demonstrated. STREAMLINE DEAE has been shown to be successful in achieving isolation of G6PDH from an unclarified homogenate with a purification factor of 12 and yield of 98% in a single step process. This ion exchange adsorbent is readily cleaned using simple cleaning-in-place procedures without affecting either adsorption or the bed expansion properties of the adsorbent after many cycles of operation. The ability of combining clarification, capture, and purification in a single step will greatly simplify downstream processing flowsheets and reduce the costs of protein purification. (c) 1996 John Wiley & Sons, Inc.     

Simple one-step purification of nisin Z from unclarified culture broth of Lactococcus lactis subsp. lactis A164 using expanded bed ion exchange chromatography

A simple one-step purification method, using expanded bed, ion-exchange chromatography, for the fractionation of nisin Z produced by Lactococcus lactis subsp. lactis A164 was developed. The highest dynamic binding capacity (0.92) of the adsorbent was obtained at a superficial velocity of 367 cm h(-1), resulting in approx. 2.7-fold bed expansion. The range of pH for the maximum adsorption was 3-4. The isocratic elution with 0.15 M NaCl led to approx. >90% recovery. Single-step purification of nisin Z from unclarified A164 culture broth resulted in 31-fold purification with a 90% yield.     

Development of an expanded bed technique for an affinity purification of G6PDH from unclarified yeast cell homogenates

The use of an expanded bed of STREAMLINE Red H-7B for the purification of the intracellular glycolytic enzyme glucose 6-phosphate dehydrogenase (G6PDH) directly from untreated preparations of disrupted yeast cells has been investigated. Small-scale experiments, carried out in packed beds, have shown that the optimal pH for adsorption is 6.0 and have enabled optimization of elution conditions using a series of eluents. The dynamic capacity of the adsorbent for G6PDH was determined in a small expanded bed to be 28 units/mL. These results were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 expanded bed column. G6PDH was purified directly from an unclarified yeast homogenate in 99% yield with an average purification factor in the eluted fraction of 103. Cleaning-in-place (CIP) procedures using 0.5 M NaOH and 4M urea in 60% (v/v) ethanol have demonstrated that the adsorbent can be regenerated with no loss of adsorption capacity of alteration of bed expansion characteristics after many cycles of operation. (c) 1995 John Wiley & Sons, Inc.     

Cell/adsorbent interactions in expanded bed adsorption of proteins

Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from unclarified feedstock. A method is presented which allows a qualitative and quantitative understanding of the main mechanisms governing the interaction of biomass with fluidised resins. A pulse response technique was used to determine the adsorption of various cell types (yeast, Gram positive and Gram negative bacteria, mammalian cells and yeast homogenate) to a range of commercially available matrices for EBA. Cells and cell debris were found to interact with the ligands of agarose based resins mainly by electrostatic forces. From the adsorbents investigated the anion exchange matrix showed the most severe interactions, while cation exchange and affinity adsorbents appeared to be less affected. Within the range of biologic systems under study E. coli cells had the lowest tendency of binding to all matrices while hybridoma cells attached to all the adsorbents except the protein A affinity matrix. The method presented may be employed for screening of suitable biomass/adsorbent combinations, which yield a robust and reliable initial capture step by expanded bed adsorption from unclarified feedstock.     

Direct recovery of glutathione S-transferase by expanded bed adsorption: anion exchange as an alternative to metal affinity fusions

The use of expanded beds of ion-exchange adsorbents for the direct recovery of a recombinant intracellular protein, glutathione S-transferase (GST), from unclarified Escherichia coli homogenates is described. The results form the basis for a comparison between this approach for purifying GST and a chelating fusion strategy and highlight the need to consider the additional costs entailed by these more-complicated approaches. The separation performance was investigated with respect to choice of anion or cation exchanger, adsorption pH, load volume, sample preparation, and stepwise elution protocol. Anion exchange was found to be more appropriate than cation exchange, as the low pHs involved in the latter caused a loss of activity. The optimal pH for adsorption was found to be 9 with a dynamic capacity from clarified homogenate in packed mode of 112 U mL(-1) (11.2 mg GST mL(-1)). As increasing volumes of unclarified homogenate were applied to the expanded bed, the yield of GST in the eluate decreased, and the purification factor was found to increase and then decrease. This was due to the displacement of weakly bound proteins by GST and then its displacement by even more strongly binding proteins. The dynamic capacity of the anion exchanger, STREAMLINE DEAE, from unclarified homogenate in expanded mode decreased slightly to 85 U mL(-1) (8.5 mg GST mL(-1)). The elution protocol for GST from the anion exchanger was then adjusted to try to maximize the degree of purification. Anion exchange expanded bed adsorption of GST from unclarified E. coli homogenate gave an eluted yield of 95.7% and 1.64-fold purification. Interestingly, a decrease in the expression level of GST in the feedstream from 23 down to 13% caused a decrease in the dynamic capacity from 85 to 14.5 U mL(-1) whereas the degree of purification remained similar.     

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.     

Dye–ligand expanded bed adsorption of G6PDH from highly dense unclarified yeast extract

The application of dye–ligand expanded bed chromatography adsorption (EBA) of glucose-6-phosphate dehydrogenase (G6PDH) from unclarified yeast extract was undertaken by using a commercially available expanded bed column (20 mm i.d.) and UpFront adsorbent (ρ = 1.5 g/mL) from UpFront Chromatography. The influence of biomass concentration on the adsorption capacity was explored by employing yeast extracts containing various biomass concentrations (5–30%, w/v). It was demonstrated that the biomass concentration had little effect on G6PDH adsorption performance. Feedstock containing 15% (w/v) biomass gave a relatively high recovery yield (>90%) of G6PDH compared to feedstock containing 30% (w/v) biomass, which gave a recovery of 75% G6PDH. Nevertheless, the enzyme specific activity of 7 U mg⁻¹ with a purification factor of 6 was achieved in the feedstock containing biomass concentration of 30% (w/v). The generic applicability of dye–ligand as an affinity tool in expanded bed chromatography is discussed.     

Facilitated downstream processing of a histidine-tagged protein from unclarified E. coli homogenates using immobilized metal affinity expanded-bed adsorption

The facilitated downstream processing of an intracellular, polyhistidine-tagged protein, glutathione S-transferase [GST-(His)(6)], direct from unclarified E. coli homogenates using expanded beds of STREAMLINE chelating, has been investigated. A series of pilot experiments were used to develop preparative-scale separations of GST-(His)(6), initially in packed and then in expanded beds. Packed beds of Ni(2+)-loaded STREAMLINE chelating proved to have the highest 5% dynamic capacity for GST-(His)(6), of 357 U mL(-1) (36 mg mL(-1)). When using immobilized Cu(2+) or Zn(2+), metal ion transfer was observed from the iminodiacetate ligands to the high-affinity chelator, GST-(His)(6). The subsequent metal affinity precipitation of this homodimer resulted in operational problems. An equilibrium adsorption isotherm demonstrated the high affinity of GST-(His)(6) for immobilized Ni(2+), with a q(m) of 695 U mL(-1) (70 mg mL(-1)) and a K(d) of 0.089 U mL(-1) (0.0089 mg mL(-1)). Ni(2+)-loaded STREAMLINE chelating was therefore selected to purify GST-(His)(6) from unclarified E. coli homogenate, resulting in an eluted yield of 80% and a 3.34-fold purification. The high dynamic capacity in the expanded mode of 357 U mL(-1) (36 mg mL(-1)) demonstrates that this specific interaction was not affected by the presence of E. coli cell debris.     

Evaluation of the effect of in-bed sampling on expanded bed adsorption

An expanded bed adsorption (EBA) column (5 cm diameter) has been modified to allow the abstraction of liquid samples from various positions along the height of an expanded bed. As the adsorbent particles were fluidized, in-bed monitoring of key component concentrations during feedstock application, washing and elution was achieved by the withdrawal of liquid samples from the voids within the expanded bed through ports along the wall of the column. Component levels in the withdrawn streams can be assayed using on-line analytical chromatography or samples can be collected and assayed off-line. On-line monitoring can be used to control the duration of the loading stage and as a tool to provide information about the hydrodynamic and adsorption/desorption processes that occur during expanded bed adsorption. Studies of residence time distributions indicated that the modifications to the column do not significantly affect liquid dispersion. Using the adsorption of glucose-6-phosphate dehydrogenase from yeast homogenate on Streamline DEAE as a model system, comparison of breakthrough curves for runs when in-bed monitoring was and was not performed also suggested that separation efficiency is not appreciably affected by in-bed sampling.     

Direct coupling of expanded bed adsorption with a downstream purification step

In the course of developing a cost-effective, scaleable process for the purification of a recombinant protein from Chinese hamster ovary (CHO) suspension cell culture, we investigated direct capture of this molecule using expanded bed adsorption (EBA). EBA combines clarification, purification, and concentration of the product into a single step. The unclarified bioreactor material was directly applied to a STREAMLINE 25 column containing an affinity STREAMLINE adsorbent. This work focused on simplifying the EBA operations and minimizing the overall processing time by running the EBA column unidirectionally, eluting in the expanded bed mode, and coupling the EBA column directly with ion exchange or hydrophobic interaction chromatography. Unidirectional EBA was clearly a simpler unit operation and did not require the use of specialized equipment. The increase in the elution pool volume was insignificant, especially when the EBA column was eluted directly onto the downstream column. Scale-down was simple and could be automated. Coupling of unidirectional EBA with a downstream purification step reduced processing time, equipment requirements and cost.     

Influence of the extent of disruption of Bakers' yeast on protein adsorption in expanded beds

Expanded bed adsorption chromatography is used to capture the protein product of interest from a crude biological suspension directly, thereby eliminating the need for the removal of the cell debris. While this technique may replace three or four unit operations in a typical downstream process for biological product recovery, the adsorption process is influenced by the interaction between the microbial cells or cell debris and the adsorbent as well as the presence of contaminating solutes. The influence of the extent and nature of disruption of Bakers' yeast on the adsorption of the total soluble protein and alpha-glucosidase was investigated in this study. Two different techniques were used for cell disruption: high pressure homogenisation and hydrodynamic cavitation. Two different adsorbents were chosen: anionic Streamline DEAE and cationic Streamline SP. The settled bed height and the superficial velocity were constant across all experiments. The feedstock was characterised in terms of viscosity, pH, conductivity, particle size distribution of the cell debris and the extent of protein and alpha-glucosidase released. The performance of the adsorption process was found to be influenced by the electrostatic interactions of cell debris with the anionic adsorbent Streamline DEAE and the intraparticle diffusional resistance inside the pores of the adsorbent matrix. The increase in the intensity of disruption resulted in an increase in the dynamic binding capacity (10% feed) of both the total soluble protein and the alpha-glucosidase. However, the increase in the DBC of protein and alpha-glucosidase were not proportional. The amount of protein that could be adsorbed per ml of adsorbent from the samples subjected to a lower intensity of disruption was found to exceed that obtained at a higher disruption intensity on increasing the volume of feed suggesting multilayer adsorption. In this case, selective adsorption of the model protein alpha-glucosidase was reduced, illustrating the compromise of maximising protein recovery through non-specific binding. The study illustrates the need for an interrogation of the intensity of disruption needed and a rigorous understanding of the influence of cell debris and adsorbent-protein interaction, in optimising the selective recovery of intracellular products by EBA.     

An improved method for purification of recombinant truncated heme oxygenase-1 by expanded bed adsorption and gel filtration

Recombinant truncated human heme oxygenase-1 (hHO-1) expressed in Escherichia coli was efficiently separated and purified from feedstock by DEAE-ion exchange expanded bed adsorption. Protocol optimization of hHO-1 on DEAE adsorbent resulted in adsorption in 0 M NaCl and elution in 150 mM NaCl at a pH of 8.5. The active enzyme fractions separated from the expanded bed column were further purified by a Superdex 75 gel filtration step. The specific hHO-1 activity increased from 0.82 ± 0.05 to 24.8 ± 1.8 U/mg during the whole purification steps. The recovery and purification factor of truncated hHO-1 of the whole purification were 72.7 ± 4.7 and 30.2 ± 2.3%, respectively. This purification process can decrease the demand on the preparation of feedstock and simplify the purification process.