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

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 μg 106 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 °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 °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 °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 ∼13 to ∼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”.