Maduramycin, a novel glycosylation modulator for mammalian fed-batch and steady-state perfusion processes.

Controlling high-mannose (HM) content of therapeutic proteins during process intensification, reformulation for subcutaneous delivery, antibody-drug conjugate or biosimilar manufacturing represents an ongoing challenge. Even though a range of glycosylation levers to increase HM content exist, modulators specially increasing M5 glycans are still scarce. Several compounds of the polyether ionophore family were screened for their ability to selectively increase M5 glycans of mAb products and compared to the well-known α-mannosidase I inhibitor kifunensine known to increase mainly M8-M9 glycans.

Generation of site-distinct N-glycan variants for in vitro bioactivity testing.

Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a Fc-fusion antibody comprizing three different N-glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N-glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms.

Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation.

Glycosylation plays a pivotal role in pharmacokinetics and protein physiochemical characteristics. In particular, effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) can be desired, and it has been described that high-mannose species exhibited enhanced ADCC. In this work we present the trisaccharide raffinose as a novel cell culture medium supplement to promote high mannose N-glycans in fed-batch cultures, which is sought after in the development of biosimilars to match the quality profile of the reference medicinal product (RMP) also.