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.