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A modified crystallohydrodynamic approach introduced in 2001 is applied to two human IgG4 constructs from mouse IgG1. The constructs were point mutants of the chimeric antibody molecule cB72.3(gamma4): cB72.3(gamma4A), devoid of inter-chain disulfide bridging, and cB72.3(gamma4P), which has full inter-chain bridging. As before, the known crystallographic structures for the Fab and Fc domains were combined with the measured translational frictional ratios to obtain an estimate for the apparent time-averaged hydration of the domains and hence for that of the intact molecule. The original approach was modified with the hydrated dimensions of the domains being applied, rather than the anhydrous crystallographic dimensions, for assessing the inter-domain orientations using the algorithms HYDROSUB and SOLPRO. Both chimeric IgG4 molecules were found to have open, rather than compact, structures, in agreement with the previous study on wild-type human IgG4. The insertion of a frictionless connector between the domains was necessary, however, for representing the cB72.3(gamma4A) chimera. It therefore appears that the inter-chain disulfide bonds act as physical constraints in the cB72.3(gamma4P) chimera, forcing the antibody domains together and producing a less elongated structure than that of cB72.3(gamma4A). The open structures produced for the two IgG4 chimeras showed similarity to those structures identified for murine IgG1 and IgG2a molecules through X-ray crystallography.

Abstract.A potentially fruitful but hitherto relatively unexplored application of the analytical ultracentrifuge is the investigation of interactions in pharmaceutical polymer drug carrier systems. In this study we investigate the interactions of the cationic material chitosan with gastric mucin: 1) The physicochemical properties of gastric mucin and chitosan (highly deacetylated chitosan "Sea Cure + 210") in solution are summarised; 2) We described how the gastric mucin (from pig) can be purified by a combination of preparative isopycnic density gradient ultracentrifugation and gel chromatography, and how its purity and structural integrity can be checked by analytical isopycnic density gradient ultracentrifugation (on a MSE Centriscan 75 Analytical Ultracentrifuge) and on line GPC/MALLS (gel permeation chromatography coupled on-line to a multi-angle laser light scattering photometer); 3) Using co-sedimentation experiments with the appropriate controls in an XL-A Ultracentrifnge (absorption optics) and an MSE Mk II Analytical Ultracentrifuge (Schlieren optics) a definite interaction between chitosan and pig gastric mucin was demonstrated, with complexes sedimenting faster than 1000 S, depending on the amount of the mucin used.