Control of Culture Environment for Improved Polyethylenimine-Mediated Transient Production of Recombinant Monoclonal Antibodies by CHO Cells

摘要

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 IgG_4 monoclonal antibody (Mab)production in agitated shake flask cultures to be a DNA concentration of 1.25 mu g 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 deg 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 deg 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 deg 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 centre dot 13 to centre dot 39 mg L~(-1).Under these conditions,Mab glycosylation at Asn_(297)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".