title: Purification optimization for a recombinant single-chain variable fragment against type 1 insulin-like growth factor receptor (IGF-1R) by using design of experiment (DoE).
abstract: Design of experiment (DoE) is a statistics-based technique for experimental design that could overcome the shortcomings of traditional one-factor-at-a-time (OFAT) approach for protein purification optimization. In this study, a DoE approach was applied for optimizing purification of a recombinant single-chain variable fragment (scFv) against type 1 insulin-like growth factor receptor (IGF-1R) expressed in Escherichia coli. In first capture step using Capto L, a 2-level fractional factorial analysis and successively a central composite circumscribed (CCC) design were used to identify the optimal elution conditions. Two main effects, pH and trehalose, were identified, and high recovery (above 95%) and low aggregates ratio (below 10%) were achieved at the pH range from 2.9 to 3.0 with 32-35% (w/v) trehalose added. In the second step using cation exchange chromatography, an initial screening of media and elution pH and a following CCC design were performed, whereby the optimal selectivity of the scFv was obtained on Capto S at pH near 6.0, and the optimal conditions for fulfilling high DBC and purity were identified as pH range of 5.9-6.1 and loading conductivity range of 5-12.5 mS/cm. Upon a further gel filtration, the final purified scFv with a purity of 98% was obtained. Finally, the optimized conditions were verified by a 20-fold scale-up experiment. The purities and yields of intermediate and final products all fell within the regions predicted by DoE approach, suggesting the robustness of the optimized conditions. We proposed that the DoE approach described here is also applicable in production of other recombinant antibody constructs.
authors: The Affiliated Hospital of Hebei University of Engineering, Handan, China; Medical College, Hebei University of Engineering, Handan, China.
authors: Yong-Hong
authors: YH
authors: Song
authors: Medical College, Hebei University of Engineering, Handan, China.
authors: Xue-Wen
authors: XW
authors: Sun
authors: Cixian People's Hospital, Handan, China.
authors: Bo
authors: B
authors: Jiang
authors: Handan Central Hospital, Handan, China.
authors: Ji-En
authors: JE
authors: Liu
authors: The Affiliated Hospital of Hebei University of Engineering, Handan, China; Medical College, Hebei University of Engineering, Handan, China. Electronic address: Xianhui_Su@yeah.net.
authors: Xian-Hui
authors: XH
authors: Su
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D017526:Receptor, IGF Type 1 / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012107:Research Design; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D012995:Solubility
chemical_list: D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D017526:Receptor, IGF Type 1
keywords: Central composite circumscribed design; Design of experiment; DoE; Fractional factorial analysis; IGF-IR; ScFv