Mild hypothermia: Insights and implications for productivity in mammalian and insect cell cultures

Between 1977 and 1979, the first genetically modified organisms were created, leading to the large-scale production of insulin, somatostatin, and growth hormone (Demain et al., 2017). However, these early genetically modified organisms were of prokaryotic origin, which limited their use to generating simple, non-glycosylated proteins (O'Flaherty et al., 2020). Protein glycosylation plays a crucial role in multiple functional aspects, including immune response, protein folding, aggregation, stability, and transport (Lalonde and Durocher, 2017; O'Flaherty et al., 2020; J.-H. Zhang et al., 2022). Unlike yeasts, insects, and plants, mammalian cells possess a glycosylation pattern very similar to that of humans. This similarity has made mammalian expression systems the preferred choice for producing many complex therapeutic proteins, despite their slower growth and higher production costs compared to other systems (Noh et al., 2019; J.-H. Zhang et al., 2022). As a result, the first biological drug produced using recombinant mammalian cells was approved in 1986 (Bielser et al., 2018; Demain et al., 2017; Dumont et al., 2015). Since then, more than 207 biopharmaceutical products derived from mammalian cell cultures have been approved in the EU and the USA between 1987 and 2020 (Al-Majmaie et al., 2021). Between 2016 and 2020, over 80 % of recombinant proteins approved for pharmaceutical use in Europe and the US were produced in mammalian cell cultures (Al-Majmaie et al., 2021). Chinese Hamster Ovary (CHO) cells remain the dominant platform in large-scale biopharmaceutical manufacturing, accounting for 81 % of total production, followed by murine myeloma cells from NS0 (8 %) and Sp2/0 tumor cells (4 %), Baby Hamster Kidney (BHK) cells (3 %), Human Embryonic Kidney cells (HEK-293) (2 %), and other human-derived lines (2 %) (Al-Majmaie et al., 2021; Noh et al., 2019).

Over the past decades, insect cell lines derived from Spodoptera frugiperda (Sf-9 and Sf-21), Trichoplusia ni (Tn-368 and High-Five), and Drosophila melanogaster (S2) have emerged as a viable alternative to mammalian cell lines for recombinant protein (r-protein) production (Drugmand et al., 2012; O'Flaherty et al., 2020). The first system developed for r-protein expression in insect cells was the insect cell–baculovirus expression vector system (IC-BEVS), introduced by Smith et al. (1983), which remains one of the most widely used platforms in biotechnology for producing vaccines, therapeutic proteins, and recombinant viral particles. More recently, the development of recombinant insect cell lines, capable of stable protein expression without baculovirus infection, has proven to be an effective alternative, enabling high-level expression and secretion of complex therapeutic proteins (Fernandes et al., 2020, Fernandes et al., 2021, Fernandes et al., 2022; Rossi et al., 2012; Swiech et al., 2008). Insect cells offer several advantages, including high protein yields in serum-free media, excellent scalability, and cost-effective production (Drugmand et al., 2012). Additionally, the non-infectious nature of baculoviruses in vertebrates contributes to the safety of this expression system. However, the platform does present some limitations. Insect cells typically generate a high proportion of potentially immunogenic glycan structures, such as oligomannose and paucimannose glycans, and lack the enzymatic machinery to produce sialylated glycans, an essential post-translational modification for many therapeutic proteins (Drugmand et al., 2012; O'Flaherty et al., 2020).

Significant advances in mammalian and insect cells cultivation have been achieved through cell line engineering, the development of chemically defined media, and improved characterization of recombinant products (Drugmand et al., 2012; McKee and Chaudhry, 2017; O'Flaherty et al., 2020). However, maximizing r-protein productivity remains a major challenge in biopharmaceutical production, particularly in the development of new vaccines and complex therapeutic proteins, in an increasingly competitive market. To address this challenge, several optimization strategies have been developed to enhance r-protein production, including: cellular engineering, optimization of media and culture conditions, design and optimization of bioprocess strategies (Al-Majmaie et al., 2021; Drugmand et al., 2012; Mark et al., 2022; O'Flaherty et al., 2020) and the use of low culture temperatures (Bedoya-López et al., 2016; Chen et al., 2021; Fernandes et al., 2020; Johari et al., 2021; Kaisermayer et al., 2016; Rossi et al., 2012; Torres et al., 2018; Zhu et al., 2023).

In mammalian cell, low culture temperature in the range of 28 to 34 °C, referred to as mild hypothermia (MH), has been widely explored for its ability to improve cell viability and increase r-protein yields (Bedoya-López et al., 2016; Mellahi et al., 2019; Sou et al., 2017). The first studies evaluating the impact of temperature shifts on CHO cell growth and r-protein productivity were conducted by Jenkins and Hovey (1993), who demonstrated that reducing the temperature to 34 °C improved cell viability and increased r-protein yields by 35 % compared to cultures maintained at 37 °C. However, earlier studies in mouse-mouse hybridoma cell lines showed that although MH improved cell viability, it reduced volumetric productivity for monoclonal antibody (mAb) production (Reuveny et al., 1986). These findings, along with more recent studies, indicate that the effect of MH is highly cell-line dependent and varies according to the r-protein being expressed (Jang et al., 2021; McHugh et al., 2020). Additionally, several critical factors influence the overall effect of MH, including: promoter, temperature, mechanism to reduce temperature, temperature shift time, medium composition, pH and culture mode (Hennicke et al., 2019; Kaisermayer et al., 2016; McHugh et al., 2020).

Regarding insect cells, the available information on the application of MH is limited compared to that for mammalian cells. Consequently, further research is needed to optimize its application in these expression platforms. Identifying optimal conditions, along with the key influencing factors, is essential for the effective implementation of MH in both mammalian and insect cell cultures. Additionally, gaining a deeper understanding of the molecular mechanisms underlying MH responses is critical, as this knowledge holds significant implications for cell engineering strategies. Such insights are particularly valuable for improving cell culture performance, especially in the production of complex therapeutic proteins, where both productivity and product quality are crucial.

This review explores the application of MH in mammalian and insect cell cultures within the framework of biomanufacturing. Understanding each of these elements is key to assess the impact of MH on cell metabolism, productivity and product quality. This work examines the principal factors influencing cellular responses to MH, investigates the molecular mechanisms involved, and identifies key elements associated with temperature shift responses and their potential effects on recombinant protein (r-protein) productivity. Furthermore, it analyses the impact of MH on cell metabolism, r-protein expression, and the quality of r-proteins produced under mild hypothermia conditions.