Kinetics of Whole Serum and Prepurified IgG Digestion by Pepsin for F(ab')_2 Manufacture

摘要

An alternative route for the production of polyclonal F(ab')_2 fragments that might be adopted for the facile preparation of antivenoms is assessed in this work. The method involves the digestion of whole serum by free pepsin, which results in reduction of the numberof processing steps commonly in use, because it avoids the initial purification of IgG's prior to their proteolytic cleavage by the enzyme. Digestion kinetics of whole serum and caprylic acid prepurified IgG using free pepsin were monitored with SDS-PAGE followed by densitometric analysis and antigen binding activity assay of the digested samples. It was observed that with equal units of pepsin activity, caprylic acid prepurified IgG was digested more rapidly than whole serum but that the overall retention of antigen binding activity was significantly greater in the latter case. The estimated first-order digestion rate parameters were 11.8 and 4.42 muM min~(-1) digestion was 33 muM and that for pure IgG digestion was 43.5 muM. Calibration with using SDS-PAGE followed by image analysis. A linear relationship was observed between the protein concentration and the respective band intensity within the range of concentrations investigated (0.63-31.2 muM IgG concentration). This technique proved to be relatively rapid, rerpoducible and more precise than size-exclusion chromatography as a result of its F(ab')_2/IgG resolving power. Staining and destaining protocols were reproduced in terms of staining and destaining times, volumes added, and compositions. Furthermore, all digestion experiments were performed in duplicate sets to mintor the extent of variation of the digestion kinetic parameters measured by this method. The results obtained from this technique confirm and quantify previous observations that pepsin digestion of whole serum is slower and easier to control than digestion of pure IgG and results in higher recovery of antigenic binding activity.