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.     

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.     

Cost Comparison of Protein Capture from Cultivation Broths by Expanded and Packed Bed Adsorption

In the downstream processing of recombinant protein production, the reduction of unit operations required for product capture and purification, is of the utmost priority due to its cost diminishing effect. In this regard, target protein capture from cell suspensions in a fluidized bed of affinity particles with different sizes (expanded bed adsorption (EBA) with classified particles), presents an efficient tool since EBA may substitute cell disintegration, separation by centrifugation or filtration, and packed bed adsorption. However, as illustrated by experiments with the BSA/yeast cells system, the entire broth processing used in EBA also has detrimental influences due to the cell (or cell debris) binding on the affinity carrier. In particular, external mass transfer may become more dominant, and the lifetime of the affinity particles may reduce as a result of other cleaning procedures. Using simulations performed with a commercial software package, the cost superiority of alternate process routes (EBA or packed bed adsorption with preceding steps) can be evaluated. This elucidates the favorable application range for each route.     

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.     

A Study of the interaction of HEK-293 cells with streamline chelating adsorbent in expanded bed operation

Expanded bed adsorption (EBA) is an efficient protein purification process reducing time and steps of downstream processing (DSP) since nonclarified culture media can be processed directly without prior treatments such as filtration or centrifugation. However, cells and debris can interact with the adsorbent and affect bed stability as well as purification performance. To optimize EBA operating conditions these biomass/adsorbent interactions have to be understood and characterized. The adsorption of Human Embryonic Kidney cells (HEK 293) on unprimed and nickel-primed metal affinity adsorbent was studied in a closed loop EBA setup. With the unprimed adsorbent, the overall level of interaction observed was nonsignificant. With the nickel-primed adsorbent and an initial cell concentration ranging from 0.08 x 10(6) to 0.2 x 10(6) cells/mL, biomass/adsorbent interaction was found to be moderate and the adsorption apparent first-order kinetic rate constant was determined to be k = 0.009 to 0.011 min(-1).     

The influence of homogenisation conditions on biomass-adsorbent interactions during ion-exchange expanded bed adsorption

Expanded bed adsorption (EBA) is an integrative step in downstream processing allowing the direct capture of target proteins from cell-containing feedstocks. Extensive co-adsorption of biomass, however, may hamper the application of this technique. The latter is especially observed at anion exchange processes as cells or cell debris are negatively charged under common anion exchange conditions. The restrictions observed under these conditions are, however, directly related to processing steps prior to fluidised bed application. In this study, it could be shown that the effective surface charge of cell debris obtained during homogenisation is closely related to the debris size and thus to the homogenisation method and conditions. The amount and thus effect of cells binding to the adsorbent could be significantly decreased when optimising the homogenisation step not only towards optimal product release but towards a reduction of debris size and charge. The lower size and charge of the debris results not only in a reduced retention probability but also, in a lower collision probability between debris and adsorbent. The applicability was shown in an example where the homogenisation conditions of E. coli were optimised towards EBA applications. In a previous report (Reichert et al., 2001) studying the suitability of EBA for the capture of formate dehydrogenate from E. coli homogenate the pseudo affinity resin Streamline Red was identified as the only suitable adsorbent. The new approach, however, led to a system where anion exchange as capture step became possible, however, to the cost of binding capacity.     

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.     

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.     

Separation of nattokinase fromBacillus subtilis fermentation broth by expanded bed adsorption with mixed-mode adsorbent

Mixed-mode hydrophobic/ionic matrices exhibit a salt-tolerant property for adsorbing target protein from high-ionic strength feedstock, which allows the application of undiluted feedstockvia an expanded bed process. In the present work, a new type of mixed-mode adsorbent designed for expanded bed adsorption, Fastline PRO^®, was challenged for the capture of nattokinase from the high ionic fermentation broth ofBacillus subtilis. Two important factors, pH and ion concentration, were investigated with regard to the performance of nattokinase adsorption. Under initial fermentation broth conditions (pH 6.6 and conductivity of 10 mS/cm) the adsorption capacity of nattokinase with Fastline PRO was high, with a maximum capacity of 5,350 U/mL adsorbent. The elution behaviors were investigated using packed bed adsorption experiments, which demonstrated that the effective desorption of nattokinase could be achieved by effecting a pH of 9.5. The biomass pulse response experiments were carried out in order to evaluate the biomass/adsorbent interactions betweenBacillus subtilis cells and Fastline PRO, and to demonstrate a stable expanded bed in the feedstock containingBacillus subtilis cells. Finally, an EBA process, utilizing mixed-mode Fastline PRO adsorbent, was optimized to capture nattokinase directly from the fermentation broth. The purification factor reached 12.3, thereby demonstrating the advantages of the mixed-mode EBA in enzyme separation.     

Human chymotrypsinogen B production from Pichia pastoris by integrated development of fermentation and downstream processing. Part 2. Protein recovery

The purification of human chymotrypsinogen B (hCTRB) after expression and secretion by the yeast Pichia pastoris is described based on two different approaches using integrated initial recovery. Extraction employing aqueous two-phase systems (ATPS) from poly(ethylene glycol) and sodium sulfate allows direct processing of cell containing yeast suspensions of 50% wet weight. The target protein is obtained partially purified in the top phase while cells and cell debris are partitioned to the bottom phase of the system. hCTRB is further purified by adsorption from the top phase to the cation exchanger SP Sepharose Big Beads and elution in a salt step. The single step isolation of hCTRB is possible by expanded bed adsorption (EBA) using a fluidized cation exchanger (Streamline SP XL). A design strategy is shown taking both target protein binding and stable fluidization of the stationary phase in cell containing suspensions into consideration. For the example of hCTRB isolation from cell containing P. pastoris suspensions, a successful use of this strategy is demonstrated. Both initial recovery strategies deliver a product that can be further purified and formulated by ultrafiltration/diafiltration followed by lyophilization, resulting in a homogeneous product. Scale-up to 30-90 L of culture suspension was shown for both methods, resulting in a product of similar quality. Comparing both strategies reveals that the two-step ATPS route is better suited for high cell density cultures, while the single step EBA method is preferred for cultures of moderate cell density. This is due to the fact that application of EBA is restricted to suspensions of 10-12.5% wet weight cell concentration, thus necessitating dilution of the original broth prior to sample application. The data presented show that integrated recovery operations are a valuable alternative to traditional processing for systems that are problematic during initial solid-liquid separation.     

The use of rapid on-line monitoring of products and contaminants from within an expanded bed to control separations exhibiting fast breakthrough characteristics and to maximize productivity

Conventional control of expanded-bed adsorption (EBA), like that of packed-bed chromatography, is based upon off-line measurements of the column eluant. The relatively high-void volumes in EBA systems means that this approach can lead to significant performance losses caused by the inability to achieve tight control of breakthrough. This problem is made worse if the product has a fast breakthrough characteristic or if it is necessary to operate to low levels of product loss. In this article we examine the utility of constant on-line monitoring from within the expanded bed using stopped-flow analysis (SFA) to provide data for the control of the expanded-bed operation. A modified Streamline 50 column with side ports that enable sampling along the expanded axis of the bed was used. Comparisons between off-line and on-line measurements are presented, showing how the advanced monitoring method can lead to better control and to an analysis of breakthrough development within the bed. The expanded bed was used to purify alcohol dehydrogenase from homogenized suspensions of bakers' yeast. Accurate control of breakthrough to 10% of the target enzyme was achieved using a SFA control system with a response time of 40 seconds. On-line data compared well to assays carried out off-line on the outlet stream for both the product enzyme (ADH), total protein, RNA, and cell debris levels (via UV 650 nm). This information was used to generate a series of graphs with which to track the EBA process in real-time. Results showed that bed utilization was not linear along the bed axis so that, for example, 60% of ADH is bound in the bottom 33% of the column during loading.     

Monitoring, modeling, and control strategies for expanded-bed adsorption processes

Expanded-bed adsorption (EBA) is a technique for the purification of proteins from cellular debris in downstream processing. An expanded bed presents the possibility of protein recovery in a single step, eliminating the often costly clarification processing steps such as ultrafiltration, centrifugation, and precipitation. An obstacle to the successful commercialization of this technology is the inability to accurately monitor and control the bed height in these systems.In this paper, we present an overview of work in our laboratory addressing monitoring, modeling, and control strategies as applied to EBA. First, we present the development of a level measurement technique based upon ultrasonics. It is shown that this technique has great promise for bed-height measurement in EBA systems. Second, we present modeling strategies for bed-height dynamics due to flow rate and fluid property changes, and lastly, we show how monitoring and modeling information can be used for the control and regulation of bed expansion.     

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.     

Thymol-triggered lysis of Escherichia coli expressing Lactobacillus phage LL-H muramidase

Effective disruption of Escherichia coli cells is achieved by the intracellularly accumulated recombinant murein hydrolase (Lactobacillus bacteriophage LL-H muramidase) after the addition of 5 mM thymol. Thymol destroys the integrity and electric potential of the cytoplasmic membrane, and as a consequence the muramidase can access and hydrolyze the cell wall murein leading to cell lysis. Lysis occurred within 5 min after the addition of thymol and seemed to be efficient at high culture densities. This lysis method does not require cell harvesting or addition of other cell wall weakening substances or exogenous enzymes. As a cell disruption method, thymol-triggered lysis is as efficient as sonication in the presence of 1% Triton. Furthermore, thymol did not interfere with the purification steps of Mur by expanded bed adsorption chromatography (EBA), suggesting that the lysis method presented here is well suited for large-scale production and purification of intracellular proteins of E. coli. Received 21 April 1998/ Accepted in revised form 5 December 1998     

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.     

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.     

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.     

The influence of complex biological feedstock on the fluidization and bed stability in expanded bed adsorption

The stability of expanded bed adsorption systems (EBA) was studied in biomass containing culture broth by residence time distribution (RTD) experiments, using pulse inputs of fluorescent molecules as tracers. Different commercial adsorbents (Streamline DEAE, SP, Phenyl, Chelating, and AC) were tested at various biomass concentrations (2.5–12 %, wet weight) of whole (Saccharomyces cerevisiae) yeast, yeast cell homogenate, and Escherichia coli homogenate. Analyzing the RTD according to the PDE model (PDE: axially dispersed plug‐flow exchanging mass with stagnant zones) allowed the calculation of three parameters: the number of transfer units for mass exchange between mobile and stagnant fraction (N), the Peclet number for overall axial dispersion (P), and the mobile fraction of the liquid in axially dispersed plug flow (φ). When fluidization was performed in particle‐free buffer the normalized response signal (after perfect input pulse) was symmetric (N:0; P: 50–100; φ: 1), thus, demonstrating the formation of a homogeneous fluidized (expanded) bed. Upon application of suspended biomass the RTD was skewed, depending on the adsorbent used and the type and level of biomass present in the sample. This situation leads to three different characteristic pictures: the well‐fluidized system (N: ≥ 7–10; P: ≥ 40; φ: 0.80–0.90), the system exhibiting bottom channeling (N: < 1–2; P: ≥ 40; φ: 0.5–0.7) and, the system where extensive agglomeration develops (N: 4–7; P: 20–40; φ: < 0.5). These results demonstrate that changes in the hydrodynamics of EBA already take place in the presence of moderate concentrations of biomass. Furthermore, those changes can be quantitatively described mainly in terms of the fraction of stagnant zones in the system, which are formed due to the interaction of biomass and adsorbent. The technique described here can be used to evaluate a certain combination of adsorbent and biomass with regard to its suitability for expanded bed adsorption from whole broth. © 1999 John Wiley & Sons, Inc. Biotechol Bioeng 64: 484–496, 1999.