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.
