ANALYSIS OF RECOMBINANT HUMAN PLATELET-DERIVED GROWTH-FACTOR BY REVERSED-CHARGE CAPILLARY-ZONE-ELECTROPHORESIS

Reversed-charge capillary zone electrophoresis (RC-CZE) has been devel oped as a clipping (proteolysis) assay for homodimeric protein recombi nant human platelet-derived growth factor (rhPDGF-BB), a major serum m itogenic factor involved in subcutaneous wound healing. When expressed in yeast, the protein is excreted as a fully folded homodimeric prote in consisting of two antiparallel B chains held together by two interc hain disulfide bonds. During fermentation, internal proteolysis (clipp ing between residues Arg(32) and Thr(33)) and C-terminal truncation (A rg(32) and Thr(109)) may occur. Internal proteolysis yields three pote ntial forms of rhPDGF-BB: intact (both B chains are intact), single-cl ipped (one B chain is clipped), and double-clipped (both B chains are clipped). Clipping also creates new C-terminal sites for further C-ter minal truncations and leads to a very complex mixture of isoforms. Rou tine 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; h owever, only the level of total clipping is identified, Since RC-CZE s eparation relies upon differences in molecular charge/size ratio, it c an resolve the three rhPDGF-BB forms differing in the additional expos ed residues. The choice of reversed-charge CZE columns (amine-coated c olumn) allows proteins of high pi such as rhPDGF-BB (pl > 10) to be re adily analyzed while minimizing protein loss from column adsorption. T o simplify the electropherogram of clipped forms, the sample is treate d first with carboxypeptidase B to reduce the charge microheterogeneit y of partial Arg(32) truncation. Analysis of rhPDGF-BB by RC-CZE yield s a baseline separation between the three forms, intact and single- an d double-clipped rhPDGF-BB.