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

•NANOBODYⓇ molecules with pI ranging from 5.0 to 10.0 were evaluated.•Experimental evaluation of CEX and AEX separation mechanisms was performed using real-time pH monitoring tool.•NANOBODYⓇ molecule elution characterized by charge plateau where net charge remains constant over ∼ pH 5.5 to pH 8.0.•Molecule specific charge-threshold identified to further define NANOBODYⓇ retention.•Theoretical approach based on protein charge plots implemented to accelerate the targeted development of IEX methods for NANOBODYⓇ molecules. 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. [Display omitted]