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Fast Analysis of Biomolecules by Using Smaller and Innovative Particles
Daniel Eßer, Ann Marie Rojahn, Mathias Hehn, YMC Europe GmbH, Dinslaken, Germany Abstract
Modern stationary phases for BioLC have to offer high resolutions, fast methods, high robustness and high lot-to-lot reproducibility for reliable results. Furthermore, the separation modes typically used in BioLC present different challenges. These requirements can be fulfilled by state-of- the-art particles of optimised sizes and types. Selected solutions for different biological substances are described in this article.
Introduction
Requirements in BioLC differ from those encountered in small molecule analyses. For example, analytes usually have a higher molecular weight (MW) and a greater hydrodynamic volume. Due to their increased size, these substances such as proteins or antibodies also are extremely hydrophobic. Furthermore, closely related compounds have to be separated from each other including monomers from aggregates or fragments, native compounds from post translational modified ones, or target compounds from side products such as those with additional/missing amino acids or nucleotides.
The demand for BioLC products is increasing thanks to ever-expanding research activities with all types of biomolecules as well as the plethora of biological compounds entering the commercial market which must be analysed in quality control labs (QC) [1]. Consequently, faster analytical solutions to enhance the sample throughput are desired. As encountered in small molecule QC labs, reliable, robust and reproducible (ultra) high performance liquid chromatography ((U)HPLC) products are required to ensure high-quality standards for these products entering the commercial space.
Over the past several years the UHPLC column market for BioLC has grown according to the needs of users in both the R&D and QC environments [2]. Solutions for the different types of biomolecules are now available: peptides and proteins, monoclonal antibodies (MAbs) and
Figure 1: Symmetrical peaks for challenging MAbs using UHPLC column YMC-Triart Bio C4 (1.9 µm, 300 Å, 50 x 2.1 mm ID) at an elevated temperature of 90 °C. Eluent: A) 0.1 % TFA in water, B) 0.1 % TFA in acetonitrile; gradient: 25–50 % B (0-4 min); flow rate: 0.4 mL/min; detection: fluorescence ex 280 nm; em 350 nm; injection: 0.5 µL. Courtesy of University of Geneva, School of Pharmaceutical Sciences, Department of Analytical Pharmaceutical Chemistry.
antibody-drug-conjugates (ADCs) as well as oligonucleotides and nucleic acids. To analyse these compounds, products for the various separation modes are available: reversed phase (RP), size exclusion (SEC), ion exchange (IEX) and hydrophobic interaction (HIC). Small particles with different base materials ensure quick runs and different detection options, such as UV, MS or MALLS (multi-angle laser light scattering).
UHPLC columns for RP based on hybrid particles
In RP, sub-2 µm particles for UHPLC applications have been a standard for
several years. As biomolecules have different requirements than small molecules, larger pore sizes and higher temperature tolerances are crucial. This permits the analysis of large proteins, antibodies, and strongly hydrophobic compounds, which require the lower hydrophobicity provided by wide pore phases with short chain modifications. In addition, particle robustness and mechanical stability are mandatory, which is very challenging in UHPLC due to the high pressures encountered.
In general, employing higher temperatures for analysis often has a beneficial effect on the chromatographic results; such as
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