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Techniques & Tools Pharma & Biopharma, Technology

The Biopharma Multi-tool

sponsored by Tosoh

Quick and straightforward, size-exclusion chromatography (SEC) has become a Swiss Army knife for the analysis of protein aggregates. It is a “mild” technique that usually preserves biological activity and structural integrity – and could almost be considered a platform technology. Coupling of SEC with various advanced detectors, such as a mass spectrometry, light scattering or surface plasmon resonance, makes it a versatile tool for numerous applications beyond aggregates. With new biopharmaceutical formats in the pipeline – bispecific monoclonal antibodies, antibody–drug conjugates and virus-like particles, for example – rapid and thorough characterization will be even more important.

Timelines in analytical laboratories are increasingly tough – on the other hand, there is a strong drive to explore and understand biologics in more detail. In separation science, this means higher resolution and plate counts in shorter analysis times.

Small is beautiful

Over the past 30 years, miniaturization has been the key trend both in stationary phases and instrumentation. Smaller particles provide better separation performance, so we can either shorten the required separation path lengths, or provide better performance at the same path length. The use of smaller particles also pushed the optimum column dimensions towards smaller values, with the welcome side-effect of saving solvent.

When it comes to biopharmaceuticals, HPLC and UHPLC cannot be as strictly segregated as in other disciplines. In this setting, UHPLC is defined as “ultra-high performance LC” rather than the more common definition – “ultra-high pressure LC”. Especially in SEC, the small (2 μm) particles of UHPLC do increase the pressure – but rarely exceed the pressure limits of modern HPLC. Any higher, and the resulting frictional heating and shear forces would damage the macromolecules and lead to artefacts. Instead, bio-UHPLC is focused on optimization of valves, capillaries and flow cells to decrease dead volumes and subsequent Eddy diffusion – which also holds true for conventional HPLC columns.

Upgrading biopharma’s Swiss Army knife

UHPLC is already a standard technology in the analysis of small-molecule drugs so it is no surprise that biopharmaceutical manufacturers are now looking to follow suit. Our TSKgel 3000SWXL column is a standard tool for QC aggregate analysis by SE-HPLC, and we noticed an increasing demand for a 2 µm-particle column that enables easy method transfer to UHPLC.

With new biopharmaceutical formats in the pipeline, rapid and thorough characterization will be even more important.

There were many challenges in developing such a column. The minimum particle size for HPLC is believed to be 1 µm (1), so at 2 μm we are skirting the edge of what is possible – and that’s never easy going. But chromatographers really appreciate the benefits in terms of speed and resolution.

Of course, UHPLC is not a magic bullet. Coupling a SE-UHPLC column to a non-optimized system with dead volumes is an unhappy marriage. The smaller internal diameters result in lower volumetric flow rates, which leave more room for undesirable Eddy diffusion. In some cases, this may result in worse separation performance than with a conventional SE-HPLC column, so it’s important to consider the system as a whole.

Thorough characterization of every new drug is crucial for patient safety – and complex next-generation biopharmaceuticals present a challenge. I expect to see diversification of columns – columns capable of delivering high performance for fundamental research and columns dedicated to rapid in-process control and drug release. With regard to instrumentation, the use of LC-MS and other hyphenated techniques will continue to grow.

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  1. I Halász et al., “Ultimate limits in high-pressure liquid chromatography” J Chromatogr A, 112, 37–60 (1975). Available at: bit.ly/2p4pVfh. Accessed May 9, 2017.

About the Author

Judith Vajda

Senior Laboratory Specialist, TOSOH BIOSCIENCE GmbH, Germany.

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