Analysis of Recombinant Human Platelet-Derived Growth Factor by Reversed-Charge Capillary Zone Electrophoresis

Abstract

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.

Separation of Recombinant Human Erythropoietin Glycoforms by Capillary Electrophoresis Using Volatile Electrolytes. Assessment of Mass Spectrometry for the Characterization of Erythropoietin Glycoforms

“…Polybrene (PB) is a polycationic polymer composed of quaternary amines that strongly adsorbs to the fused-silica inner surface and enables the EOF to be reversed toward the anode. The net positively charged amine layer formed on the capillary wall reversed the EOF, and the field polarity used for the separation must be also reversed in order to ensure that the positively charged analytes migrated toward the detector. − Several authors have also pointed out that PB and other related compounds may act as pseudostationary phases promoting a reversible partitioning mechanism that would also affect the selectivity of separations and the resolution of rHuEPO glycoforms. , The effects of various factors such as volatile buffer type and composition, buffer pH, voltage, capillary length, and temperature on glycoform separation were studied. The influence of the type of running electrolyte used and its pH was initially investigated in the pH-interval values near the pI of the EPO glycoforms (assumed to be between 3.5 and 5.0 19,37 ), using 100 mM FA-NH 4 F, 100 mM HAc−NH 4 Ac, 100 mM MES−NH 4 MES, and 100 mM Tricine-TricineAc.…”

Ionene‐dynamically coated capillary for analysis of urinary and recombinant human erythropoietin by capillary electrophoresis and online electrospray ionization mass spectrometry

“…After activation, capillaries were coated and equilibrated by flushing them first for 45 min with an aqueous solution of 5% w/v ionene and 2% v/v ethylene glycol, and then with separation buffer for 45 min [28][29][30][31]. Between runs, the capillary was rinsed for 2 min with 0.1 M NaOH, 2 min with water, 2 min with ionene solution, and 5 min with separation buffer.…”

“…Three kinds of ionenes (Table 1) synthesized are polycationic polymers composed of ammonium function that strongly adsorbs to the fused-silica inner surface which prevents adsorption of rhEPO on the inner walls of bare fused-silica capillaries, and reverses the EOF toward the anode [17,[29][30][31].…”

Capillary electrophoresis for monitoring bioprocesses

“…Human platelet‐derived human growth factor (hGF) is a major serum factor involved in subcutaneous wound healing and three potential forms of recombinant hGF (rhGF) produced by fungal fermentation of S. cerevisiae .…”