Introduction
Mammalian cell cultures are widely used for the production of protein biopharmaceuticals because of their capacity for complex and physiologically relevant post-translational modifications, often required for therapeutic efficacy (Varki et al., 2009). Chinese Hamster Ovary (CHO) cells have become the most used cell line for industrial production of recombinant glycoproteins due to their in-depth characterization and capacity to produce human-like glycosylation (Hamaker and Lee, 2018; Hong et al., 2018).
In the past decades, there has been an increasing demand for large-scale production of biopharmaceuticals, especially antibodies, from mammalian cell culture processes (Lalonde and Durocher, 2017). More than 90% of all antibodies on the market are produced in mammalian cells (Ecker et al., 2014). Over the years, cell culture engineering has made significant advances with improved growth media, culture condition control and cell lines (Butler and Meneses-Acosta, 2012; Kelly et al., 2018). In media development, formulations based on cellular metabolic needs (Ghaffari et al., 2020), as well as the addition of small molecules such as butyrate (Mimura et al., 2001) or butyrated ManNAc (Yin et al., 2018), have increased recombinant protein production. Improvements in cell line engineering include the development of superior mammalian expression systems (Bebbington et al., 1992; Lucas et al., 1996; Hamaker and Lee, 2018; Amann et al., 2019; Bulté et al., 2020) and the inclusion of anti-apoptotic genes to improve cell survival (Kim and Lee, 2002; Mastrangelo et al., 2000; Tey et al., 2000; Figueroa et al., 2007) with the goal of enhancing specific and volumetric productivity.
While apoptosis caused by nutrient limitations is the most well-known cell death mechanism impacting bioprocesses (Kim et al., 2012), autophagy (“self-eating”) is being studied in this context as well, often functioning as a pivotal tipping point between cell survival and death (Doherty and Baehrecke, 2018). Indeed, through vacuolar engulfment, degradation, and recycling of large protein complexes to entire organelles, autophagy is a critical mitigating response to metabolic stressors including nutrient depravation, thereby maintaining the cell’s energetic needs and homeostasis (Hwang and Lee, 2008; Zustiak et al., 2008; Mizushima and Klionsky, 2007; Mizushima et al., 2008; Doherty and Baehrecke, 2018). We previously reported that the addition of 3-Methyl Adenine (3-MA), a reputed autophagy inhibitor, increased recombinant protein productivity in CHO cell fed-batch cultures (Jardon et al., 2012). This phenomenon was further studied by Baek et al. (2016), testing 3-MA and eight other chemical inhibitors of autophagy, and their respective effects on cell culture productivity. An increased specific productivity was confirmed for 3-MA, and also observed in response to two other reputed autophagy inhibitors including dorsomorphin (AMP-activated protein kinase inhibitor), and SP600125 (c-Jun NH2-terminal kinase inhibitor). However, Baek et al. (2016) recognized that these agents did not inhibit autophagy but rather appeared to induce it, thereby linking 3-MA-enhanced autophagic flux rather than inhibition with increased bioprocess productivity. This induction of autophagy in response to 3-MA, particularly under nutrient-rich culture conditions, is consistent with previous studies by Wu et al. (2010).
In this study, a novel autophagy-inducing peptide was tested to further investigate the influence of this cellular process on secreted protein production. While the addition of chemicals to induce autophagy in cell culture is possible, their use comes with the risks of confounding effects (e.g. 3-MA (Klionsky et al., 2016, Wu et al., 2010)). The use of an alternative, more specific agent that could be more readily incorporated into an industrial bioprocess and at lower concentrations is preferable. Indeed, current media formulations often contain peptides as part of hydrolysates added to the medium to increase growth and productivity (Spearman et al., 2014). This made the use of a unique and specific cell-permeable, autophagy-inducing peptide (AIP), derived from the autophagy protein Beclin 1 (Shoji-Kawata et al., 2013), suitable for this purpose. Therefore, we evaluated and developed the capacity of this AIP to enhance therapeutic protein productivity in CHO cells maintained in batch culture as well as in the more industrially relevant fed-batch culture. In addition to therapeutic protein productivity, we determined the impact of the AIP on cell culture growth and viability.