Chemical inhibition of autophagy: Examining its potential to increase the specific productivity of recombinant CHO cell lines

Abstract

Chinese hamster ovary (CHO) cells activate and undergo apoptosis and autophagy for various environmental stresses. Unlike apoptosis, studies on increasing the production of therapeutic proteins in CHO cells by targeting the autophagy pathway are limited. In order to identify the effects of chemical autophagy inhibitors on the specific productivity (qp ), nine chemical inhibitors that had been reported to target three different phases of autophagy (metformin, dorsomorphin, resveratrol, and SP600125 against initiation and nucleation; 3-MA, wortmannin, and LY294002 against elongation, and chloroquine and bafilomycin A1 against autophagosome fusion) were used to treat three recombinant CHO (rCHO) cell lines: the Fc-fusion protein-producing DG44 (DG44-Fc) and DUKX-B11 (DUKX-Fc) and antibody-producing DG44 (DG44-Ab) cell lines. Among the nine chemical inhibitors tested, 3-MA, dorsomorphin, and SP600125 significantly increased the qp of DG44-Fc and DUKX-Fc. In contrast, for DG44-Ab, only 3-MA significantly increased the qp . The autophagy-inhibiting activity of the nine chemical inhibitors on the rCHO cell lines was evaluated through Western blot analysis and flow cytometry. Unexpectedly, some chemical inhibitors did not exhibit any apparent inhibition activity on autophagy. The chemical inhibitors that enhanced the qp , 3-MA, dorsomorphin, and SP600125, exhibited instead an increased autophagic flux. Taken all together, the chemical inhibition of autophagy was not effective in increasing the qp in rCHO cell lines and the positive effect of 3-MA, dorsomorphin, and SP600125 on the qp was not due to the inhibition of autophagy. Biotechnol. Bioeng. 2016;113: 1953-1961. © 2016 Wiley Periodicals, Inc.

Cell culture media for recombinant protein expression in Chinese hamster ovary (CHO) cells: History, key components, and optimization strategies

“…Apoptosis and Autophagy are programmed cell death processes, which can be triggered by nutrient depletion or environmental stress, potentially decreasing overall cell health and cell culture performance. Supplementation with apoptosis inhibitors and autophagy inhibitors has been evaluated, in some cases improving recombinant protein expression. Most of these supplements are not widely used due to the cost at manufacturing scale.…”

Types of cell death and apoptotic stages in Chinese Hamster Ovary cells distinguished by Raman spectroscopy

“…A particularly successful example was the transfection of two inducible anti‐apoptotic genes into Chinese Hamster Ovary (CHO) cells that enabled increased fed‐batch viable cell concentrations as well as recombinant protein production (Figueroa et al, ). More recently, factors that modulate autophagy have also been used to increase CHO cell protein production up to ∼3‐fold in fed‐batch, in particular using 3‐methyladenine (a putative inhibitor that can also induce autophagy with prolonged exposure) (Baek, Kim, Kim, Lee, & Lee, ; Jardon et al, ; Nasseri et al, ). Combined strategies to inhibit apoptosis and induce autophagy have also been developed (Lee, Ha, Park, & Lee, ).…”

The Expression Pattern of the Phosphoproteome Is Significantly Changed During the Growth Phases of Recombinant CHO Cell Culture

“…Two of the pathways that were found to be highly enriched in the exponential and stationary phases were the mTOR and autophagy pathways. The mTOR pathway has been manipulated previously in CHO cells to improve production efficiencies, and recently autophagy in CHO cells have gained attention as an opportunity for controlled cell death in bioprocess . In our study, we identified 18 phosphoproteins from the mTOR pathway, including the components of both mTORC1 and mTORC2 complexes.…”

“…Pathway analysis of phosphoproteomic data also revealed a significant enrichment of autophagy‐related proteins with differential expression between Day 2 and Day 4 of the batch suspension culture. Recently autophagy has gained importance in bioprocess as an opportunity to genetically and chemically control cell survival and productivity . It is therefore important to have a detailed understanding of the autophagic pathway in order to select relevant genes and chemical targets to manipulate autophagy in CHO cells to enhance the production of biotherapeutics .…”