SEC Based Method for Size Determination of Immune Complexes of Therapeutic Antibodies in Animal Matrix

Figure 3

(a) Fab488-huDVD-ADA complexes prepared with ADAs purified from rabbit serum. 0.83 μM huDVD, 678 nM Fab488, and 0.75, 1.5, 2, and 3.5 μM ADA were incubated in 20% cynoserum as described in Section 2 and separated by size exclusion chromatography with fluorescence detection. Peak 1 is the free dye (DyLight 488), peak 2 is Fab488, peak 3 is the complex of Fab488 and huDVD, peak 4 is the complex of Fab488 and huDVD and one ADA, peak 5 is the complex of Fab488, one ADA, and two huDVD, and peaks under number 6 represent bigger complexes containing Fab488, huDVD, and ADA. (b) Quantification of fluorescence signal recovery in ADA complexes. Summed peak areas from (a) containing ADA (peaks 4–6) were set in relation to the fluorescence intensity of the whole elution profile and are shown as % complex. (c) Supernatant pellet experiment with ADA complexes. Coomassie stained SDS-PAGE showing the pellet (p) and the supernatant fraction (s) of samples containing 0, 1.5, or 3.5 μM ADA, Fab488, and huDVD prepared as in (a). The samples were treated as described in Section 2. The pellet fraction contained 20% of the reaction volume, whereas the supernatant fraction contained 80% of the reaction volume. Aggregation occurs when more than 20% of total protein amount are in the pellet fraction.

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