Purification platform for bispecific antibodies

A method of producing a protein comprising the steps of:

• a. loading a first affinity matrix comprising a Protein A ligand affixed to a substrate with a mixture of multimeric proteins, the mixture comprising:
• i. a first homodimer comprising two copies of a first polypeptide,
• ii. a heterodimer comprising the first polypeptide and a second polypeptide, and
• iii. a second homodimer comprising two copies of the second polypeptide;
• b. washing the second homodimer from the first affinity matrix in a wash buffer;
• c. eluting and collecting the first homodimer and the heterodimer from the first affinity matrix;
• d. loading a second affinity matrix comprising a Protein A ligand affixed to a substrate with a mixture comprising the first homodimer and the heterodimer collected at step (c);
• e. eluting and collecting the heterodimer from the affinity matrix in a buffer having a first pH range and comprising CaCl2 or MgCl2,
• wherein, the first homodimer elutes from the affinity matrix in the buffer at a second pH range, and
• wherein the first affinity matrix, the second affinity matrix, or both comprise a Protein A ligand affixed to the substrate, wherein the substrate is a particle and the first affinity matrix, the second affinity matrix, or both comprise a multiplicity of particles of a mean diameter of 45 μm and comprising pores having a mean diameter of 1100 Å.