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Biotechnology and Applied Biochemistry (2003) 37, (31–38) (Printed in Great Britain)

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Mammalian cell production and purification of progenipoietin, a dual-agonist chimaeric haematopoietic growth factor

David C. Wood¹, Karl J. Mathis, William D. Joy, John C. Minnerly, Lyle E. Pegg and Joseph K. Welply

Biotechnology Department, Pharmacia Corporation, 700 Chesterfield Parkway N., St Louis, MO 63198, U.S.A.

Key words: antibody affinity, circular permutation, fusion protein, preparative chromatography, process development.

Abbreviations used: AEX, anion exchange; BHK, baby-hamster kidney; CEX, cation-exchange chromatography; CV, column volumes; Flt3, fetal liver tyrosine kinase-3; G-CSF, granulocyte colony stimulating factor; HIC, hydrophobic interaction chromatography; PNGase F, peptide: N-glycosidase F; ProGP, progenipoietin; RP-HPLC, reversed-phase HPLC; SPR, surface plasmon resonance; TFA, trifluoroacetic acid; TMP, total media protein.

¹To whom correspondence should be addressed (e-mail david.c.wood@pharmacia.com).

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.

Received 30 May 2002/2 October 2002; accepted 9 October 2002

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