Production of heterodimeric proteins

An in vitro method for production of a heterodimeric antibody comprising the following steps:

• a) incubating a first homodimeric antibody with a second homodimeric antibody under reducing conditions sufficient to allow reduction of the inter-chain disulfide bonds in the hinge region, and
• wherein said first homodimeric antibody comprises an Fc region of an immunoglobulin, said Fc region comprising a first human IgG1 CH3 region, and said second homodimeric antibody comprises an Fc region of an immunoglobulin, said Fc region comprising a second human IgG1 CH3 region, wherein said first homodimeric antibody has a K409R amino acid substitution (numbering according of the EU Index), and said second homodimeric antibody has an amino acid substitution selected from the group consisting of:
• L368A, L368D, L368E, L368G, L368H, L368I, L368N, L368Q, L368R, L368S, L368T, L368V, L368W, D399A, D399F, D399H, D399K, D399R, D399Y, F405A, F405D, F405E, F405H, F4051, F405K, F405L, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, F405Y, Y407G, Y407L, Y407M, and Y407W (numbering according to the EU Index),
• b) subjecting the composition obtained from step a) to oxidizing conditions sufficient to allow oxidation of cysteines in the heterodimeric antibody to inter-chain disulfide bonds, and
• c) obtaining the heterodimeric antibody.