Capillary electrophoresis coupled to fluorescence spectroscopy for protein characterisation

Abstract

Proteins are essential molecules in all living organisms. Their involvement in numerous biological processes has led to the development of protein-based medicines (biopharmaceuticals). For good understanding of the properties and function of endogenous proteins and biopharmaceuticals, extensive protein characterisation is required. This involves assessment of features such as purity, heterogeneity, stability, activity and conformation of the protein. For instance, an altered protein conformation may influence the protein function and activity, which could have serious health implications. Clearly, there is a demand for suitable analytical methods that allow assessment of protein identity, purity and conformation. Capillary electrophoresis (CE) coupled to fluorescence spectroscopy provides high separation efficiency and selective spectroscopic detection that can be very useful for protein characterisation. Protein modifications, like glycosylation and deamidation, may involve changes in the net charge of a protein and, thus, of its electrophoretic mobility. Moreover, protein conformational changes, like unfolding or aggregation, are often accompanied by a change in protein molecular radius, which is reflected in the electrophoretic mobility as well. Fluorescence spectroscopy can provide information on the conformational state of a protein by monitoring changes in local tryptophan environments. This thesis describes the development and evaluation of novel fluorescence (Flu) and wavelength-resolved fluorescence (wrFlu) detection systems in CE for analysis of intact proteins. In order to accomplish effective Flu detection, a detector set-up was selected that incorporated a lamp as excitation source, a dedicated fluorescence detection cell and a photomultiplier detector. The analytical characteristics of the selected CE-Flu set-up and its suitability for native protein detection were tested using tryptophan and some model proteins. Protein detection limits were 7-33 nM, which was a factor of 25 better than UV absorbance detection at 280 nm, and comparable to UV detection at low-UV wavelengths. In order to obtain conformational information on separated proteins, employment of wavelength-resolved fluorescence detection was required. Therefore, the photomultiplier detector was replaced by a spectrograph equipped with a sensitive charge-coupled device (CCD) as detector. CE-wrFlu of intact proteins allowed acquisition of protein emission spectra ‘on-the-fly’. Detection limits were 6-32 nM, which is comparable to Flu detection. Analysis of model proteins in native (folded) and denatured (unfolded) state showed that protein conformational changes can be monitored via two independent parameters, the position of maximum emission wavelength and the effective electrophoretic mobility. The usefulness of CE-wrFlu for the study of protein unfolding pathways was investigated by measuring non-reduced and reduced β-lactoglobulin B (β-LGB) in several stages of unfolding. Non-reduced β-LGB showed two distinct unfolding pathways, that showed fast and slow interconversion kinetics between folded and unfolded species, respectively. Reduced β-LGB showed only one unfolding pathway, and unfolded at much lower denaturant concentration. These results suggest that disulphide bonds might be a prerequisite for the observed unfolding behaviour of non-reduced β-LGB. The potential applicability of native CE-Flu for the profiling of biopharmaceuticals was evaluated by the analysis of different products of human recombinant erythropoietin. Distinct and repeatable peak profiles were obtained, which allowed discrimination of different products based on the obtained glycoform patterns.