title: Protein-free transfection of CHO host cells with an IgG-fusion protein: selection and characterization of stable high producers and comparison to conventionally transfected clones.
abstract: In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 microg of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 pg x cells(-1) x day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 pg x cells(-1) x day(-1) in serum-dependent cultivation, a significant decrease that resulted in the range of q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development.
authors.affiliation: Austrian Center of Biopharmaceutical Technology, Muthgasse 18, 1190 Vienna, Austria. christine.lattenmayer@malteser.at
authors.forename: Christine
authors.initials: C
authors.lastname: Lattenmayer
authors.forename: Martina
authors.initials: M
authors.lastname: Loeschel
authors.forename: Kornelia
authors.initials: K
authors.lastname: Schriebl
authors.forename: Willibald
authors.initials: W
authors.lastname: Steinfellner
authors.forename: Thomas
authors.initials: T
authors.lastname: Sterovsky
authors.forename: Evelyn
authors.initials: E
authors.lastname: Trummer
authors.forename: Karola
authors.initials: K
authors.lastname: Vorauer-Uhl
authors.forename: Dethardt
authors.initials: D
authors.lastname: Müller
authors.forename: Hermann
authors.initials: H
authors.lastname: Katinger
authors.forename: Renate
authors.initials: R
authors.lastname: Kunert
mesh_terms: D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004921:Erythropoietin / Q000235:genetics; D018628:Gene Dosage; D007074:Immunoglobulin G / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
chemical_list: D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin