Limitations to the rates of recombinant protein production by mammalian cells

Mammalian cells have been genetically engineered to produce a large number of recombinant proteins for research, diagnostic and therapeutic applications. However, low cellular production rate generally limits production yields and increases production costs. To select strategies to maximize production, it is important to identify the intracellular limitations of the mammalian cell production rates. Recombinant human activated protein C (APC) and tissue plasminogen activator (t-PA) have served as low and high producing systems for this investigation, respectively. The transcription, translation, and secretory efficiencies were analyzed in clones with wide ranges of APC and t-PA productivities. A structured kinetic model was used to quantify the changes in intracellular parameters when recombinant protein expression became limiting. The production rate of APC by baby hamster kidney (BHK) cells was increased 35- fold by increasing the cDNA copy number per cell from 50 to 240. In this range, the transcription efficiency (APC mRNA per cDNA) was not constant, as had been expected, but instead increased 7 fold. This apparent cooperative effect of multiple cDNA copies could be explained by their integration in tandem. For cDNA copy numbers higher than 240, the transcription efficiency decreased dramatically, possibly due to cosuppression. Two strategies were employed to maximize APC mRNA levels and APC production rate. Sodium butyrate treatment or re-transfection of an APC producing cell line with a vector containing additional APC cDNA resulted in over 2-fold higher mRNA levels and cell specific APC production rates. At high mRNA levels, the APC secretion rate but not translation efficiency was decreased, revealing a saturation of the secretory pathway. In batch cultures of a mRNA limited clone, the levels of total cellular RNA, APC mRNA and β-actin mRNA were relatively stable while cells were in the exponential growth phase., but rapidly decreased during the stationary phase. Decreasing APC mRNA level was correlated with a decline in APC secretion rate, indicating that the mRNA levels limited the rates of APC production beyond the exponential phase, into the declining growth and stationary phases. The ƴ-carboxylation of glutamic acid residues, a post-translational modification required for APC biological activity, was also analyzed. The proportion of APC that was fully ƴ -carboxylated decreased as batch cultures progressed and in clones with increased APC production rates. The production of recombinant t-PA in Chinese hamster ovary (CHO) cells was increased by cDNA amplification using stepwise adaptation to increasing methotrexate (MTX) concentrations. Subcloning of the amplified cells showed no apparent correlation between t-PA production rate and cell specific growth rate. The highest producing clones were isolated at 5 μM MTX and yielded 26,000 U/10⁶cells-day (-43 μg/10⁶cells-day) of t-PA. In the absence of MTX, an up to 90% decline in t-PA production rate was observed within 40 days, which could be explained by an up to 60% loss of cDNA copies. In longterm serum-free culture without MTX (108 days), the maximum t-PA production rate obtained (for 320 days) was 7,000 ± 750 U/10⁶cells-day (-12 ± 1 μg/10⁶cells-day*). This stable level of production was significantly lower than the unstable levels of production that CHO cells attained under selective pressure.Applied Science, Faculty ofChemical and Biological Engineering, Department ofGraduat