• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.776-780
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• 2001

Based on the static and dynamic adsorption of Streamline DEAE, a modified tank-in-series model including particle size distribution was used to describe the adsorption performance of bovine serum albumin in an expanded bed. The calculated results indicated that the suggested model was able to simulate breakthrough curves under various conditions.

• KSBB Journal
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• v.16 no.2
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• pp.146-152
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• 2001

To avoid the intrinsic problem of aggregation associated with the traditional solution-phase refolding process, we propose a solid-phase refolding method integrated with expanded bed adsorption chromatography. The model protein used was a fusion protein of recombinant human growth hormone and a glutathione S transferase fragment. It was demonstrated that the EBA-mediated refolding technique could simultaneously remove cellular debris and directly renature the fusion protein inclusion bodies in the cell homogenate with much higher yields and less agregation. To demonstrate the applicability of the method, we successfully tested the three representative types of starting materials, i. e., rhGH monomer, washed inclusion bodies, and the E. coli homogenate. This direct and simplified refolding process could also reduce the number of renaturation steps required and allow refolding at a higher concentration, at approximately 2 mg fusion protein per ml of resin. To the best of our knowledge, it is the first approach that has combined the solid-phase refolding method with expanded bed chromatography.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.3
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• pp.280-283
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• 2005

The adsorption of a model protein bovine serum albumin (BSA) in expanded bed chromatography was undertaken by exploiting a commercially available expanded bed column (20 mm i.d.) from UpFront Chromatography and Streamline DEAE $(\rho=1.2g/cm^3)$ from Amersham Pharmacia Biotechnology. The influence of whole yeast cells on the adsorption capacity of column was explored by employing yeast cells in a concentration ranged of 0 to $15\%(w/v)$. Equilibrium isotherms for adsorption of BSA on Streamline DEAE were correlated by using Langmuir equation. The presence of yeast cells resulted in decreased of BSA binding capacity in both batch binding and expanded bed chromatography. Results indicated that the yeast cells act as competitor for proteins to bind to the sites on adsorbents.

• KSBB Journal
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• v.18 no.6
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• pp.500-505
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• 2003

‘LK (lipoprotein kringle) 68’is a polypeptide of a modified ansiostatin consisting of three kringle structures that might be clinically useful as a potential cancer therapeutics. It can be produced by overexpressing it as inclusion body in recombinant E. coli. In this study, solid-phase refolding processes using packed bed adsorption (PBA) and expanded bed adsorption (EBA) column were carried out to compare their refolding yields with that of the conventional, solution-phase refolding process, For the solution-phase and the PBA-mediated processes employing Q-Sepharose, washed inclusion body was used as the starting material, whereas both washed inclusion body and E. coli homogenate were used for the EBA-mediated process employing streamline DEAE. On the final recovery LK68 per unit mass of wet cell basis, the EBA- and PBA-mediated processes showed about 2.7- and 1.5-fold higher yields, respectively, than the solution-phase refolding method. The solid-phase refolded LK68 demonstrated the same Iysine binding bioactivity and the retention time in the RP-and SEC-HPLC as those of the native protein.

• Journal of Microbiology
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• v.42 no.3
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• pp.228-232
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• 2004

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

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.2
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• pp.128-135
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• 2005

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 feedstock via an expanded bed process. In the present work, a new type of mixed-mode adsorbent designed for expanded bed adsorption, Fastline $PRO^{\circledR}$, was challenged for the capture of nattokinase from the high ionic fermentation broth of Bacillus subtilis. Two important factors, pH and ion concentration, were investigated with regard to the performance of nattokinase ad-sorption. 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 between Bacillus subtilis cells and Fastline PRO, and to demonstrate a stable expanded bed in the feedstock containing Bacillus 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.

• Journal of Microbiology and Biotechnology
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• v.19 no.4
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• pp.416-423
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• 2009

Hepatitis B core antigen(HBcAg) is an important serological marker used in the diagnosis of hepatitis B virus(HBV) infections. In the current study, a fast and efficient preparative purification protocol for truncated HBcAg from Escherichia coli disruptate was developed. The recombinant HBcAg was first captured by anion exchange expanded bed adsorption chromatography integrated with a cell disruption process. This online capture process has shortened the process time and eliminated the "hold-up" period that may be detrimental to the quality of target protein. The eluted product from the expanded bed adsorption chromatography was subsequently purified using size-exclusion chromatography. The results showed that this novel purification protocol achieved a recovery yield of 45.1% with a product purity of 88.2%, which corresponds to a purification factor of 4.5. The recovered HBcAg is still biologically active as shown by ELISA test.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.3
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• pp.268-272
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• 2006

A dense, pellicular UpFront adsorbent ($p=1.5 g/cm^3$, UpFront Chromatography, Cophenhagen, Denmark) was characterized in terms of hydrodynamic properties and protein adsorption performance in expanded bed chromatography. Cibacron Blue 3GA was immobilised into the adsorbent and protein adsorption of bovine serum albumin (BSA) was selected to test the setup. The Bodenstein number and axial dispersion coefficient estimated for this dense pellicular adsorbent was 54 and $1.63{\times}10^{-5}m^2/s$, respectively, indicating a stable expanded bed. It could be shown that the BSA protein was captured by the adsorbent in the presence of 30% (w/v) of whole-yeast cells with an estimated dynamic binding capacity $(C/C_o = 0.01)$ of approximately 6.5 mg/mL adsorbent.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.6
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• pp.552-555
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• 2005

The influence of whole yeast cells $(0{\sim}15%\;w/v)$ on the protein adsorption performance in dye-ligand chromatography was explored. The adsorption of a model protein, bovine serum albumin (BSA), was selected to demonstrate this approach. The UpFront adsorbent $(p=1.5\;g/cm^3)$ derivatised with Cibacron Blue 3GA and a commercially available expanded bed column (20 mm i.d.) from UpFront Chromatography, Denmark, were employed in the batch binding and expanded bed operation. The BSA binding capacity was demonstrated to not be adversely affected by the presence of yeast cells. The dynamic binding capacity of BSA at a $C/C_0=0.1$ biomass concentration of 5, 10, 15% w/v were 9, 8, and 7.5mg/mL of settled adsorbent, respectively.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.5
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• pp.466-469
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• 2006

The bed stability of Streamline DEAE (p = 1.2 g/mL) in a 20mm (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).