The effect of the autophagy-inducing peptide on cell growth and
viability in batch culture
Given the encouraging productivity results, it was important to consider
the effects of the AIP on the growth and viability of the CHO cells in
culture. The results obtained with the AIP were compared to two control
cultures containing either no peptide or the scrambled negative control
peptide that did not induce autophagy (Figure 1B). Compared to the two
control cultures, with the addition of increasing AIP concentrations,
there was an increasing lag in growth over the first 4-8 days of culture
(Figure 2A). Subsequently, the AIP-containing cultures had similar rapid
growth phases, resulting in a ~5 day extended batch
culture duration. These cultures reached maximum cell densities from 8.3
± 0.1 to 9.9 ± 0.6 x 106 cells/mL, with Integral
Viable Cell Density (IVCD) values from 26.2 ± 0.3 to 40.9 ± 0.3 x
106 cells-day/mL. After reaching the maximum, the
viable cell densities consistently declined rapidly over the ensuing 2
days. Notably, the addition of 2-4 µM AIP to the cultures resulted in
significantly lower (ANOVA, P < 0.05) maximum cell densities
and IVCDs compared to the control and 1 µM AIP-containing cultures.
These differences, along with the more pronounced lag phases at higher
AIP concentrations, are likely the result of the complex control exerted
over cell survival and death by the autophagy pathway (Doherty and
Baehrecke, 2018).
The batch culture viability profiles are shown in Figure 2B. The control
cultures maintained a >90% viability until day 6, before a
rapid viability decline. For all cultures treated with AIP, the lag in
growth was accompanied by an initial drop in viability observed on day
2. Similarly, the extent of the drop was AIP concentration dependent,
with 4 µM of AIP exerting the largest, >90% decrease in
cell viability. This decrease in viability was not observed in the
control peptide culture, nor would it be expected from peptide
hydrolysate medium supplementation. Also, the decrease in viability was
not attributable to the protein transduction domain of the tat motif
present in both peptides to facilitate their uptake into the cell (Herce
and Garcia, 2007). The day 2 decreased viability likely resulted from
the induction of autophagic cell death or “autosis”, which can be a
consequence of autophagic flux in response to the AIP, as previously
reported (Liu et al., 2013). Interestingly however, the viability in all
AIP-treated cultures recovered rapidly after day 2, reaching over 90%
by day 6. After day 8, another rapid decrease in viability was observed
as these batch cultures entered the decline phase. It appears that the
autophagic flux in response to the AIP, the ensuing cell death, and
rapid subsequent expansion of surviving cells, creates conditions and/or
selects for cells that have superior protein production capabilities.
This merits further investigation, especially in the context of the
fed-batch processes most commonly used for monoclonal antibody
manufacturing.