Elsevier

Journal of Biotechnology

Volume 294, 20 March 2019, Pages 49-57
Journal of Biotechnology

Physiological alterations of GS-CHO cells in response to adenosine monophosphate treatment

https://doi.org/10.1016/j.jbiotec.2019.01.016Get rights and content

Highlights

  • AMP treatment causes reduced proliferation, accumulated S-phase cells, increased cell size and specific productivity.

  • Total intracellular protein content is considered the key determinant in increasing cell size in this case.

  • Different mechanisms are likely responsible for improved total intracellular protein content in G1 and S phase arrest.

Abstract

Growth-arrested strategies (e.g. hypothermia and hyperosmolarity) have been widely employed to enhance cell-specific productivity (qP) in mammalian cell culture bioprocess. In addition to enhanced qP, alterations in cell physiology, such as cell size and cell cycle phase, have also attracted extensive attention under growth-arrested conditions. However, to date, very few reports on associations between physiological changes in growth-inhibiting approaches have been published. In this study, we explored associations between the physiological changes of GS-CHO cells in response to adenosine monophosphate (AMP) treatment. In dose response studies, AMP treatment resulted in suppressed proliferation, accumulated S-phase cells, increased cell size and enhanced qP. Subsequently, six GS-CHO clones exhibited the physiological alterations in varying degrees when treated with 7 mM AMP. But more importantly, a significant positive correlation between total intracellular protein content and mean electronic volume, an indicator of cell size (P < 0.01) was found, indicating that total intracellular protein was the determining factor in increasing cell size in this growth-arrested strategy. Besides, our results provide additional evidence that treatment with growth-arrested agents may increase cell size; the agent per se did not cause the increased productivity.

Introduction

In mammalian cell culture, two major strategies have been employed to enhance cell-specific productivity (qP): either to develop high-producing cell lines through expression vector engineering and clone screen technologies (Browne and Al-Rubeai, 2007; Kim et al., 2012) or to enhance qP in cultivation at the cost of reduced cell growth. The latter can be accomplished in three ways: by manipulating culture conditions (mild hypothermia and amino acid deprivation), by adding cytostatic chemical agents, and by using genetic manipulation to control proliferation (Sunley and Butler, 2010).

Inducing cell cycle arrest in the G1 or S phase by adding growth-arrested agents has been widely shown to increase recombinant protein production (Al-Rubeai et al., 1992; Hendrick et al., 2001; Wang et al., 2007). Dimethyl sulfoxide (DMSO), a well-known anti-inflammatory and bacteriostatic agent as well as a cryoprotectant for preserving cells in freeze-thaw processes, has been used to induce a reversible arrest in the G1 phase for a number of cell lines (Fiore et al., 2002; Naciri et al., 2009; Sawai et al., 1990). As a result of increased hepatitis B antigen (HBsAg) gene transcription, Wang and coworkers (2007) reported that the overall yield and specific productivity of HBsAg was elevated in recombinant Chinese hamster ovary (CHO) cells in response to DMSO treatment. Similar outcomes of recombinant protein production accompanied by S-phase arrest have been noted in a hybridoma cell line exposed to excess thymidine (Al-Rubeai et al., 1992). Additionally, small molecule cell cycle inhibitors have been taken into account to improve the performance of production cell lines. Recently, a chemically synthesized pyridopyrimidine-type molecule was employed to manipulate cell proliferation by selectively inhibiting the function of cyclin-dependent kinases (CDK) 4/6 in multiple recombinant CHO cell lines. In the meanwhile, the treatment led to greatly increased qP and improved glycan processing (Du et al., 2015). Unfortunately, despite a remarkably beneficial impact on recombinant protein production, the application of the growth-arrested agents to biopharmaceutical manufacturing processes is severely hampered by the regulatory requirement to remove all traces of such cytostatic compounds in the interest of patient safety.

Cell size is a critical parameter for the regulation of cell cycle transition, and is maintained in a homeostatic process throughout the cell cycle in proliferating cells (Echave et al., 2007; Fingar et al., 2002; Tzur et al., 2009). However, in terms of cell growth, an inverse relationship between cell size and growth rate has typically been reported in different cell types (Fernandez-Martell et al., 2018; Kang et al., 2014) or cell cultures supplemented with extra nutrients (Shridhar et al., 2017). The biological control mechanisms underpinning the inverse relationship are not yet elucidated. With regard to recombinant protein production from cultivated mammalian cells, cell size has been proven to be a strong indicator of qP (Kim et al., 2001; Lloyd et al., 2000). In particular, Lloyd et al. (2000) reported that the synthesis of recombinant proteins was not specifically related to any cell cycle phase — regardless of expression system, host cell type, and the recombinant gene expressed — but such synthesis increased with cell size. The positive relationship between cell size and qP has also been observed in production cell lines simultaneously overexpressing proto-oncogenes (Krampe et al., 2011; Kuystermans and Al-Rubeai, 2009). Besides the standardization culture conditions, an increase in cell size is accompanied by increased qP under growth-arrested conditions, such as hyperosmotic pressure and overexpression of CDK inhibitors (Bi et al., 2004; Carvalhal et al., 2003a; Oh et al., 1995; Ozturk and Palsson, 1991; Ryu et al., 2001; Sun et al., 2004).

This study investigated associations between physiological alterations by exposing a panel of six GS (glutamine synthetase)-CHO clones with varying growth characteristics and qP to adenosine monophosphate (AMP). AMP, a purine nucleotide metabolic intermediate, has been shown to inhibit cell growth while increasing qP and maintaining a high cell viability (Carvalhal et al., 2003b; Hugo et al., 1992).

Section snippets

Materials and methods

All reagents were purchased from Sigma-Aldrich (Poole, UK) unless otherwise stated.

Effects of AMP treatment on cell culture in a dose-dependent manner

AMP treatment of clone 47 suppressed proliferation and viability in a dose-dependent manner. While growth at 1 mM AMP was similar to the control, maximum viable cell densities were reduced by 81 ± 1% and 93 ± 0.4% at 4 and 7 mM, respectively (Fig. 1a). Cell viability followed a similar trend; the largest difference were seen at day 4, with viabilities of 98 ± 0.2%, 93 ± 0.9%, and 83 ± 2% for 1, 4, and 7 mM AMP, respectively; however, for all cultures, viability remained above 90% for the first

Discussion

Growth-arrested strategies have been commonly employed to increase qP in recombinant protein production from cultivated mammalian cells (Sunley and Butler, 2010). In the meanwhile, alterations in cell physiology have also been noted to depict effects of growth-arrested manipulations on mammalian cells and explore the mechanism of improvement in recombinant protein expression. Unfortunately, whether there are any associations between physiological changes still remains poorly understood. In

Conclusion

In this work, we investigated the effects of AMP treatment on cell physiology and productivity of GS-CHO cells. In dose response studies by using a single clone, AMP treatment led to suppression of cell proliferation, increases in S-phase cells, cell size and qP in a dose-dependent manner. Similar physiological and productivity changes were subsequently observed in other five clones response to 7 mM AMP treatment. Importantly, a significant positive correlation between total cellular protein

Declaration of interests

The authors declare no conflict of interest.

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