The CHO miRNA Transcriptome - MicroRNAs as Tools in Biopharmaceutical Production - page 60

compared to negative controls and significantly delayed the onset of apoptosis, prob-
ably due to increased levels of several anti-apoptotic targets of miR-466h (Druz et al.
2011 ). Thus, miR-466h is the first cgr-apoptomiR that can decide over death and
survival of CHO cells that has been identified.

4.4.4 miRNA Expression Signatures That Support Recombinant

Protein Production

Besides enhanced growth and survival under stressful conditions, high recombinant
protein productivity is an equally desirable trait of an industrially used cell line. With
respect to CHO cells, media and process optimizations have lead to the most signif-
icant increases in recombinant protein titers without much increase in the specific
protein production rates (qP) that commonly range between 25 and 50 pg/cell/day
for monoclonal antibodies. However, since it is known that reduced temperature
in biphasic bioprocesses or small molecules such as sodium butyrate can improve
specific protein production rates, transcriptomics studies have been performed to
identify gene expression changes under these conditions (Yee et al. 2008 , 2009 ).
While these studies gave valuable insights into the cellular pathways that might con-
tribute or stabilize a cellular state of high recombinant protein productivity, they
failed in pinning down individual candidates for engineering productivities.
Knowing that single miRNAs can be potent regulators of CHO cell growth and
survival, the next step was to see whether miRNA expression signatures exist in CHO
cells that can drive or support a state of high protein production and secretion. Lin
et al. addressed this question with an initial comparison of miRNA expression be-
tween recombinant DG44 clones—amplified and non-amplified using methotrexate
(MTX)—and the respective DG44 host cell line (Lin et al. 2010 ): based on the re-
sults from a cross-species miRNA microarray platform a panel of 16 miRNAs was
selected for qPCR analyses in four IgG producing subclones as well as their respec-
tive parental cell line. Results of qPCR analysis showed that 8 out of 16 miRNAs
exhibited a fold change > 1.5 in at least two out of four recombinant subclones, with
two miRNAs—miR-221 and miR-222—being more than two-fold down-regulated
and miR-17 being up-regulated in all four subclones.
To determine whether expression of these miRNAs correlated with recombi-
nant IgG-productivities, MTX amplification was performed and changes in specific
productivities (qP) were analyzed. In one of the clones that demonstrated a dose-
dependent increase in qP with increasing MTX concentrations, the levels of
miR-221/222 were analyzed at four stages during MTX amplification, however,
no significant correlation between their expression and qP could be detected.
Nevertheless, in the light of an observation that was made based on miRNA-seq
data from recombinant and host CHO cell lines (Hackl et al. 2011 ), the regulation of
the mir-221 hairpin and its processing in respect to recombinant protein productivity
is of interest: Dicer1 processing of miRNA hairpins (denoted as mirs) results in a
short duplex sequence with 3 overhangs of which one strand is often (in ∼ 50%of