Oligonucleotide Purification by Ion Exchange Chromatography: A Step-by-Step Guide to Process Understanding, Modeling, and Simulation

Oligonucleotides have emerged as a promising class of pharmaceuticals, leading to significantly increased demand. Oligonucleotides are typically produced by solid-phase synthesis and then purified by ion exchange or reverse-phase chromatography. Predictive simulation is a valuable tool to help reduce process development times, secure scale-up, and decrease waste generation. In this paper, we disclose for the first time a cutting-edge mechanistic model describing oligonucleotide purification by ion exchange chromatography. The novel aspect of the model and focus of this paper is the thermodynamic description of large, highly charged molecules, which includes both solution chemistry and the ion exchange mechanism with the chromatographic medium. The different retention of such molecules depending on their sequence length, charge state, and interaction strength with the resin is accurately predicted. Thanks to a meaningful description of the underlying physical and chemical phenomena, the model also has highly predictive capabilities outside of the experimentally studied parameter ranges. It can be used to predict the outcome of changes to the operating conditions and experimental protocol, like the pH or ionic strength of buffer solutions, the number of washing steps, the loaded sample quantity, and more. The model can also account for a change of configuration from a single column to a multicolumn system. The step-by-step methodology to implement this model is presented and illustrated with examples from three leading pharmaceutical companies in the field. This methodology has been shown to lead to a significant process understanding with minimal experimental effort.