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Biotechnology
Year: 2013  |  Volume: 12  |  Issue: 2  |  Page No.: 74 - 80

Efficient Chromatographic Processes for Elevated Purification of Antibody Fragment (Fab D1.3) from Crude Escherichia coli Culture

Reza Jalalirad    

Abstract: Purification of antibody fragments (Fabs) from Escherichia coli crude extracts is more difficult than purification of whole molecule of antibody, owing to lack of the Fc constant regions in Fabs. This study compares the capacity and performance of different chromatographic processes for purification of Fab D1.3 from Escherichia coli culture broth samples. Affinity and ion exchange chromatography techniques were utilized as purification techniques. In the first purification experiment, direct loading of the culture broth sample, having high conductivity of ~40 m sec cm-1, on a HiTrap protein G column led to a low Fab D1.3 binding (<1%). Chicken Egg White Lysozyme (HEWL) was coupled with CNBr-sepharose 4 Fast Flow matrix and employed for capturing the Fab from culture broth sample. By doing this chromatography process, 17% of the existing Fab in the sample was captured on the lysozyme column and 48% Fab purity was yielded. Further purification from the lysozyme column’s eluates on HiTrap protein G yielded 97% purity. The overall yield of the target Fab in the sequential lysozyme affinity-protein G affinity chromatography route was 17%. The performance of this sequential method was compared to the sequential CEX-protein G affinity chromatography process. Despite lower purity achieved (14%) in the eluates of the column, capture of the Fab from the culture broth sample was very efficient (>97%) by CEX chromatography, in comparison to the HEWL-sepharose column. The overall yield of the target Fab in the sequential CEX-protein G affinity chromatography route was 83% which was much higher than the figure (18-33%) achieved from the sequential lysozyme affinity-protein G affinity chromatography.

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