Results and Discussion Autophagy enhances recombinant antibody production in batch cultures
Our initial investigations evaluated whether addition of the autophagy-inducing peptide (AIP), derived from the autophagy protein Beclin 1 (Shoji-Kawata et al., 2013), could enhance monoclonal antibody production in batch cultures. CHO cells expressing a human anti-IL1β IgG1 (CHO-aIL1β) were cultured in medium supplemented with AIP at 0, 1, 2, 3 and 4 µM concentrations, and IgG production was evaluated by ELISA, as described in Materials and Methods. Figure 1A compares the specific and volumetric productivities in the control- and AIP- treated cultures from a representative batch induction experiment. The specific productivity for the AIP-treated cultures increased from 11.0 ± 0.9 to 19.2 ± 0.7 µg/106 cells/day at 1 and 4 µM concentrations, respectively. The specific productivities observed for the 2, 3, and 4 µM AIP-containing cultures were significantly increased (ANOVA, P < 0.05) relative to the control culture (10.6 ± 0.6 µg/106cells/day). Similarly, the volumetric productivity increased from 404.9 ± 28.3 at 1 µM AIP to 565.5 ± 39.6 µg/mL at 4 µM AIP. However, the increase in volumetric productivity was statistically significant only for the 4 µM AIP concentration, as compared to the control culture (ANOVA, P < 0.05). Thus, substantial increases were observed, up to 82% and 46% in specific and volumetric productivities, respectively.
Peptide hydrolysates have been added to cell culture manufacturing media to increase cell growth and productivity (Franek et al., 2003; Franek and Fussenegger, 2008). To determine whether the impact of the AIP was indeed due to its capacity to induce autophagy, a negative control peptide containing the same amino acids, but in a scrambled arrangement, was also tested. Figure 1B shows that 3 or 4 µM concentrations of the negative control peptide had little if any influence on the protein productivity compared to the control culture containing 0 µM peptide. Furthermore, it was confirmed that the addition of the AIP to the culture medium induced cellular autophagy in CHO-aIL-1β cells, while the control peptide did not, as evaluated by cellular p62 protein degradation in Western blot analyses (Shoji-Kawata et al., 2013 and data not shown). Therefore, the increase in productivity observed was linked to the induction of autophagic flux in the cell culture, and this is consistent with our previous findings (Jardon et al., 2012) as interpreted and confirmed by Baek et al. (2016). Shoji-Kawata et al. (2013) have shown that the AIP used in these experiments specifically triggers the autophagy cascade by promoting the release of Beclin 1 from one of its inhibitors, the Golgi-associated plant pathogenesis-related protein 1 (GAPR-1). However, the molecular mechanism by which autophagic flux, in response to the AIP, leads to enhanced protein productivity in culture supernatants is unclear at present. It is interesting to note that the increased autophagy and CHO cell productivity in response to 3-MA was associated with the induction of the unfolded protein response (UPR) at the transcriptional level (Baek et al. 2018). These authors further suggested that UPR induction may enhance endoplasmic reticulum (ER) capacity and thereby the cellular productivity, a possibility that requires further investigation.