Matthew A. Lauber , Stephan M. Koza and Kenneth J. Fountain
Objective <br> demonstrated ACQUITY UPLC®M-Class systems and supporting columns for microliter 2D-RP / peptide Performance RP chromatography and reproducibility.
Background information <br> microliter LC-MS method has been increasingly widely used in the field of proteomics. Recently, this method has been paid attention to and applied to the analysis of residual host cell protein (HCP) in biopharmaceuticals as a complementary technology to Elisa immunoassay. Using a 300 μm narrow-diameter column allows for a wealth of information from a relatively small number of samples. At the same time, peak capacity separation of peptides is required in such work, as higher separation efficiencies make it easier to detect analytes.
Multi-dimensional chromatography can increase the peak capacity of peptide analysis, and the combination of two-dimensional orthogonal chromatographic separation methods can provide better separation ability. Inverting/reverse phase (2D-RP/RP) two-dimensional chromatography has special advantages by using an ethylene bridge hybrid silica particle stationary phase (BEH TechnologyTM) with excellent chemical and mechanical stability. The one-dimensional high-pH reverse phase separation is carried out, and then the peptide mixture is subjected to on-line enrichment to carry out the second-dimensional low-pH reverse phase gradient separation on the sub- 2 μm particle analysis column (for the specific operation, please refer to the previous application data).
Complex peptide analysis with the ACQUITY M-Class system configured with 2D-RP/RP and matched columns for higher peak capacity and better reproducibility.
In this briefing, we further extend the application and performance of this technology by using the ACQUITY UPLC M-Class System and the ACQUITY UPLC M-Class 300 μm ID column. By testing different column sets and lifetimes, it can be seen that this system not only has excellent separation capabilities, but also achieves excellent reproducibility and reliability. On-line two-dimensional chromatographic separation using the ACQUITY UPLC M-Class System and column 2D RP/RP will be an ideal method for studying complex peptide mixtures such as residual host cell proteins in complex samples with high peak capacity and stable and reliable performance.
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