Electrophoresis of megaDalton proteins inside colloidal silica

Abstract

With the growth of the biopharmaceutical industry, there is a need for rapid size-analysis of proteins on the megaDalton scale. The large pore sizes needed for such separations cannot be easily reached by pushing the current limits of size-exclusion chromatography or gel electrophoresis. The concept detailed here is the formation of arbitrarily wide pores by packing nonporous colloidal silica in capillaries. This method can be called packed-capillary electrophoresis, or "pCE". Electrophoresis of protein standards (11-155 kDa) by pCE, using 345 nm diameter particles in 100 μm diameter capillaries, gives 2x higher resolution than a typical PAGE gel in 1/6 of the time. The electropherograms show that pCE is highly efficient, with half-micrometer plate heights for all seven standards, giving 10⁵ plates for a 50 mm length. The large pore radius of 65 nm enables baseline resolution of proteins of 0.72, 1.048 and 1.236 MDa in less than 15 min. The short separation time of pCE is attributed to the absence of small pores that restrict protein migration in gels. The pCE separation is applied to the analysis of a stressed pharmaceutical-grade IgG4 sample, giving unprecedented baseline resolution of monomer, dimer, trimer and tetramer in less than 10 min.

Keywords: Aggregation; Complexes; IgG; MDa; Separation.