ReviewTrace metals in cellular metabolism and their impact on recombinant protein production
Introduction
Chinese Hamster Ovary (CHO) cells are the most common mammalian host for manufacturing of recombinant biotherapeutics. Serum-free and chemically defined medium formulations are now widely employed for commercial manufacturing using CHO cells. Metal ions are indispensable constituents of such cell culture medium formulations, impacting cell proliferation, viability and product quality. They can also affect long term cell stability by their effect on genomic stability [1]. Some metal ions such as sodium (Na+), potassium (K+), magnesium (Mg2+), calcium (Ca2+) are constituted at millimolar concentrations, while many other ‘trace’ metals have sub-micromolar concentrations. In addition to metal ions added deliberately as components of culture medium, trace metals can also be inadvertently introduced as contaminants from other medium components. Such unintended additions can lead to variability in the trace metal concentrations across different lots of medium. With the growing application of chemically defined medium formulations, the protective effect of serum is no longer available [2], increasing the susceptibility of such formulations to variations in trace metal concentrations. Largely, it is important to examine the impact of trace metals on cellular metabolism and recombinant protein quality to help understand their effect on protein production and anticipate changes that could be introduced due to their variability.
Table 1 lists the intracellular total and free ion content of trace metals in mammalian cells. Trace metal homeostasis in cells is maintained by metalloregulatory systems that control the labile pool of metal ions to favour binding of a preferred metal cofactor [3,4]. These systems import and mobilise metal ions under metal limiting conditions, while they efflux and sequester metal ions by binding or storage under excess metal conditions. Stability of metal and ligand binding in biological systems follow the Irving–Williams series in the order Mg2+ < Mn2+ < Fe2+ < Co2+ < Ni2+ < Cu2+ > Zn2+ with maximum stability with Cu2+ [5,6]. Consistent with this, the total amount of available metal ions like Cu2+ and Zn2+ in the cytosol are tightly regulated in cells compared to Mg2+ or iron (Fe2+) [5]. This contributes to the binding of correct cofactor and ensuring biological function. Changes in extracellular concentrations of trace metals can affect their own and possibly other trace metals’ availability and homeostasis, and impact cell physiology. Such instances of inter-metal competition and impact on metal homeostasis have been reported and reviewed in bacterial systems [4].
In this review, we provide an overview of the use of trace metals in cell culture medium formulations over the years. This is followed by the role of d-block metal ions manganese (Mn2+), copper (Cu2+) and zinc (Zn2+) in the metabolism of mammalian cells, primarily in central carbon metabolism. The importance of iron in cellular metabolism has been widely recognised [7,8] and is not discussed in this review. Further, reports of the effects of trace metals specifically on CHO cell metabolism are reviewed. Metabolic changes can affect several aspects of culture performance, such as growth, longevity and product quality. We further review the known effects of trace metals on important product quality attributes, including glycosylation of recombinant proteins expressed in CHO cells. Metal trafficking and sequestration is an important facet of regulation of metal ion homeostasis. Recent developments in Mn, Cu, and Zn trafficking and homeostasis in eukaryotic systems have been discussed by [[9], [10], [11]] and are not reviewed here. The effects of trace metals can depend on the availability of other nutrients, including other trace metals in the cell culture medium. Such interactions are discussed to highlight the interdependent nature of trace metal transport and binding.
Section snippets
Evolving role of trace metals in medium development- serum containing to chemically defined medium
Cell culture medium formulations for bioprocess applications have come a long way from the initial work on understanding how serum, plasma or tissue extracts supported in vitro cell culture. Serum supplementation was universally used initially to maintain cells and support growth. The usage of serum however introduces variability and raises ethical concerns, increases cost and the risk of undesirable contaminants like viruses and prions. Bioprocess applications now widely use serum-free
Variability in trace metal content of cell culture media in a biomanufacturing process can arise from multiple sources
Trace metal concentrations in medium can vary from batch-to-batch [28,29]. Such variability has been reported in both basal medium formulations like MEM, DMEM [21] and serum-free media formulations [21,30]. Trace element impurities can be introduced from any medium components. Cu impurities identified in yeast hydrolysate and sodium bicarbonate have been reported to affect cell growth and protein quality [21,31,32]. Trace metal contaminants were also identified in other components like amino
Activity of metabolic enzymes in central carbon metabolism is modulated by trace metal availability
A number of enzymes involved in glycolysis, TCA cycle and oxidative phosphorylation require metal cofactors. Mg2+ is an important cofactor for ADP/ATP-dependent enzymes involved in glycolysis and TCA cycle, while Ca2+ regulates mitochondrial dehydrogenases and ATP synthase [36,37]. Other than the kinases, enolase requires two Mg2+ bound at the active site [38]. Apart from Mg2+ and Ca2+, which are present in most medium formulations at millimolar concentration, enzymes in these pathways also
Impact of trace metals on CHO cell metabolism and quality of recombinant proteins
Supplementation of trace metal ion mixtures to CHO cell cultures has been demonstrated to boost culture performance by improving cell growth, culture longevity and recombinant protein productivity [[76], [77], [78], [79], [80], [81], [82], [83]]. In shake flasks as well as production scale reactors, hypoxia or oxidative stress arising from different sources can affect culture performance [31,81,84]. This was shown to be mitigated by supplementation of metal ions like Fe and Cu and thereby
Trace metal ion effects can depend on the availability of other metal ions and other nutrients
The impact of metal ions on culture process and protein quality attributes is responsive to the presence of other metal ions as well as other medium components. The final outcome of these interactions can be a result of synergistic or antagonistic activities. Zn2+ and Cu2+ are both known to influence basic variants in recombinant mAbs produced in CHO cells by influencing terminal lysine processing. Carboxypeptidases involved in C-terminal lysine processing require Zn2+ as a cofactor. Varying
Conclusion and future perspectives
Availability of trace metal ions can alter cellular metabolism and growth, and hence, their optimal supplementation is essential, especially in chemically defined medium formulations. Cataloguing the effects of trace metals on culture performance and product quality would help in root-cause analysis and to ensure consistency and reproducibility in manufacturing. However, factors such as the ability of some metal ions to interfere with the binding of native metal cofactors to enzymes and shared
Declaration of Competing Interest
The authors declare a patent application on zinc supplementation decreasing galactosylation of recombinant proteins. There is no other conflict of interest to declare.
Acknowledgements
AP acknowledges senior research fellowship from Council of Scientific & Industrial Research, India. MG acknowledges funding from Department of Biotechnology.
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