Raffinose acts as a competitive inhibitor for a specific transferase in the Golgi, which slows, rather than blocks, the glycan branching process. This results in the enrichment of Manos-5 species, a different outcome than the Manos-8/9 glycans produced by a complete block with inhibitors like kifunensine.
Using raffinose to adjust glycosylation is a regulatory-friendly strategy. Since it is a simple media component adjustment, not an enzyme inhibitor or genetic modification, it aligns with standard process development activities. This avoids intense scrutiny and justification required for more complex methods, simplifying the CMC package.
Initial experiments failed because adding raffinose also increased osmotic stress, killing cells. The breakthrough was maintaining constant osmolality by adjusting NaCl as raffinose was added. This isolated the specific effect of raffinose on glycan profiles, revealing a clear dose-response relationship without harming cell viability.