Hydrophobic Interaction Capillary Electrochromatography of Protein Mutants. Use of Lipid-Based Liquid Crystalline Nanoparticles as Pseudostationary Phase Christian Nilsson, †,‡ Kristian Becker, † Ian Harwigsson, § Leif Bu ¨ low, † Staffan Birnbaum, ‡ and Staffan Nilsson* ,† Pure and Applied Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden, Biopharmaceuticals Support QC, Manufacturing Development, Novo Nordisk A/S, Brogårdsvej 66, DK-2820 Gentofte, Denmark, and Camurus AB, So ¨lvegatan 41, Ideon Science Park Gamma 1, SE-223 70 Lund, Sweden Nanoparticle-based hydrophobic interaction-capillary elec- trochromatography was utilized for separation of proteins with similar mass-to-charge ratio at neutral pH without organic modifier. Lipid-based liquid crystalline nanopar- ticles were prepared and used as pseudostationary phase, benefiting from their high biocompatibility, ease of prepa- ration, and suspension stability at high concentrations. Use of laser-induced fluorescence enabled detection at high nanoparticle concentrations. Green fluorescent pro- tein (GFP) and mutants of GFP harboring single or double amino acid substitutions with the same charge were separated in the described system but not in conventional capillary electrophoresis. Separation was achieved by increasing the salt concentration to promote hydrophobic interactions by shielding of the repulsive electrostatic interactions. In addition, the method was adapted to a capillary with an effective length of 6.7 cm, enabling fast separations and future applications on chip. The demand for new analytical techniques for the characteriza- tion of isoforms of proteins is rapidly increasing, for example, in the case of biological drugs for which high and consistently well- documented homogeneity is required, and in the increasing use of protein isoforms for diagnostic purposes, for example, cancer 1 and haemostasis studies or human biopsies following onset of Alzheimer’s disease 2 or type 2 diabetes. 3 Protein separation techniques that are highly efficient, sample conserving, fast, and widely applicable using physiologically relevant conditions are of great value. A hydrophobic interaction-capillary electrochromatography (HI-CEC) method using nanoparticles as pseudostationary phase (PSP) for resolution of protein mutants of similar mass-to-charge ratio under native conditions is presented. These proteins are not resolved with conventional capillary electrophoresis (CE). CEC is usually performed using packed, 4,5 open tubular 6,7 or monolithic 11 columns. Alternatively, a PSP consisting of submi- crometer particles, that is, nanoparticles suspended in the elec- trolyte, can be used, in so-called PSP-CEC. 9-12 Nanoparticles possess a favorable surface-to-volume ratio, providing enhanced mass transfer, and their use as PSP minimizes carry-over effects and enables fast column regeneration. In addition, nanoparticles are difficult to pack in traditional column CEC and liquid chromatography (LC). 13 PSP-CEC is preferably performed using a continuous full filling approach 14,15 to maximize the amount of nanoparticles, that is, the surface area. The use of UV detection is restricted because of the light scattering properties of the nanoparticles. To circumvent this drawback, laser-induced fluo- rescence (LIF) detection, 16 mass spectrometry (MS) detection, 14,15 or a partial filling approach 17 combined with UV detection can be used. 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Chem. 2009, 81, 315–321 10.1021/ac8020533 CCC: $40.75  2009 American Chemical Society 315 Analytical Chemistry, Vol. 81, No. 1, January 1, 2009 Published on Web 12/02/2008