Separation of charge variants during purification of monoclonal antibodies is important for obtaining the desired distribution of charge variants in the final drug product. This is of particular significance to manufacturers of biosimilars as they are mandated by regulatory bodies to match charge variant distribution with the innovator product. This typically requires separation of charge variants on cation exchange chromatography (CEX) using a pH or conductivity based linear gradient elution. Although an effective approach, this is challenging particularly during continuous processing as creation of linear gradient during continuous processing adds to process complexity and can lead to deviations in product quality on slight change in gradient formation. Moreover, the long length of elution gradient along with the required peak fractionation makes process integration difficult. This study, therefore proposes an alternative method for separation of charge variants during continuous CEX chromatography. The proposed method separates the mAb acidic and basic variants from the main species by utilizing a combination of displacement mode chromatography and pH step elution. In the method, separation of acidic variants is achieved using sample displacement by overloading the columns in periodic counter current mode, whereas basic variants are separated using a pH step elution. The established CEX method was tested with varying load amounts and an empirical model was developed to predict the acidic species concentration in the elute as a function of protein load amount. Further, in order to showcase the amenability of the designed CEX method in continuous operation, the method was utilized in our in-house continuous mAb platform. During the continuous operation acidic variants (3.5 % ± 0.3 %), main species (67.5 % ± 0.5 %), and basic variants (29 % ± 0.4 %) were found to be controlled within the acceptable range.

Novel method for continuous chromatographic separation of monoclonal antibody charge variants by combining displacement mode chromatography and step elution

ACS Spring 2022

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