Multiproduct High-Resolution Monoclonal Antibody Charge Variant Separations by pH Gradient Ion-Exchange Chromatography

In the biotechnology industry, ion-exchange chromatography is widely used for profiling the charge heterogeneity of proteins, including monoclonal antibodies. Ionic strength based ion exchange separations, while having excellent resolving power and robustness, are product specific and time-consuming to develop. In the present work, a pH gradient based separation using a cation exchange column is described and shown to be a multiproduct charge sensitive separation method for monoclonal antibodies. Simple mixtures of defined buffer components were used to generate the pH-gradients that separate closely related antibody species. The form of the pH gradient was controlled and optimized by the pump as well as the buffer composition if necessary. During this work, the buffer compositions for the separation were optimized in parallel for several MAbs. The data shows that the multiproduct method is optimal for all of the MAbs studied. Operational aspects of the separation such as column chemistry, column length, and sample matrix indicate a very robust method. The pH gradient ion-exchange method is demonstrated to have significant resolving power and peak capacities far in excess of what we would expect for ionic strength elution ion-exchange. Data obtained demonstrates that the separation is relatively insensitive to column length. Direct analysis (no buffer exchange) of samples in matrixes consistent with in-process manufacturing pools is demonstrated. Such a capability is extremely useful for the high throughput evaluation of in-process and final product samples.