Abstract
Straight‐through chromatography, wherein the eluate from one column passes directly onto another column without adjustment, is one strategy to integrate and intensify manufacturing processes for biologics. Development and optimization of such straight‐through chromatographic processes is a challenge, however. Conventional high‐throughput screening methods optimize each chromatographic step independently, with limited consideration for the connectivity of steps. Here, we demonstrate a method for the development and optimization of fully integrated, multi‐column processes for straight‐through purification. Selection of resins was performed using an in silico tool for the prediction of processes for straight‐through purification based on a one‐time characterization of host‐cell proteins combined with the chromatographic behavior of the product. A two‐step optimization was then conducted to determine the buffer conditions that maximized yield while minimizing process‐ and product‐related impurities. This optimization of buffer conditions included a series of range‐finding experiments on each individual column, similar to conventional screening, followed by the development of a statistical model for the fully integrated, multi‐column process using design of experiments. We used this methodology to develop and optimize integrated purification processes for a single‐domain antibody and a cytokine, obtaining yields of 88% and 86%, respectively, with process‐ and product‐related variants reduced to phase‐appropriate levels for nonclinical material.
Highlights
Biopharmaceutical companies have increasingly diverse pipelines of recombinant protein‐based medicines including single‐domain antibodies, bi‐specific constructs, and enzyme replacements (Morrison, 2020)
We developed integrated processes for the purification of two different classes of proteins, a single‐domain antibody and a cytokine, with overall purification yields of 88% and 86% through two and three stages of chromatography, respectively, and process‐related impurities reduced below regulatory guidelines for nonclinical development
We have presented a methodology for optimizing the buffer conditions used in integrated, straight‐through chromatographic processes, including conditions for the bridging buffer, utilizing high‐throughput screening technologies and design of experiments
Summary
Biopharmaceutical companies have increasingly diverse pipelines of recombinant protein‐based medicines including single‐domain antibodies, bi‐specific constructs, and enzyme replacements (Morrison, 2020). High‐throughput screening has become a common method to develop and optimize processes for the purification of new products These methods enable screening large numbers of resins and conditions in parallel using miniature columns or 96‐well plates (Łacki, 2012). The data from such screens often inform the optimal resin and operating conditions for an individual step in a multi‐stage sequence, but offer little consideration on the requirements for the connectivity of this step with other steps in the final process. Developing new techniques that consider the complete connectivity of steps in the final process could allow for more intensified processes by reducing unnecessary intermediate steps
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