title: Purification of recombinant human growth hormone from CHO cell culture supernatant by Gradiflow preparative electrophoresis technology.
abstract: Purification of recombinant human growth hormone (rhGH) from Chinese hamster ovary (CHO) cell culture supernatant by Gradiflow large-scale electrophoresis is described. Production of rhGH in CHO cells is an alternative to production in Escherichia coli, with the advantage that rhGH is secreted into protein-free production media, facilitating a more simple purification and avoiding resolubilization of inclusion bodies and protein refolding. As an alternative to conventional chromatography, rhGH was purified in a one-step procedure using Gradiflow technology. Clarified culture supernatant containing rhGH was passed through a Gradiflow BF200 and separations were performed over 60 min using three different buffers of varying pH. Using a 50 mM Tris/Hepes buffer at pH 7.5 together with a 50 kDa separation membrane, rhGH was purified to approximately 98% purity with a yield of 90%. This study demonstrates the ability of Gradiflow preparative electrophoresis technology to purify rhGH from mammalian cell culture supernatant in a one-step process with high purity and yield. As the Gradiflow is directly scalable, this study also illustrates the potential for the inclusion of the Gradiflow into bioprocesses for the production of clinical grade rhGH and other therapeutic proteins.
authors.affiliation: Bioengineering Centre, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
authors.forename: Dallia
authors.initials: D
authors.lastname: Catzel
authors.forename: Helen
authors.initials: H
authors.lastname: Lalevski
authors.forename: Chris P
authors.initials: CP
authors.lastname: Marquis
authors.forename: Peter P
authors.initials: PP
authors.lastname: Gray
authors.forename: Derek
authors.initials: D
authors.lastname: Van Dyk
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Mahler
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D017077:Culture Media, Conditioned / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation / Q000379:methods*; D013006:Growth Hormone / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; D013006:Growth Hormone