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.