High molecular weight aggregates are considered a product-related impurity due to their potential immunogenicity in patients. Aggregates are typically removed from a monoclonal antibody product stream via various modes of chromatography, including cation exchange (CEX) and hydrophobic interaction chromatography (HIC). However, formation of aggregates during downstream processing due to various stresses (shear, interfacial stress, pH, etc.) can also be regulated and/or prevented in order to minimize the total levels in bulk drug substance. Using both differential scanning fluorimetry and a measurement of aggregation kinetics, it was determined that the Affinity Chromatography step was contributing towards the denaturation of the mAb and causing a subsequent aggregation event during the low pH virus inactivation step. Implementation of a milder elution pH buffer resulted in less formation of aggregate across these unit operations. This work demonstrates how the optimization of the existing two column process can be achieved to reduce aggregation while negating the need for a third chromatography step and the necessary capital expenditure in the New Product Introduction (NPI) facility.

Investigation and prevention of on-column denaturation and aggregation during an affinity chromatography step

ACS Fall 2021

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