Preparation And Separation Of Fab And Fc Fragments From Human Immunoglobulin G

For the preparation of Fab and Fc fragments, human immunoglobulin G (hIgG) can be treated with papain. Fab and Fc fragments of hIgG are immunochemical tools in physiological and clinical applications. In addition, investgetion into the interaction between the fragments of antibodies and the chromatographic ligands can support the optimization of antibody purification and the design of ligands by experiments. In the present work, the preparation of Fab and Fc fragments of hIgG and the chromatography of them by mixed-mode media are studied. Main results were listed as follows.Firstly, the concentration of cystein, papain addition, pH and the incubation time were optimized to improve the digestion efficiency with papain. Digestion of human IgG with papain was performed in0.1M sodium phosphate buffer (pH7.6) containing lOmM cysteine and2mM EDTA and incubated for4h to prepare Fab and Fc fragments. The additional of IgG was10mg/ml and of papain was0.5mg/ml.The conversion of IgG could reach more than98%. Then Protein A affinity chromatography and DEAE anion-exchange chromatography were used to separate the Fab and Fc fragments from the digestion mixture of hIgG. Based on the SEC-HPLC analysis, the purity of Fab fragment was96.6%with the yield of40.1%, while the purity of Fc fragment was95.7%with the yield of72.6%.Then the adsorption of home-made Fab and Fc fragments on mixed-mode chromatography colume such as MEP HyperCel, Capto adhere, and TOYOPEARL MX-Trp-650M was investigated. Capto adhere, a strong anion-exchanger with a phenyl group for hydrophobic interactions and a hydroxyl group for hydrogen bonding, was found of certain selectivity for Fab and Fc fragments. Fc fragment can be capture entirely at pH7to pH8, while Fab fragment little. MEP HyperCel, which is famous for the hydrophobic charge-induction chromatography, and TOYOPEARL MX-Trp-650M, a mixed-mode cation-exchanger, present. less selectivity for Fab and Fc fragments.Considering the digestion conditions and the adsorption difference among these mixed-mode adsorbents, mixed-mode adsorbent Capto adhere was adopted to isolate Fab and Fc fragments from the papain-digested hIgG at pH7.5. Based on the Protein A analysis, Fc fragment was almost completely captured by the media, and the breakthrough fractions of Capto adhere contained only Fab fragment. The high-performance separation of antibody fragments was achieved.Generally, in the present work, the conditions for the digestion of human IgG by papain were optimized, and Fab and Fc fragments were isolated by Protein A affinity chromatography and DEAE anion-exchange chromatography. The adsorption of Fab and Fc fragments on different mixed-mode chromatograhy columes was investigated, and the mixed-mode strong anion-exchanger, Capto adhere, was adopted to purification Fab and Fc fragments from the papain-digested IgG. The results revealed that mixed-mode adsorbents could be used for the purification of Fab and Fc fragments of human IgG instead of Protein A, and the chromatography of Fab and Fc fragments on mixed-mode adsorbents could be effective tools in the optimization of ligands design and studying the mechanism of the interaction between the antibodies and the mixed-mode ligands.