Human IgG4 antibodies are dynamic molecules that in vivo exchange half-molecules to become bispecific antibodies. Here we show that IgG4 antibodies and IgG4 Fc fragments similarly exchange resulting in hybrid antibodies (a single Fab+Fc) with a molecular weight of ca. 100kDa. These antibodies can be separated from IgG4 and IgG4 Fc using size-exclusion chromatography and non-reducing SDS-PAGE. This method does not rely on a cross-linking immunoassay with its potential for false-positive results due to aggregation and it unambiguously demonstrates that the 'Fab-arm' exchange process depends entirely on the Fc part (hinge+CH2+CH3).
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