Analysis of recombinant human platelet-derived growth factor by reversed-charge capillary zone electrophoresis

Reversed-charge capillary zone electrophoresis (RC-CZE) has been developed as a clipping (proteolysis) assay for homodimeric protein recombinant human platelet-derived growth factor (rhPDGF-BB), a major serum mitogenic factor involved in subcutaneous wound healing. When expressed in yeast, the protein is excreted as a fully folded homodimeric protein consisting of two antiparallel B chains held together by two interchain disulfide bonds. During fermentation, internal proteolysis (clipping between residues Arg32 and Thr33) and C-terminal truncation (Arg32 and Thr109) may occur. Internal proteolysis yields three potential forms of rhPDGF-BB: intact (both B chains are intact), single-clipped (one B chain is clipped), and double-clipped (both B chains are clipped). Clipping also creates new C-terminal sites for further C-terminal truncations and leads to a very complex mixture of isoforms. Routine baseline resolution of these three forms by various modes of HPLC proved unsuccessful. When the disulfide bonds of antiparallel chains are reduced, the complex peptide mixture can be analyzed by RP-HPLC; however, only the level of total clipping is identified. Since RC-CZE separation relies upon differences in molecular charge/size ratio, it can resolve the three rhPDGF-BB forms differing in the additional exposed residues. The choice of reversed-charge CZE columns (amine-coated column) allows proteins of high pI such as rhPDGF-BB (pI > 10) to be readily analyzed while minimizing protein loss from column adsorption. To simplify the electropherogram of clipped forms, the sample is treated first with carboxypeptidase B to reduce the charge microheterogeneity of partial Arg32 truncation. Analysis of rhPDGF-BB by RC-CZE yields a baseline separation between the three forms, intact and single- and double-clipped rhPDGF-BB.