Bivalent bispecific antibodies (BiS) are produced by recombinantly expressing a whole monoclonal antibody (mAb) genetically fused to two single chain variable fragments (scFv) using flexible polypeptide linkers. Three purified homologous BiS samples with identical peptide sequences and varying only by scFv attachment location studied in this work show reversible, three-peak elution profiles on ProPac WCX-10 and Source 15S cation exchange columns. Collection, dilution, and reinjection of any of the three peaks results in the same three-peak elution profile. This behavior is seen for all pH values tested in the range 5-8.5. In-line Dynamic Light Scattering showed that all peaks have a monomer-sized hydrodynamic radius of 6.2 nm. Unlike normal chromatographic behavior, decreasing flow rate decreased resolution between peaks. At the lowest flow rate tested, only a single symmetric peak was observed. This peak eluted between the first and second peak of the higher flow rate elution profiles. A hold step at strong binding conditions followed by gradient elution with high flow rate enriched the third, most retained, peak. Biophysical measurements including in-line fluorescence, in-line DLS, chemical cross-linking, hydrogen deuterium exchange mass spectrometry, enzyme digestion to determine domain contributions, and a mechanistic column model reveals that the three-peak elution is caused by conformational interconversion occurring on time sales comparable with chromatographic separation. From the peptide sequence, the scFvs carry a net positive charge. When the scFv is folded onto the base mAb weaker binding to the resin occurs whereas if the scFv is outstretched a more favorable interaction and stronger binding occurs on the resin surface. The three peaks from weakest to strongest retention are: both scFvs interacting with base mAb, one scFv interacting with base mAb and one outstretched, and both scFvs outstretched. Equilibrium favors the tight conformation in solution and outstretched while adsorbed. The highly flexible linkers allow slow changes in tertiary structure responsible for reversible, three-peak elution behavior.

Chromatographic behavior of bivalent bispecific antibodies on cation exchange columns

2019 Spring National Meeting

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