Mammalian cell production and purification of progenipoietin, a dual-agonist chimaeric haematopoietic growth factor.
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 (NH4)2SO4 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.
Wood
David C
DC
Biotechnology Department, Pharmacia Corporation, 700 Chesterfield Parkway N., St Louis, MO 63198, USA. david.c.wood@pharmacia.com
Mathis
Karl J
KJ
Joy
William D
WD
Minnerly
John C
JC
Pegg
Lyle E
LE
Welply
Joseph K
JK
D000818:Animals; D019149:Bioreactors; D002478:Cells, Cultured; D002845:Chromatography / Q000379:methods*; D003001:Cloning, Molecular; D003115:Colony-Stimulating Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006224:Cricetinae; D015202:Protein Engineering / Q000379:methods*; D011786:Quality Control; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000819:agonists / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D003115:Colony-Stimulating Factors; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins