| Summary: | 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.
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