Use of Ultrashort Columns for Therapeutic Protein Separations. Part 1: Theoretical Considerations and Proof of Concept.

Due to the particular elution mechanism observed with large solutes (e.g., proteins) in liquid chromatography, column length has less impact in controlling their retention compared to small solutes. Moreover, long columns-in theory-just broaden the peaks of large solutes since a great part of the column only acts as void (extra) volume. Such a theory suggests that using very short columns should result in comparable separation quality versus using long columns and make it possible to perform faster (high-throughput) analyses. Therefore, the elution behavior of various therapeutic monoclonal antibodies and their fragments (25-150 kDa) has been investigated using modern instrumentation and column formats. The possibilities offered by narrow-bore columns packed with state-of-the-art 2.7 μm superficially porous particles with 5, 50, 100, and 150 mm lengths have been compared. In particular, the impact of gradient steepness and column length on separation efficiency was evaluated. Using 5 mm × 2.1 mm columns, it has become possible to separate antibody fragments and antibody-drug conjugate species in less than 30 s. Such fast methods can be very useful for high-throughput screening purposes in biopharmaceutical industries.