FcγRIIIa chromatography to enrich a-fucosylated glycoforms and assess the potency of glycoengineered therapeutic antibodies.

Therapeutic antibodies can elicit an immune response through different mechanisms, either cell independent via complement activation (CDC) or through activation of immune-effector cells (such as macrophages and NK cells). After target binding, the Fc part of the antibody will interact with Fc receptors on the surface of effector cells, leading to activation and lysis of the target cells by a mechanism called antibody-dependent cell-mediated cytotoxicity (ADCC). The ADCC of an antibody can be increased by modifying the carbohydrates on the Fc part. If the fucose on the first N-acetylglucosamine is absent, the affinity for the FcγRIIIa is increased and the ADCC enhanced. We describe the development of a chromatography method that is based on the differential affinity of the Fc receptor FcγRIIIa (high affinity V158 variant) for fucosylated and a-fucosylated antibodies. Immobilized FcγRIIIa can be used for the separation of immunoglobulins carrying these glycosylation variants for both, analytical and preparative purposes. The biological activity and fucose content of three pools enriched for fully fucosylated, mono-fucosylated or a-fucosylated carbohydrates could be characterized. Mono-fucosylated and a-fucosylated immunoglobulins have the same enhanced biological activity compared to fully fucosylated IgGs. A direct, label- and modification-free analytical method for screening of IgGs from culture supernatant was developed and was amenable to high-throughput screening. Clones producing antibodies with a high content of a-fucosylated oligosaccharides could be successfully selected.

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