Theoretical charge plots as a tool for targeted and accelerated ion exchange chromatography method development of...

NANOBODY^Ⓡ molecules are an innovative class of biotherapeutics based on heavy chain only VHH immunoglobulins. Much like canonical antibodies, they are prone to the formation of charge variants and other post-translational modifications, which can potentially impact their critical quality attributes. Therefore, establishing high-resolution product-specific methods, such as IEX chromatography, is essential for evaluating the purity of these molecules. However, due to the lower surface charge of NANOBODY^Ⓡ molecules, their charge-based elution behavior can differ considerably from that of classical antibodies, resulting in a more extensive method development set-up for these smaller molecules. Using an initial pH screening gradient based on theoretical protein charge plots, we investigated the IEX retention behavior of eight NANOBODY^Ⓡ molecules with a wide range of pI values (pI 5.0 to 10.0). Our findings reveal that the charge-based chromatographic behavior of NANOBODY^Ⓡ molecules cannot be solely attributed to the isoelectric point (pI) of the protein. Rather, a molecule-specific charge threshold was identified as a critical parameter for NANOBODY^Ⓡ molecule retention. Furthermore, the protein charge plot also showed that NANOBODY^Ⓡ molecule elution can be characterized by a charge plateau where the net charge of the protein remains constant over a certain pH range (∼ pH 5.5 to pH 8.0), further challenging the paradigm that elution pH and pI are fixed values. The application of this theoretical approach using protein charge plots to define NANOBODY^Ⓡ molecule charge threshold and charge plateau parameters, can reduce overall IEX method development turnaround time by at least 2-fold.