Successful Analysis of Protein Biopharmaceuticals Using Ultra-Short Columns

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Davy Guillarme from the University of Geneva, Switzerland, presented a talk called “Why Not Using Ultra-Short Columns for the Chromatographic Analysis of Protein Biopharmaceuticals?” in a keynote lecture at the 51st International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2023) in Düsseldorf this week (1).

Liquid chromatography (LC) and mass spectrometry (MS) are the most commonly used analytical approaches for monoclonal antibody (mAb) characterization. Guillarme proposed a novel approach that leverages state-of-the-art, ultra-short reversed-phase, hydrophilic interaction liquid chromatography (HILIC-), and ionic exchange (IEC-) column formats, ranging from 5 to 20 mm in length, to further improve the capabilities of chromatographic techniques for protein analysis.

Short columns have demonstrated the ability to achieve separation efficiency comparable to standard-sized columns but with significantly reduced analysis times. This is attributed to the high S values (slope of the log k relation relative to the percentage of acetonitrile) exhibited by large proteins and the use of an on-off retention mechanism known as bindelute. Notably, this approach has successfully analyzed mAbs, bispecific antibodies (bsAbs), and immunoconjugates (ADCs) within a few tens of seconds with various chromatographic modes.

However, it is evident that the current ultrahigh-performance liquid chromatography (UHPLC) instrumentation is not yet optimized to fully exploit the benefits of these ultra-short columns, particularly when coupled with MS detection, according to Guillarme. Further advances are needed to enhance the performance of UHPLC instruments, enabling the realization of the full potential of ultra-short columns in mAb analysis.


Guillarme concluded the research highlights the potential of using ultra-short columns in chromatographic techniques for the analysis of therapeutic proteins. By leveraging these advanced column formats, significant improvements in analysis time and separation efficiency can be achieved. Guillarme added that optimization of UHPLC instrumentation is essential to fully exploit these benefits, especially in relation to MS detection.


(1) Guillarme, D. Why Not Using Ultra-Short Columns for the Chromatographic Analysis of Protein Biopharmaceuticals? Presented at: HPLC 2023. June 18–22, 2023. Düsseldorf, Germany. KN17.