Sialylation of therapeutic monoclonal antibodies (mAbs) and glycoproteins, or total sialic acid (TSA) content, plays a crucial role in many bioprocesses and can have a significant impact to protein stability, as well as many other biophysical and biochemical properties. Enhanced sialylation has even been shown to drastically impact the pharmacokinetics (PK) of mAbs and Fc-fusion proteins, making it an essential quality attribute to monitor and control throughout the downstream purification process. While separation by degree of sialylation through both anion exchange (AEX) chromatography and custom affinity columns (utilizing immobilized serotonin) has been previously reported, the selectivity has yet to be extensively studied on Protein A chromatographic resins. For an Fc-fusion protein (FC1), a pH-dependent separation of sialylated species was observed on various Protein A resins when a two-step elution system was applied. This data suggests that higher specificity could exist for sialylated FC1 species and that understanding this phenomenon is crucial for maintaining process performance and product quality. A decrease in the dynamic binding capacity (DBC) was also observed for higher TSA containing load material, indicating that sialylation of FC1 could potentially impact its binding behavior and influence the overall yield of the Protein A capture step. To gain mechanistic understanding of this phenomenon, column loading and de-sialylation of FC1 were investigated to determine any impacts to binding and elution behavior. In summary, this study gives insight into the mechanism of separation of sialylated FC1 species during Protein A column chromatography and a deeper understanding of how sialylation affects downstream process performance.

Investigation of pH-dependent protein-A chromatography separation of sialylated species for an Fc-fusion protein

ACS Spring 2022

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