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Mammalian cell production and purification of progenipoietin, a dual‐agonist chimaeric haematopoietic growth factor
Author(s) -
Wood David C.,
Mathis Karl J.,
Joy William D.,
Minnerly John C.,
Pegg Lyle E.,
Welply Joseph K.
Publication year - 2003
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20020046
Subject(s) - affinity chromatography , ion chromatography , haematopoiesis , biology , gel permeation chromatography , chromatography , fast protein liquid chromatography , hydrophilic interaction chromatography , receptor , biochemistry , chemistry , high performance liquid chromatography , microbiology and biotechnology , stem cell , enzyme , organic chemistry , polymer
One member of the progenipoietin (ProGP) family of engineered proteins, ProGP‐2, is a chimaeric dual cytokine receptor agonist, expressed in mammalian cells, that stimulates both human fetal liver tyrosine kinase‐3 (Flt3) and the granulocyte‐colony‐stimulating‐factor (G‐CSF) receptor. The production of ProGP‐2 on a small and large scale using either anti‐(Flt3 ligand) antibody‐affinity chromatography, or a combination of (NH 4 ) 2 SO 4 fractionation, anion‐exchange chromatography, hydrophobic‐interaction chromatography and preparative reverse‐phase chromatography is described. ProGP‐2 was produced in hollow‐fibre reactors containing stably transfected NS0 cells. The productivity of ProGP‐2 was initially high, but was found to decrease 3–4‐fold over time. When the yield of ProGP‐2 decreased, the combination of three conventional chromatography steps was required to meet protein purity similar to that achieved by the anti‐(Flt3 ligand) chromatography method. In addition, a protease activity was observed in conditioned media from the hollow‐fibre reactors that resulted in increased degradation of ProGP‐2 that was removed by hydrophobic‐interaction chromatography at higher pH. Together the results demonstrated a method for production and purification of ProGP‐2 for additional studies on its haematopoietic activity.