Expression and Purification of Recombinant Human ...

Expression and Purification of Recombinant Human Granulocyte ColonyStimulating Factor in Fed-Batch Culture of Escherichia coli Chang-Kyu Kim, Jun-Ha Choi, SeungBae Lee, Sang-Mahn Lee & Jae-Wook Oh

Applied Biochemistry and Biotechnology Part A: Enzyme Engineering and Biotechnology ISSN 0273-2289 Volume 172 Number 5 Appl Biochem Biotechnol (2014) 172:2425-2435 DOI 10.1007/s12010-013-0708-y

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Author's personal copy Appl Biochem Biotechnol (2014) 172:2425–2435 DOI 10.1007/s12010-013-0708-y

Expression and Purification of Recombinant Human Granulocyte Colony-Stimulating Factor in Fed-Batch Culture of Escherichia coli Chang-Kyu Kim & Jun-Ha Choi & Seung-Bae Lee & Sang-Mahn Lee & Jae-Wook Oh

Received: 24 October 2013 / Accepted: 25 December 2013 / Published online: 5 January 2014 # Springer Science+Business Media New York 2014

Abstract Granulocyte colony-stimulating factor (G-CSF) is a cytokine that has multiple roles in hematopoietic cells such as the regulation of proliferation and differentiation. Here, we describe fed-batch culture, refolding, and purification of rhG-CSF. The suitability of urea or sarcosine for solubilizing inclusion bodies (IBs) was tested. It was observed that urea is more efficient for solubilizing and refolding IBs than sarcosine is. The purity of rhG-CSF and the removal percentage of the rhG-CSF isoforms during purification were increased by pH 5.5 precipitation. The purity and the yield of purified rhG-CSF were 99 % and 0.5 g of protein per liter culture broth, respectively. Our protocols of recombinant protein purification using ion exchange chromatography and semipreparative high performance liquid chromatography of pH-precipitated refolded solution may be informative to the industrial scale production of biopharmaceuticals. Keywords Human granulocyte colony-stimulating factor . Fed-batch . Inclusion bodies . Refolding buffer . Recombinant protein purification

Introduction Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein that is secreted in response to specific stimulation by a variety of cells including macrophages, fibroblasts, endothelial cells, and bone marrow stroma [1, 2]. Upon binding to the cognate membrane receptor on cells, G-

Electronic supplementary material The online version of this article (doi:10.1007/s12010-013-0708-y) contains supplementary material, which is available to authorized users.

C.30 % saturation dissolved oxygen (DO), 1 vvm air flow, and internal pressure of 0.5 kgf/cm2 at 400 rpm. The pH of the culture broth was maintained at 7.0 by ammonia water. As the DO value decreases, the agitation speed, airflow rate, and internal pressure were increased up to 600 rpm, 2 vvm, and 1.0 kgf/cm2, respectively. Substrate was fed using the glucose control method based on pH stat. A peristaltic pump was used to control the addition of feeding media to maintain the glucose concentration of the culture broth at 0.1 %. IB Solubilization and Refolding of rhG-CSF The cells treated with CaCl2 were harvested by centrifugation at 5,000×g for 20 min. Cell pellets were suspended in lysis buffer and disrupted using a homogenizer at 9,000 psi for three cycles. IBs were washed twice with buffer and the washed IBs including rhG-CSF were solubilized in two different buffers: 50 mM Tris (pH 8.0) containing 2 % sarcosine and 8 M urea containing 10 mM DTT, 1 mM EDTA, and 50 mM glycine (pH 8.0), respectively, and then diluted in refolding buffer (50 mM Tris, 20 mM CuSO4 5H2O) to a final protein concentration of 1 mg/mL. The diluted solution was refolded at 25 °C for 16 h. After refolding, the pH of the solution was adjusted to 5.5 with 1 M H3PO4. The supernatant containing the refolded rhG-CSF after centrifugation was recovered for the next experiments. rhG-CSF Purification Ion Exchange Chromatography Acetic acid (3 %) was added to the recovered supernatant for maximum binding to the cation exchange resins, and the supernatant was concentrated using hollow fiber ultrafiltration (M.W.C.O. 10 K). One gram of the concentrated supernatant was loaded on 150 mL of source S resins (Amersham Bioscience, Piscataway, NJ, USA) and sulfopropyl (SP)-sepharose, respectively. Chromatography was performed at a flow rate of 5 mL/min at 4 °C using AKTA Prime chromatographic system (GE Healthcare Biosciences, Piscataway, NJ, USA). During the SPsepharose chromatography, fractions were collected by 0–1 M NaCl gradient elution method. In the case of source S resins, chromatography was carried out by pH gradient elution method. Semipreparative High Performance Liquid Chromatography (Semiprep HPLC) The refolded rhG-CSF was loaded on the column (Vydac Protein C4 # 214 TP 1010— 250 mm×10 mm) and the fractions containing rhG-CSF were collected. Each fraction was diluted with distilled water (pH 3.5) and was concentrated by Amicon ultrafiltration system. Fractions were eluted at a flow rate of 2 mL/min at 40 °C, with a linear gradient of acetonitrile (ACN) for 140 min using solvent A (22.5 % ACN and 0.1 % trifluoroacetic acid (TFA)) and solvent B (90 % ACN and 0.1 % TFA). Absorbance was measured at 280 nm. Eluted fractions were changed with a buffer (10 mM sodium acetate, pH 4.0, 0.0004 % polysorbate 80, 5 % D-mannitol) by Amicon ultrafiltration system. Analytical Methods The pattern of cell growth was controlled by reading absorbance at a wavelength of 600 nm using a spectrophotometer. A glucose analyzer (YSI 2700 STAT, YSI, Yellow Springs, OH, USA) was used to monitor the residual glucose concentration in the culture medium. rhG-CSF

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was analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight marker was SeeBlue Standard from Novex by Life Technologies. Isoelectric focusing (IEF) was performed with pH 3–10 IEF gel (Invitrogen, Grand Island, NY, USA). Reverse phase (RP)-HPLC was further carried out using reverse phase C4 column (250×4.6 mm, Bio-Rad). Elution was carried out at a flow rate of 0.8 mL/min at 40 °C with a linear gradient of ACN (42–76 %) for 75 min. Absorbance was monitored at 215 nm. The protein concentration was measured by the BCA method [19]. Cell Proliferation Assay of Purified rhG-CSF The bioactivity of rhG-CSF was assayed based on the proliferative activity of murine myeloblastic NFS-60 cells [20]. G-CSF-dependent NFS-60 cells were washed three times with sterile PBS and then resuspended in RPMI 1640 medium supplemented with L-glutamine (200 mM) and sodium pyruvate (1 mM) at a concentration of 5.0×103 cells/mL in a 96-well culture plate (SPL, Pocheon, South Korea). Purified rhG-CSF was added to the growth medium and incubated at 37 °C under 5 % CO2 humidified condition for 48 h. Then, 10 μL of cell proliferation reagent WST-1 (Roche Applied Science, Indianapolis, IN, USA) was added and incubated further for 4 h. The absorbance was determined at 450 nm using an automatic microplate reader (Bio-Tek, Winooski, VT, USA). The absorbance of the blank was subtracted from the absorbance of each sample. Statistical Analysis Student’s t test was used to assess significant differences among treatment groups. The criterion for statistical significance was set at p