Streamline SP Research Articles

Purification of lysozyme from chicken egg white by high-density cation exchange adsorbents in stirred fluidized bed adsorption system

Lysozyme from crude chicken egg white (CEW) feedstock was successfully purified using a stirred fluidized bed adsorption system ion exchange chromatography where STREAMLINE SP and SP-XL high density adsorbents were selected as the adsorption carrier. The thermodynamic and kinetic studies were carried out to understand the characteristics of lysozyme adsorption by adsorbents under various conditions, including adsorption pH, temperature, lysozyme concentration and salt concentrations. Results showed that SP and SP-XL adsorbents achieved optimum lysozyme adsorption at pH 9 with capacity of ~139.77 and ~251.26 mg/mL, respectively. The optimal conditions obtained from batch studies were directly employed to operate in SFBA process. For SP-XL adsorbent, the recovery yield and purification factor of lysozyme were 93.78% and ~40 folds, respectively. For SP adsorbent, lysozyme can be eluted ~100% with purification factor of ~26 folds. These two adsorbents are highly suitable for use in direct recovery of lysozyme from crude CEW.

Purification of Peroxidase from Rice Bran Using Expanded-Bed Ion-Exchange Chromatography

Peroxidase catalyzes the oxidation of various substrates at the expense of hydrogen peroxide. Among the various techniques used for the purification of enzymes, expanded-bed adsorption chromatography is particularly popular because it offers several advantages, such as greater interactions between adsorbents and target molecules, increased overall yield, potential for a scale-up and shorter process times. It relies on the interaction between charged molecules in the mobile phase (i.e. buffer and sample) and oppositely charged groups coupled to the resin in the expanded-bed form. Other chromatographic techniques are also commonly used for peroxidase purification and characterization; however, there are no reports in the literature about the use of expanded-bed adsorption chromatography for this purpose. In this paper, the purification of peroxidase from rice bran using ion-exchange chromatography (expanded-bed column) was investigated. Chromatographic assays were carried out using STREAMLINE SP cationic re...

Modeling and simulation of breakthrough curves during purification of two chitosanases from Metarhizium anisopliae using ion-exchange with expanded bed adsorption chromatography

A mathematical model was developed to predict breakthrough curves during purification of the two chitosanases from Metarhizium anisopliae by expanded bed adsorption, taking into account the axial dispersion of liquid and using Streamline DEAE and SP XL adsorbents, anion and cation exchange resins, respectively. All the experiments were performed without clarification (with cells) aiming at the reduction of unit operations in future projects of separation processes, thereby reducing capital and operating costs. Chitosanases are enzymes that hydrolyze the carbohydrate chitosan, resulting in oligosaccharides that have many remarkable biological activities, such as anti-cancer, anti-HIV and antioxidant activities. The two adsorbents had similar performance in relation to hydrodynamics and mass transfer. The results of the parametric sensitivity analysis agree with the literature, and the model was validated with an average high degree of fit (94.68%) between simulated and experimental data obtained in this work.

Mathematical modeling and simulation of inulinase adsorption in expanded bed column

A mathematical model for an expanded bed column was developed to predict breakthrough curves for inulinase adsorption on Streamline SP ion-exchange adsorbent, using a crude fermentative broth with cells as the feedstock. The kinetics and mass transfer parameters were estimated using the PSO (particle swarm optimization) heuristic algorithm. The parameters were estimated for each expansion degree (ED) using three breakthrough curves at initial inulinase concentrations of 65.6 U mL −1. In sequence, the model parameters for an ED of 2.5 were validated using the breakthrough curve at an initial concentration of 114.4 U mL −1. The applicability of the validated model in process optimization was investigated, using the model as a process simulator and experimental design methodology to optimize the column and process efficiencies. The results demonstrated the usefulness of this methodology for expanded bed adsorption processes.

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.

A permeabilization effect of organic solvents on cell bound penicillin v acylase activity of Erwinia aroideae (DSMZ 30186)

Methanol limited fed-batch cultivation was applied for production of a plant derived β-glucosidase by Pichia pastoris. The β-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 β-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 β-glucosidase on Streamline Direct HST1 (74%) was significantly higher than on Streamline SP (48%). Furthermore, β-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 β-glucosidase from unclarified P. pastoris high-cell-density cultivation broth.

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.

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 α-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 α-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 α-glucosidase. However, the increase in the DBC of protein and α-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 α-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.

Method development for direct recovery of lysozyme from highly crude chicken egg white by stirred fluidized bed technique

An improved procedure was developed involving a stirred fluidized bed (SFB) adsorption for direct capture of lysozyme from unclarified preparations of chicken egg whites (CEW). The maximal binding capacity of Streamline SP adsorbent for lysozyme was observed at pH 4. A typical adsorption isotherm curve for lysozyme in crude CEW was well correlated with the Freundlich model. For an initial concentration of 20 mg CEW/ml, adsorption approached equilibrium after 30 min. Determination of dynamic binding capacity for lysozyme in a stirred fluidized bed column (i.d. 2.5 cm) was carried out using frontal analysis. The maximal loading volume of crude CEW feedstock (20 mg/ml) at 1% breakthrough for lysozyme was approximately 5, 22, and 28-fold settled bed volumes, corresponding to 256, 2173, and 4125 ml CEW, respectively. Optimal conditions for elution scheme were determined in small packed bed experiments conducted with clarified CEW. Further experiments were carried out to assess the feasibility of using Streamline SP adsorbent and SFB column for direct recovery of lysozyme from highly crude CEW (20 mg/ml in 20 mM sodium acetate buffer at pH 4). It was found to be successful in achieving purification of lysozyme in a high yield of 98% with a purification factor of 11 in a single step. The specific activity (7.80 × 10 4 U/mg) was higher than that obtained with a commercially available pure sample of lysozyme (Sigma, 7.30 × 10 4 U/mg).

On-column refolding of recombinant human interferon-γ inclusion bodies by expanded bed adsorption chromatography

A refolding strategy was described for on-column refolding of recombinant human interferon-gamma (rhIFN-gamma) inclusion bodies by expanded bed adsorption (EBA) chromatography. After the denatured rhIFN-gamma protein bound onto the cation exchanger of STREAMLINE SP, the refolding process was performed in expanded bed by gradually decreasing the concentration of urea in the buffer and the refolded rhIFN-gamma protein was recovered by the elution in packed bed mode. It was demonstrated that the denatured rhIFN-gamma protein could be efficiently refolded by this method with high yield. Under appropriate experimental conditions, the protein yield and specific activity of rhIFN-gamma was up to 52.7% and 8.18 x 10(6) IU/mg, respectively.

Recovery of recombinant β-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 β-glucosidase by Pichia pastoris. The β-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 β-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 β-glucosidase on Streamline Direct HST1 (74%) was significantly higher than on Streamline SP (48%). Furthermore, β-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 β-glucosidase from unclarified P. pastoris high-cell-density cultivation broth.

Predictive modeling of protein adsorption along the bed height by taking into account the axial nonuniform liquid dispersion and particle classification in expanded beds

Expanded bed adsorption (EBA) is a special chromatography technique with perfect classification of adsorbent particles in the column, thus the performance of protein adsorption in expanded beds is particular, obviously nonuniform and complex along the column. Detailed description of the complex adsorption kinetics of proteins in expanded bed is essential for better analyzing of adsorptive mechanisms, the design of chromatographic processes and the optimization of operation parameters of EBA processes. In this work, a theoretical model for the prediction of protein adsorption kinetics in expanded beds was developed by taking into account the classified distribution of adsorbent particles along the bed height, the nonuniform behaviors of axial liquid dispersion, the axial variation of local bed voidage as well as the axial changes of target component mass transfer. The model was solved using the implicit finite difference scheme combining with the orthogonal collocation method, and then applied to predict the breakthrough behaviors of bovine serum albumin (BSA) on Streamline DEAE and lysozyme on Streamline SP along the bed height in expanded beds under various conditions. In addition, the experiments of front adsorption of BSA on Streamline DEAE at different axial column positions were carried out to reveal the adsorption kinetics of BSA along the bed height in a 20 mm I.D. expanded bed, and the influences of liquid velocity and feed concentration on the breakthrough behaviors were also analyzed. The breakthrough behaviors predicted by the present model were compared with the experimental data obtained in this work and in the literature published. The agreement between the prediction and the experimental breakthrough curves is satisfied.

Variation of the local effective axial dispersion coefficient with bed height in expanded beds

Abstract The local axial dispersion characteristics are very important and fundamental for the analyzing, modeling and understanding of biomolecules adsorption performance in expanded bed adsorption. In this work, the local effective axial dispersion coefficients along the bed height of an expanded bed system with porous adsorbent particles, Streamline SP and DEAE, were determined by measuring residence time distributions (RTDs) using the tracer pulse–response method combining in-bed sampling technique. The decreasing trend of the local effective axial dispersion coefficient with the increase of bed height was identified. An empirical correlation for the variations of the local effective axial dispersion coefficient with bed height was developed by taking account of the complex effects of liquid and particle properties, liquid flow behaviors, particle movements and the local bed voidage and particles size variations.

A 3-zone model for protein adsorption kinetics in expanded beds

The adsorption behavior of expanded beds is more complex than that of fixed beds, since the adsorbent particle size, local bed voidage and liquid axial dispersion will vary axially with expanded height. Models applicable to fixed beds maybe not adequately describe the hydrodynamic and adsorption behavior in expanded beds. In this paper, a 3-zone model is developed, in which the model equations are written for the bottom zone, middle zone, and top zone of the column, respectively, and the model parameters, such as the adsorbent particle diameter, bed voidage and liquid axial dispersion coefficient, are zonal values. In-bed breakthrough curves are predicted by the 3-zone model, and tested against literature data for lysozyme adsorption on Streamline SP in an expanded bed. Model parametric sensitivity is analyzed. The effects of film mass transfer resistance, liquid axial dispersion and adsorbent axial dispersion on the breakthrough curves are weaker than that of protein intraparticle diffusion resistance for stable expanded beds. Adsorbent particle size axial distribution and bed voidage axial variation significantly affect in-bed breakthrough curves, therefore, model parameters should not be assigned uniform values over the whole column; instead the model should account for the adsorbent particle size axial distribution and bed voidage axial variation.

Ion exchange expanded bed chromatography for the purification of an extracelular inulinase from Kluyveromyces marxianus

The use of an expanded bed of Streamline SP resin (Pharmacia) for the purification of an extracellular inulinase directly from an unclarified crude broth was investigated. The purification yield achieved 74% with a purification factor of about 10.4. Adsorption were similar whether the crude medium had microbial cells or not. The influence of bed expansion on adsorption performance was determined by frontal analysis. Breakthrough curves for inulinase were compared and the process was more efficient at a bed expansion degree of 2.0 (bed voidage of 0.7).

Modeling axial distributions of adsorbent particle size and local voidage in expanded bed

Axial distribution of adsorbent particle as well as local voidage variation is very important and fundamental for the predicting and understanding of adsorptive performance in expanded bed adsorption. Based on the analysis of bed expansion and fluid hydrodynamic behaviors, a model was developed to predict the mean particle size and local voidage variations with the axial bed height under various operation conditions in expanded bed. Experimental measurements of particle size and local voidage changes with bed height were conducted in a modified glass column of 20 mm inner diameter with sampling ports. Experimental data obtained in this work and in the literature with Streamline SP, Streamline Phenyl and Streamline quartz base matrix particles in the columns of inner diameters 20– 50 mm , were used to verify the model prediction. The results were in good agreement with the experimental data.

Production of a human calcitonin precursor with Staphylococcus carnosus: secretory expression and single-step recovery by expanded bed adsorption

Recombinant human calcitonin (hCT) can be expressed by Staphylococcus carnosus as a precursor peptide being part of a secreted fusion protein which contains the propeptide of a Staphylococcus hyicus lipase as secretion carrier and multiple repeats of the hCT precursor. Fusion proteins containing two (PhCT2), four (PhCT4), or eight (PhCT8) hCT precursors were expressed. Due to the unfavourable biochemical properties of PhCT4 and PhCT8, PhCT2 was the most efficient fusion protein for a production process. Expression from a xylose-inducible promoter from Staphylococcus xylosus resulted in a maximum secretory production of PhCT2, since the amount of PhCT2 synthesized in total corresponded almost precisely to the upper limit of the capacity of the cellular protein secretion apparatus. An expanded bed adsorption procedure for the recovery of PhCT2 from unclarified culture broth was established using the cation exchanger STREAMLINE SP at pH 5. This integrated operation allows a single-step recovery of PhCT2 and was successfully transferred to a pilot scale (100 l culture broth).

Effect of Saccharomyces cerevisiae fermentation conditions on expanded bed adsorption of heterologous cutinase

AbstractThe effect of culture media composition, and fermentation conditions and strategy on the growth and cutinase production of recombinant Saccharomyces cerevisiae and subsequent cutinase purification by expanded bed adsorption (EBA) was studied. The reduction in the amount of yeast extract used as nitrogen source from 20 g dm−3 to 10 g dm−3 in batch cultures led to a 29% decrease in the heterologous cutinase production, while the 5% cutinase dynamic adsorption capacity (q5%) on the cation Streamline SP XL was increased 6.7‐fold. By dilution of the whole fermentation broth, performed with the lowest yeast extract content, which reduces conductivity, the q5% was additionally increased by 1.9‐fold. After implementation of a fed‐batch strategy the cutinase concentration, cutinase yield on carbon source, cutinase yield on nitrogen source and productivity were increased by 10.8‐, 2.9‐, 5.3‐ and 6.4‐fold, respectively, in relation to the previously‐mentioned batch fermentation. However, the increased cutinase production was compromised by heterologous protein loss during the EBA recovery operation and the cutinase dynamic adsorption capacity and purification productivity decreased by 90% and 75%, respectively. Thus, target protein production by S cerevisiae fermentation and a downstream process with EBA cannot be considered as separate entities, where the understanding of the factors that affect the interactions among them are crucial towards optimization of the overall production process of heterologous proteins.© 2002 Society of Chemical Industry

The combined use of in-bed monitoring and an adsorption model to anticipate breakthrough during expanded bed adsorption

Tighter control of expanded bed adsorption (EBA) separations is shown to be possible through the combined use of on-line monitoring of samples abstracted from within a column and an adsorption model. A 5 cm diameter EBA column was modified to allow liquid sampling from within the fluidized bed during the adsorption of pure lysozyme and bovine serum albumin (BSA) on STREAMLINE SP and STREAMLINE DEAE, respectively. The developing breakthrough curve was measured at various positions along the bed. The adsorption model incorporated terms for liquid dispersion, film mass transfer, pore diffusion and adsorption. Parameters obtained by fitting the model to the experimental in-bed breakthrough curves were subsequently used to predict breakthrough at the bed exit. Comparison of the actual breakthrough curves at the bed exit ( 40 cm sample port) with those predicted based on the fit of the model to the experimental breakthrough data obtained from the 25 cm sample port, demonstrated that on-line, in-bed monitoring of the progress of the front of non-bound protein can provide information for tighter control of EBA separations. The accuracy of the model predictions was improved by employing information on the axial variations in the bed voidage, liquid phase axial dispersion and dynamic capacity for the top and bottom zones within the column. Ten per cent breakthrough at the 40 cm sample port was predicted to within 2% and 5% of the breakthrough time observed for the experimental runs for lysozyme and BSA, respectively.

Facile F(ab′) 2 Manufacturing: Strategies for the Production of Snake Antivenoms

Asimple process for the manufacture of polyclonal F(ab′)2 fragments that might be adopted for the facile preparation of antivenoms is described. The production of polyclonal F(ab′)2 fragments from serum commonly involves the initial purification of IgG's prior to their proteolytic cleavage and further purification. To reduce the number of processing steps the authors have compared the digestion of whole serum by free and immobilized pepsin with that of pure IgG. It was observed that with equal units of pepsin activity, caprylic acid pre-purifi ed IgG was digested more rapidly than whole serum but that the overall retention of antigen binding activity was signifi cantly greater in the latter case. The effi ciencies of whole serum digestion by the same total units of free and immobilized pepsin was also compared. IgG digestion was complete after 12 hours, as was serum digestion. About 80% of the antigen binding activity was retained in either case after 24 hours though the immobilized pepsin lost about 40% of its proteolytic activity after first use. The purifi cation of F(ab′)2 on two ion exchange adsorbents (StreamlineTMSP and paramagnetic CM beads) on paramagnetic thiophilic beads and on a hydrophobic custom assembled guanidine agarose was studied. The binding capacity on CM beads was too low to be useful for F(ab′)2 preparation. Most of the F(ab′)2 bound to the Streamline SP at low pH (pH 4.0 and 3.0), as did significant amounts of low molecular weight digestion products. The F(ab′)2 fragments were eluted completely with 0.3M NaCl whilst the low molecular weight digestion products required 1M NaCl to elute them. F(ab′)2 also bound to the thiophilic beads, free from the low molecular weight digestion products, and could be eluted with low ionic strength solutions. The guanidine agarose beads showed a lower capacity than the thiophilic beads but their selectivity was comparable. Overall the thiophilic and hydrophobic matrices showed better selectivity than the cation exchange matrices.