title: Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells.
abstract: In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG4 monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 microg 10(6) cells(-1) mL(-1) at a PEI nitrogen:DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 degrees C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from approximately 13 to approximately 39 mg L(-1). Under these conditions, Mab glycosylation at Asn297 remained essentially constant and similar to that of the same Mab produced by stably transfected GS-CHO cells. From these data we suggest that the efficiency of transient production processes (protein output per rDNA input) can be significantly improved using a combination of mild hypothermia and growth factor(s) to yield an extended "activated hypothermic synthesis".
authors.affiliation: School of Engineering, University of Queensland, St. Lucia, QLD 4072, Australia.
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Galbraith
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Tait
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Racher
authors.forename: John R
authors.initials: JR
authors.lastname: Birch
authors.forename: David C
authors.initials: DC
authors.lastname: James
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D006224:Cricetinae; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D015870:Gene Expression / Q000187:drug effects; D018014:Gene Transfer Techniques; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000187:drug effects; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011094:Polyethyleneimine / Q000737:chemistry* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D011094:Polyethyleneimine; D004247:DNA; D000469:Alkaline Phosphatase