Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents.
We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG4 encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 microg mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn297 was not significantly affected by nocodazole during transient production by this method.
Tait
Andrew S
AS
School of Engineering, University of Queensland, St. Lucia, QLD 4072, Australia.
Brown
Catherine J
CJ
Galbraith
Douglas J
DJ
Hines
Michael J
MJ
Hoare
Mike
M
Birch
John R
JR
James
David C
DC
D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics; D016503:Drug Delivery Systems / Q000379:methods*; D018390:Gene Targeting / Q000379:methods; D024861:Genetic Enhancement / Q000379:methods; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics; D008870:Microtubules / Q000187:drug effects; D015739:Nocodazole / Q000008:administration & dosage*; D014677:Pharmaceutical Vehicles / Q000737:chemistry; D011094:Polyethyleneimine / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection / Q000379:methods*
D000970:Antineoplastic Agents; D007074:Immunoglobulin G; D014677:Pharmaceutical Vehicles; D011994:Recombinant Proteins; D011094:Polyethyleneimine; D004247:DNA; D015739:Nocodazole