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).

摘要: Design of experiment (DoE) is a statistics-based technique for experimental design that could overcome the shortcomings traditional one-factor-at-a-time (OFAT) approach protein purification optimization. In this study, DoE was applied optimizing recombinant single-chain variable fragment (scFv) against type 1 insulin-like growth factor receptor (IGF-1R) expressed in Escherichia coli. first capture step using Capto L, 2-level fractional factorial analysis and successively central composite circumscribed (CCC) were used to identify optimal elution conditions. Two main effects, pH trehalose, identified, high recovery (above 95%) low aggregates ratio (below 10%) achieved at range from 2.9 3.0 with 32-35% (w/v) trehalose added. second cation exchange chromatography, an initial screening media following CCC performed, whereby selectivity scFv obtained on S near 6.0, conditions fulfilling DBC purity identified as 5.9-6.1 loading conductivity 5-12.5 mS/cm. Upon further gel filtration, final purified 98% obtained. Finally, optimized verified by 20-fold scale-up experiment. The purities yields intermediate products all fell within regions predicted approach, suggesting robustness We proposed described here also applicable production other antibody constructs.