Glycan structures attached to the Fc domain of recombinant monoclonal antibodies (mAbs) affect antibody effector function, clearance, and therapeutic efficacy. Afucosylated Fc glycoforms enhance antibody-dependent cell-mediated cytotoxicity (ADCC) against target cells by increased binding to the FcγRIIIa receptor of immune cells. For many mAbs, ADCC activity is a primary mechanism of action (MOA) to achieve therapeutic efficacy. Hence, it is desirable to modulate the afucosylation level for mAb therapeutics during manufacturing processes. In this study, two small molecule fucostatin inhibitors, 6,6,6-trifluorofucose (fucostatin I) and its phosphonate analog (fucostatin II), were investigated and compared for modulation of mAb afucosylation level in the cell culture processes. Fucostatin I is highly soluble in cell culture media and was demonstrated to increase afucosylation level in a concentration-dependent manner, with minimal impact on cell culture performance. Although fucostatin I is highly potent and applicable to cell culture processes, it was incorporated into Fc glycans at low levels (&lt;1%) in place of endogenous fucose. Alternatively, fucostatin II, did not incorporate into Fc glycans but exhibited slightly lower potency and poor stability. To improve the applicability of fucostatin II, its stability in cell culture media as well as in dimethyl sulfoxide (DMSO) was characterized. It was determined that fucostatin II was stable in DMSO and daily dosing of DMSO-containing fucostatin II also modulated the afucosylation level of multiple mAbs in a concentration-dependent manner. These results suggest that fucostatin II is an ideal lever to modulate afucosylation levels for recombinant mAbs when compared to fucostatin I. Inhibitor chemical structure, modulation mechanism and an application strategy for cell culture processes will be discussed.

Modulation of the afucosylation level of recombinant antibodies with small molecule fucostatin inhibitors in cell culture processes

253rd National Meeting (2017)

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