Capillary zone electrophoretic resolution of recombinant human bone morphogenetic protein 2 glycoforms : an investigation into the separation mechanisms for an exquisite separation

Abstract Recombinant human bone morphogenetic protein 2 (rhBMP-2) is a disulfide-linked homodimeric glycoprotein ( M r = 30 000) which induces bone formation in vivo in several animal model systems. In this paper, we report the separation of a homogeneous rhBMP-2 sample into nine peaks by capillary zone electrophoresis (CZE), using a simple, pH 2.5, phosphate buffer containing no additives. The nine peaks have been identified to be glycoforms of rhBMP-2 [designated as (rhBMP-2) 2 -(GlcNAc) 4 (Man Z ), where Z varies from 10 to 18]. The difference between any adjacent pair of peaks is only one mannose residue ( M r = 162). The ability of CZE to resolve rhBMP-2 glycoforms having the same charge and differing only 0.5% in molecular mass, without resorting to chemical complexation, is both unexpected and intriguing. Possible mechanisms explaining how the additional mannose can affect the mobility of rhBMP-2 glycoforms were explored. Zeta potentials of various glycoforms were calculated from their mobilities and interpreted in light of diffuse double layer parameters. Our results suggest that CZE employing a low-pH buffer, where proteins are highly charged, may be uniquely suitable for complex protein glycoform analysis.