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Comprehensive two-dimensional liquid chromatography for the characterization of protein biopharmaceuticals at the protein level

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

Protein biopharmaceuticals are getting increasingly popular in the treatment of various diseases. Biosimilar proteins including monoclonal antibodies (mAbs) such as Herceptin are currently being used in clinical practice for the treatment of patients with cancer and immune-mediated disorders. Globally, protein therapeutics represent about 20 % of the total pharmaceutical market, and experts forecast that over 50 % of new drug approvals in the next decade will be for biologics, especially monoclonal antibodies. MAbs and related therapeutics currently have global sales of over US$ 90 billion. A number of protein biopharmaceuticals, including trastuzumab, rituximab, cetuximab, bevacizumab and adalimumab, will lose patent protection in the next few years, which will allow biosimilar mAbs to enter clinical use. They will require very accurate structural characterization and purity analysis during both the development of the new drugs, and later during manufacturing for quality control purposes.
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.
A generally accepted approach to LC×LC requires the use of very dissimilar conditions in the two columns (separation dimensions) used for the analysis of the sample components. While maximizing the differences allows effective separation of many molecules, it creates numerous practical problems. For example, the solvents used in the two dimensions might not be miscible with each other, which requires very complex and sophisticated approaches to make LC×LC practical. In addition, when the conditions in the two dimensions are very different, it is possible that compounds that interact both weakly and strongly with the stationary phase in the second column may be present in the same fraction. This requires that the composition of the solvent in the second dimension be changed very quickly during the analysis to make sure that all components leave the column before the next fraction is injected, which requires special instrumentation. The main idea of the project was the development of simple LC×LC methods for the separation of proteins using only slightly differing conditions in both dimensions. In this scenario, analytes pre-separated in the first dimension according to one property can be effectively separated in the second dimension using only slightly different conditions. In particular, the need for rapid changes in the solvent composition in the second dimension can be eliminated. This, in turn, means that the analysis can be successfully carried out using much simpler instrumentation, making it more accessible. In the initial stages of the project, different column and solvent combinations were tested in the separation of monoclonal antibody fragments using standard one-dimensional HPLC. After optimizing the conditions, the best column and solvent combinations were tested in LC×LC mode with simple ultraviolet (UV) detection. It has been demonstrated that very efficient separations can be obtained in this way without the need for very complex instrumentation. Further optimization was focused on improving the sensitivity of the method. This was accomplished through the combination of a more sensitive detector, optimization of the column geometry in the second dimension, and optimization of the solvent flow in both dimensions. Finally, the method was optimized for the coupling with high-resolution mass spectrometry. This required changes to the composition of the solvent used in the second dimension to make it compatible with sensitive MS detection. The optimized method was tested using a monoclonal antibody Trastuzumab (trade name Herceptin) in its native and oxidized forms, as well as two antibody-drug conjugates: Brentuximab vedotin (trade name Adcetris) and Trastuzumab emtansine (trade name Kadcyla).
The use of similar separation mechanisms in the two dimension of LC×LC has been discussed in the literature for some time, but the approach has not gained the traction it deserves. Recently we have demonstrated that methods based on this principle can be very powerful in the analysis of small molecule drugs. Thus far, the only LC×LC methods for protein separations utilizing similar separation mechanisms in LC×LC described in the literature required changes in acidity in the two dimensions of separation, which complicated the analysis. It has been demonstrated through this research that the same goal can be accomplished using very simple conditions and instrumentation, which will make this method accessible to a large number of laboratories that might not be equipped with the latest generation, cutting edge LC×LC equipment. Accurate and sensitive characterization of the biopharmaceutical proteins, and especially the biosimilar (generic) monoclonal antibodies expected to enter the market in the near future, is of utmost importance for the safety of the patients and confidence the society has in this new type of pharmaceuticals.
LC×LC separation of trastuzumab emtansine (Kadcyla).