Periodic Reporting for period 1 - LCxLCProt (Comprehensive two-dimensional liquid chromatography for the characterization of protein biopharmaceuticals at the protein level)
Reporting period: 2020-01-01 to 2020-12-31
Biopharmaceutical protein molecules are very large and heterogeneous, which makes their characterization very difficult. Molecules of this type are typically characterized using a combination of two techniques: high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS). HPLC allows molecules in a mixture to be separated from each other, whereas HRMS then provides accurate masses for the separated components. In HPLC, a sample is introduced into a so-called chromatographic column, which is a tube filled with very small particles (a stationary phase). The components of the sample are then eluted from the column using solvents of varying composition. Different molecules interact with the stationary phase differently, and as a result elute from the column at different times. While HPLC is a powerful separation technique, the number of components that can be separated from each other is limited when using the standard one-dimensional implementation of the technique. This makes accurate characterization of samples as complex as biopharmaceutical protein formulations very difficult, if not impossible.
The overall objective of the project was the development of new, innovative approaches to the characterization of protein biopharmaceuticals, based on comprehensive two-dimensional liquid chromatography (LC×LC). In this technique, the liquid eluting from the chromatographic column is divided in an on-line fashion into small fractions, which are then injected in quick succession into a second column with different properties for additional separation. The technique allows many more components to be separated in a single run, and makes it possible to detect traces of potentially dangerous compounds that could be covered by main components in standard one-dimensional HPLC, and thus undetectable.