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".

Effect of addition of ‘carrier’ DNA during transient protein expression in suspension CHO culture

“…Thus the average IgG yield per unit plasmid DNA concentration is 2.5 lg IgG/lg plasmid DNA with the use of carrier DNA. Galbraith et al (2006) reported an IgG yield of *1 lg/ml (*0.2 lg/ml 1 day post transfection similar to the levels achieved in control condition in this study), when transfecting CHO cells at a density of 10 6 cells/ml with branched PEI using a plasmid DNA concentration of 1.25 lg/ml. They showed that treatment with LR3-IGF resulted in a yield of *2.5 lg/ml, which is similar to the yields obtained in this study at a lower plasmid DNA concentration of 1 lg/ml with the use of carrier DNA.…”

Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection

“…The viability decreased dramatically, resulting in complete cell death when the DNA to PEI concentration exceeded a ratio of 1:48, while complexes below this level of PEI showed viabilities above 80% (Table 1). The cell number demonstrated that growth occurred soon after transfection (Table 1 column 2), while other labs dealing with transient transfection normally have to cope with low viabilities after transfection and delayed cell growth (Galbraith et al 2006). The next step was to determine the most efficient DNA to PEI ratio to gain maximal transfection efficacy.…”

Development of a generic transient transfection process at 100 L scale

“…Although scalable transient expression using CHO cells is frequently reported (Derouazi et al 2004;Tait et al 2004;Kunaparaju et al 2005;Galbraith et al 2006) the human embryonic kidney 293 cell line (HEK293) is the most widely used cell line for transient protein expression. HEK293 cells readily take up DNA using various gene transfer vehicles such as calcium phosphate (Jordan et al 1998;Meissner et al 1999;Girard et al 2001;Meissner et al 2001;Durocher et al 2002;Baldi et al 2005), XtremeGENE Ro1539 (Schlaeger et al 1998;Schlaeger et al 2003) and PEI (Schlaeger and Christensen 1999;Durocher et al 2002;Pham et al 2003;Schlaeger et al 2003;Baldi et al 2005).…”