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. 2019 Aug 8;71(5):915–924. doi: 10.1007/s10616-019-00334-1

Zinc supplementation increases protein titer of recombinant CHO cells

Berta Capella Roca 1,2,✉, Antonio Alarcón Miguez 1, Joanne Keenan 1,2, Srinivas Suda 3, Niall Barron 2,3, Donal O’Gorman 1, Padraig Doolan 1,#, Martin Clynes 1,2,#
PMCID: PMC6787129  PMID: 31396753

Abstract

In order to study the impact of zinc and copper on the titer levels of mAb and recombinant protein in CHO cells, the IgG-expressing (DP12) and EPO-expressing (SK15) cell lines were cultured in chemically defined media with increasing concentrations of either metal. Supplementation with 25 mg/l in CDM media resulted in a significant increase in EPO (1.7-fold) and IgG (2.6-fold) titers compared to control (no added zinc). Titers at this Zn concentration in CDM containing the insulin replacing agent aurintricarboxylic acid (ATA) (CDM + A) showed a 1.8-fold (EPO) and 1.2-fold (IgG) titers increase compared to control. ATA appeared to also reduce the specific productivity (Qp) enhancement induced by Zn-25, with up to 4.9-fold (DP12) and 1.9-fold (SK15) Qp increase in CDM compared to the 1.6-fold (DP12) and 1.5-fold (SK15) Qp increase observed in CDM + A. A 31% reduced Viable Cell Density (VCD) in DP12 was observed in both Zn-supplemented media (3 × 106 cells/ml vs 4.2 × 106 cells/ml, day 5), whereas SK15 Zn-25 cultures displayed a 24% lower peak only in CDM + A (2.2 × 106 cells/ml vs 3.2 × 106 cells/ml, day 5). Supplementation with copper at 13.7–20 mg/l resulted in less significant cell line/product-type dependent effects on titer, VCD and Viability. Analysis of the energetic phenotype of both cell lines in 25 mg/l Zn-supplemented CDM media revealed a twofold increase in the oxygen consumption rate (OCR) compared to non-supplemented cells. Together, these data suggest that high zinc supplementation may induce an increase in oxidative respiration metabolism that results in increased Qp and titers in suspension CHO cultures.

Electronic supplementary material

The online version of this article (10.1007/s10616-019-00334-1) contains supplementary material, which is available to authorized users.

Keywords: Zinc, Copper, Titer, Chinese Hamster Ovary cells (CHO), Erythropoietin (EPO), IgG

Introduction

Enhancement of Chinese Hamster Ovary (CHO) culture performance profiles (titer, specific productivity (Qp), peak cell density, Viability) is a key goal of the biopharmaceutical sector. Industrial production of human therapeutics requires the use of serum-free formulations, mainly protein-free and chemically defined media, due to biosafety concerns (Gstraunthaler 2003) and to facilitate downstream processing. Consequently, in order to improve CHO performance in serum-free culture, a range of additives have been evaluated in basal serum-free formulations to optimise growth and productivity profiles.

Supplementations with chemical reagents such as sodium butyrate or valeric acid (Damiani et al. 2013; Park et al. 2016) have been extensively tested, offering a chemically-defined additive alternative for improving specific productivity of several therapeutically-relevant proteins in CHO cultures. However, the specific productivity effects observed with some of these chemicals (sodium butyrate, lithium chloride, valproic acid) have also been frequently associated with apoptosis (NaBu, reviewed by Kim et al. 2013) and/or low growth profiles (Park et al. 2016).

More recently, metal supplementation has shown substantial potential in improving CHO performance features in serum-free culture. For instance, supplementation with iron-citrate has been observed to increase mAb titer by 30–40% (Bai et al. 2011), while extended lifespan of CHO cultures and increased product titer have been reported following copper supplementation (Yuk et al. 2015; Luo et al. 2012). Additionally, manganese supplementation has been related to the modulation of glycoforms patterns of several recombinant products, including mAb (Grainger and James 2013). More recently, Kim and Park (2016) reported the titer-associated benefits of high zinc supplementation of a DG44 culture growing in an in-house and commercial media, with up to 6.5-fold increase in mAb titer observed (Kim and Park 2016).

In this study, we aimed to examine the effects of supplementation of a chemically-defined media with copper or zinc on the titer, VCD and Viability profiles of two CHO cell lines producing different products in serum-free suspension culture: an IgG-expressing (DP12) and an EPO-expressing CHO-K1 (SK15).

Materials and methods

In-house chemically-defined media development

As commercially prepared media products frequently do not disclose their exact components, two chemically-defined media (CDM + A and CDM) were developed based on an in-house serum-free media formulation: DMEM-F12 (D3487) supplemented with sodium selenite (S5261), recombinant insulin (I9279), ethanolamine (E0135), ammonium iron (III) citrate (F5879), poly vinyl alcohol, l-glutamine (Gibco (Dublin, Ireland), 25030024), NEAA (Gibco (Dublin, Ireland), 11140035) and putrescine dihydrochloride (P7505). For the development of chemically-defined and protein-free formulation, insulin was replaced with 30 mg/l aurintricarboxylic acid (ATA) (CDM + A media). Due to the chelating nature of ATA, which might mask the effects of the supplemented metals, ATA-removed media (CDM) was evaluated. All supplements were purchased from Sigma Aldrich (Wicklow, Ireland) unless otherwise stated.

Cell culture

Over the course of this study, two suspension producer CHO cell lines were used: CHO-DP12 (a recombinant human anti-IL-8 producer, ATCC CRL-12445 clone#1934) and SK15 (an in-house CHO-K1 (ATCC CCL-61) derived cell line, expressing recombinant human erythropoietin (EPO) in a pcDNA3.1 vector (Invitrogen) modified with puromycin resistance as selection system) (Costello et al. 2019b). Both cell lines were maintained in in-house protein-free chemically-defined media supplemented with increasing concentrations of zinc sulphate heptahydrate (1, 10, 15, 25 and 30 mg/l) or copper sulphate pentahydrate (1, 7.5, 13.7 and 20 mg/l) (added to the basal levels found in DMEM-F12: 0.432 mg/l Zn and 1.3 µg/l Cu). Cells were routinely split and re-seeded at 2 × 105 cells/ml in 5 ml working volume. At least 2 passages were allowed for adaptation before each test. DP12 and SK15 cells were pulsed every second passage with 200 nM MTX (Sigma, M8407) (DP12) or 10 µg/ml puromycin (Gibco, A11138-03) (SK15). Viable Cell Density (VCD) and Viability were analysed in triplicate using the ViaCount on a Guava easyCyte HT benchtop cytometer (Merck Millipore, UK).

Enzyme Linked Immunosorbent Assay (ELISA)

Enzyme Linked Immunosorbent Assay (ELISA) was performed in order to determine the levels of mAb and EPO. For mAb detection, the protocol described in the Human IgG ELISA Quantitation Set from Bethyl Laboratories Inc. (E80-104) was followed. For EPO detection, the protocol previously described by Costello et al. (2019a) was followed, including modifications on incubation times for both samples (1.5 h) and capture antibody (overnight). Statistical analysis of the average of each biological triplicate of ELISA data obtained was performed in Microsoft excel software using Fischer’s Exact Test to determine variance and the two-tailed T test tools to generate p-values. Cell specific productivity (Qp; pg protein/cell/day) was determined as per Clarke et al. (2011).

RNA isolation

RNA samples from SK15 and DP12 cultures in Zn-25 and CDM control media were collected at day 5 from 30 ml cultures in shake flasks. Between 1 − 5 × 106 cells were collected, centrifuged at 1000 rpm for 5 min and resuspended in 1 ml Trizol reagent (Thermo Scientific). RNA was extracted following the Trizol protocol as per manufacturer’s instructions. Quantity and quality of the extracted samples were analysed by Nanodrop (Thermo Scientific). To remove potential genomic DNA contamination, DNaseI treatment (Sigma Aldrich) was applied as per manufacturer’s protocol.

RT-qPCR

The High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was followed as per manufacturer’s protocol to generate cDNA from total RNA samples. cDNA was used for mRNA level quantification by qPCR using Fast SYBR Green Master Mix (Applied Biosystems) in a 7500 (Applied Biosystems). 2XSYBR master mix was prepared with 400 nM primers, 200 ng cDNA and nuclease-free water made up to 20 µl per reaction well. Relative quantification was measured by the delta delta Ct method with Gapdh as an endogenous control. Each biological replicate was measured in technical triplicate wells. The sequences of the primers used were as follows (5′→3′): IgG-LC Fwd-CATGTCCCGCTCACGTTT, IgG-LC Rev-CAGGCACACAACAGAAGCA (Beckmann et al. 2012); IgG-HC Fwd-ACGGTGTCGTGGAACTCAG, IgG-HC Rev-ACGCTGCTGAGGGAGTAGAG (Haredy et al. 2013); hEPO Fwd-GCATGTGGATAAAGCCGTCA, hEPO Rev-GCAGTGATTGTTCGGAGTGG; Gapdh Fwd-TGGCTACAGCAACAGAGTGG, Gapdh Rev-GTGAGGGAGATGATCGGTGT.

Energetic phenotype: oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

An Agilent Technologies XF96 Analyzer was used to analyse the metabolic potential of both cell lines. Suspension cells were immobilised prior to analysis with the XF96 Seahorse using the ”Immobilization of non-adherent cells with Cell-Tak for Assay on the Seahorse XF/XF96″ protocol (Agilent Technologies, Technical overview, Publication Part Number: 5991-7153EN, 2016). Some modifications were applied (as per Kelly et al. 2019): 20 µl Cell-Tak was used per well and plates were placed in a non-CO2 incubator at 37 °C for 1 h. Plates were then washed twice and air-dried for an hour at room temperature before cell plating. The test used was the Agilent Seahorse XFp Cell Energy Phenotype Test Kit (Agilent Technologies, 103275-100) with a final seeding density of 20,000 cells/well and a final concentration of Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and oligomycin of 1 µM, as per manufacturer’s instructions. Basal (“Baseline levels”) levels of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) were first measured under standard conditions (no inhibitors supplemented). The cells were then “Stressed” following injection of oligomycin (ATP synthase inhibitor) and FCCP (potent uncoupler of mitochondrial oxidative phosphorylation). This supplementation results in the maximum cellular respiration state (maximum Electron Transport System) (“Stressed levels”) which corresponds to the OCR and ECAR levels of the cells attempting to restore the proton gradient loss from the mitochondrial inhibitors supplemented. Consequently, by the continuous monitoring of oxygen concentration changes in the media (OCR) and pH (ECAR), the XF96 Seahorse instrument allows direct quantification of the mitochondrial respiration and glycolysis of the cells (Plitzko and Loesgen 2018).

In order to avoid interference by dead cells, SK15 and DP12 cells were analysed at day 4. A total of 2 biological replicates for each cell line, with analysis of 11–21 wells/condition per replicate, were performed.

Results and discussion

Zinc supplementation of CDM + A results in enhanced IgG titer and Qp but lower peak VCD in CHO

Significantly enhanced EPO and IgG titer profiles were observed at Zn-25 media (Fig. 1), displaying a 40–50% increase in IgG titer (by day 2–4; Fig. 1b) and a 1.8-fold increase in EPO titer (by day 8; Fig. 1a) compared to the non-supplemented CDM + A control. No significant effects on titer were observed with any of the lower zinc supplementations at later stages of culture.

Fig. 1.

Fig. 1

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Titer (a, b), Viable Cell Density (VCD) (c, d) and Viability (e, f) of SK15 (a, c, e) and DP12 (b, d, f) cells grown in suspension in in-house chemically-defined medium CDM + A supplemented with zinc at: 1 mg/l (Zn-1), 10 mg/l (Zn-10), 15 mg/l (Zn-15) and 25 mg/l (Zn-25). Statistical differences of titer data compared to the control (CDM + A) are represented as: p-value < 0.05 (*)

Results for Qp are presented in Table 1. A 1.5-fold (SK15) and 1.6-fold (DP12) significant increase in Qp was observed at Zn-25 compared to control media (Table 1). No positive effects were displayed at lower zinc concentrations.

Table 1.

Specific productivity (Qp) (pg/cell/day) of SK15 and DP12 cell lines (day 0–day 4) in CDM + A and CDM supplemented with zinc at: 0 mg/l (Ctl), 1 mg/l (Zn-1), 10 mg/l (Zn-10), 15 mg/l (Zn-15), 25 mg/l (Zn-25) and 30 mg/l (Zn-30)

SK15 DP12
CDM + A
 Ctl 1.7 ± 0.5 3.5 ± 0.4
 Zn-1 1.1 ± 0.4 3.1 ± 0.6
 Zn-10 1.3 ± 0.3 3.6 ± 0.6
 Zn-15 1.5 ± 0.3 3.6 ± 0.4
 Zn-25 2.4 ± 1.6 5.8 ± 0.4**
CDM
 Ctl 3.5 ± 0.4 2.2 ± 0.6
 Zn-1 6.1 ± 0.2 1.6 ± 0.7
 Zn-10 8.1 ± 1.9 6 ± 0.5***
 Zn-25 11.4 ± 1* 10.4 ± 2.7**
 Zn-30 11.7 ± 2* 16 ± 7.4**
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Statistical differences of titer data compared to the respective control are represented as: p-value < 0.05 (*), < 0.01 (**), < 0.001 (***)

Decreases in peak VCD in both cell lines were observed at 25 mg/l zinc supplementation; a 31% drop in SK15 and a 24% drop in DP12 at day 5 (Fig. 1c, d). DP12 VCD was also negatively affected in Zn-15 media, with a 12% lower peak VCD observed (Fig. 1d). Supplementation at lower zinc concentrations did not display any effect on VCD in either cell line. Viability profiles were observed to be similar between supplemented and control CDM + A media at all concentrations (Fig. 1e, f).

Copper increases EPO titer in SK15 cells

Compared to the CDM + A control, supplementation with copper resulted in different effects in the two CHO cell lines; with EPO titers increased by 80–90% (at 13.7 mg/l and 20 mg/l Cu, respectively) and 65% (at 1 mg/l and 7.5 mg/l Cu) media, while IgG final titers decreased by 20% following copper supplementation at all concentrations (Supplementary Figure 1a, b).

Copper-supplemented SK15 cultures displayed similar VCD and Viability profiles to non-supplemented control CDM + A media for all concentrations; with peak VCD of 3–3.2 × 106 cells/ml and Viabilities above 91% until day 5 observed (Supplementary Figure 1c, e). Similar to the results obtained for titer profiles (Supplementary Figure 1a, b), DP12 cells were observed to be negatively affected, with peak VCD drops of 2–24% displayed as the concentrations of copper increased (Supplementary Figure 1d). While similar Viability profiles (to the CDM + A control) were observed with supplementations up to 7.5 mg/l Cu, concentrations above 13.7 mg/l resulted in detrimental effects, with a 25% drop in DP12 Viability from day 4 to day 6 (Supplementary Figure 1f).

Previous studies have reported enhanced titer and VCD profiles following copper supplementation in serum-free CHO culture (Yuk et al. 2015, Xu et al. 2016). However, similar to our observations, cell line-dependant outcomes have also been reported by Luo et al. (2012), with increased VCD and mAb titers in two (of three) DUXKB11 CHO suspension cell lines following high copper supplementation in a proprietary CDM, but zero effect on either phenotype in the 3rd subline studied. Additionally, previous studies supplementing with copper concentrations equivalent to the higher levels tested here reported induced DNA damage and reduced Viabilities of CHO-K1 parental cells in a cytotoxic study performed in serum-supplemented media (Grillo et al. 2010), which may explain the decreased Viabilities observed in the DP12 results presented here (Supplementary Figure 1f).

Removal of ATA from CDM + A further increases zinc-induced enhancement of titer and Qp

Similar to zinc, ATA has previously been used as an insulin-replacement additive in the development of PFM for CHO cells (Miki and Takagi 2015) and its molecular structure provides strong metal chelating ability (Kumar Sharma et al. 2000), acting by forming a coat on the cell surface interacting with IGF-1R (Beery et al. 2001). To avoid possible interference by chelating activity, a medium without ATA (CDM) was formulated and used in order to identify (i) whether addition of zinc by itself could replace the growth-stimulatory effects of ATA while simultaneously improving titer and (ii) if ATA was masking the maximum positive effects of zinc on product titer due to chelating interaction. Moreover, an additional 30 mg/l Zn concentration was also included to evaluate the potential for further titer enhancement at higher concentrations.

Removal of ATA resulted in enhanced EPO and IgG titer profiles following Zn supplementation in the concentration range of 10–30 mg/l; displaying a maximal increase of 1.7-fold EPO titer (in Zn-25) and 3.9-fold IgG titer (in Zn-30) compared to the non-supplemented control (Fig. 2a, b). Final IgG yield was also increased by 2.6-fold with 25 mg/l zinc supplementation (vs. 7.1 mg/l in CDM control) (Fig. 2b).

Fig. 2.

Fig. 2

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Titer (a, b) Viable Cell Density (VCD) (c, d) and Viability (e, f) of SK15 (a, c, e) and DP12 (b, d, f) cells grown in suspension in in-house chemically-defined medium CDM supplemented with zinc at: 1 mg/l (Zn-1), 10 mg/l (Zn-10), 25 mg/l (Zn-25) and 30 mg/l (Zn-30). Statistical differences of titer data compared to the control (CDM) are represented as: p-value < 0.05 (*), < 0.01 (**), < 0.001 (***)

Specific productivity of both CHO cell lines increased as the concentration of zinc increased, reaching up to 11.4 pg EPO/cell/day (SK15) and 16 pg IgG/cell/day (DP12) at Zn-30 (Table 1). Supplementation with 25 mg/l zinc resulted in a 1.9-fold (SK15) and 4.8-fold (DP12) increase in Qp compared to non-supplemented media.

VCD and Viability results for each Zn concentration tested are displayed in Fig. 2c–f. No effects on SK15 VCD (relative to CDM control) were observed at 25 mg/l zinc supplementation (Fig. 2c). For DP12, negative effects on maximal VCD were observed in Zn-25, causing a drop of 29% in peak VCD (Fig. 2d). At this zinc concentration, both cell lines displayed lower Viability profiles compared to the non-supplemented CDM control media (90–83% vs. 94–88%; Fig. 2e, f).

Interestingly, the beneficial effects of zinc supplementation on titer were also observed at the transcriptional level, with a 25.6-fold (heavy chain) and 4.3-fold (light chain) increase in IgG mRNA levels in DP12 cells and a 1.8-fold increase in hEPO mRNA levels in SK15 cells, following supplementation at 25 mg/l (Fig. 3). This result is in accordance with the IgG expression cassette used for the development of DP12 cell line, which enhances IgG heavy chain expression relative to light chain as a result of MTX selection and amplification, since the DHFR and heavy chain sequences share the same promoter (Gonzalez et al. 2000).

Fig. 3.

Fig. 3

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Relative quantification (RQ) of a heavy and light chain of IgG (anti-IL-8) antibody in DP12 cells and b hEPO mRNA levels in SK15 cells in CDM Zn25 media normalized to the mRNA levels of the cells in CDM control media. The Gapdh endogenous gene was used to standardize the results. RNA samples analysed were collected at day 5 from 30 ml cultures

Only a few studies have reported beneficial effects of high zinc supplementation on enhancing recombinant protein production in CHO. Zuquelis et al. (2006) observed an eightfold increase of IFN-β1a titer following supplementation with 150 µM zinc and a lower (twofold) increase at 25–50 µM Zn (both in adherent CHO-K1 cultures grown in 0.5% FBS) (Zuqueli et al. 2006). More recently, zinc supplementation at 30–60 µM in PFM and CDM in-house formulations increased mAb titer by a maximum of 6.5-fold and peak VCD by 1.2-fold in DG44 suspension cultures (Kim and Park 2016). Moreover, Kim and Park (2016) also reported enhanced mAb titer at 90 µM Zn supplementation in CDM, which was associated with a lower VCD peak, a finding which is similar to the results presented here at Zn-25 (equivalent to 86.93 µM Zn). However, supplementation with zinc concentrations above 100 µM in CHO suspension cultures has been shown to impact final mAb quality (reduction in galactosylation patterns), although the effects can be reversed by addition of manganese (Prabhu et al. 2018).

From the results displayed here, strategies involving supplementation of 25 mg/l zinc on commercial media may be considered as credible approaches focused on increasing titer in suspension CHO cultures utilising commercial media. However, it is important to note that the different additives present in each formulations may influence the positive effects observed with zinc (as was observed here when zinc was co-supplemented in the presence of ATA (Fig. 1a, b)). Interestingly, Kim and Park (2016) have shown increases of 1.2- to 1.5-fold in mAb titer with zinc supplementation to a range of three commercial media (Power CHO-2CD (Lonza), CDM4CHO (Hyclone) and EXCELL CD CHO (SAFC Bioscience)), although the concentrations used (60 µM, 17.25 mg/l) were lower than the optimal concentration described here. Consequently, due to the lack of disclosure on composition of commercial media formulations, it may be necessary to deploy several zinc concentration and supplementation strategies to achieve titer enhancement in other culture systems.

Zn-25 increases the oxidative respiration of DP12 and SK15 cells

Zinc is involved in the folding, stability and/or activity of hundreds of proteins, being essential for several cellular functions such as DNA and RNA synthesis, mRNA stability and protection against apoptosis. Moreover, it also participates in the activation of glutathione and antioxidant enzymes such as, superoxide dismutase and catalase, hence protecting against ROS species produced during cellular respiration (Kloubert and Rink 2015). However, little is known about its function as a possible additive for improving production of therapeutic proteins.

Batch cultures typically display a stationary phase where growth slows and a production profile is observed, correlated with a switch to oxidative respiration (Dickson 2014). A maximum induction of Oxygen Consumption Rate (OCR: indicator of mitochondrial respiration) was observed following supplementation at Zn-25, displaying a substantial 1.8-fold (SK15) and 2.1-fold (DP12) increased oxygen consumption compared to the CDM non-supplemented media (Fig. 4a, b). OCR levels were also affected at lower zinc concentrations, although to a lesser degree; with an increase of 1.2-fold in Zn-10 supplemented SK15 and a 1.3-fold (Zn-1)—1.4-fold (Zn-10) increase in DP12. Cellular Extracellular Acidification Rate (ECAR: indicator of glycolysis) levels were not substantially affected following Zn supplementation under stressed conditions (Fig. 4c, d).

Fig. 4.

Fig. 4

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Oxygen Consumption Rate (OCR) (a, b) and Extracellular Acidification Rate (ECAR) (c, d) of SK15 (a, c) and DP12 (b, d) cells in CDM supplemented with zinc at: 0 mg/l (CDM), 1 mg/l (Zn-1), 10 mg/l (Zn-10) and 25 mg/l (Zn-25) at day 4. “Baseline” indicates OCR and ECAR levels measured under normal growing conditions; “Stressed” indicates OCR and ECAR levels measured following supplementation of FCCP and oligomycin, which induces maximum cellular respiration state (maximum Electron Transport System) of the cells by blocking mitochondrial function. Consecutively the mitochondrial respiration and glycolysis of the cells are quantified by the changes on the oxygen consumption and pH in both Baseline and Stressed levels. OCR are indicated as pmol Oxygen consumed per min whereas ECAR is indicates as mpH/min

Disrupted homeostasis by high zinc concentrations results in the sequestration by metallothionein or internalization into organelles, including mitochondria (Qiping et al. 2016). Zinc has been observed to stimulate oxidative phosphorylation and the electron transport chain (ETC) in rat hepatic mitochondria (Masayoshi et al. 1982). Increased ATP production and mitochondrial biogenesis has been also displayed in melanocytes after zinc supplementation (Rudolf and Rudolf 2017). However, divergent observations have been also reported in rat neurons and prostate epithelial cells, with reduced mitochondrial energy production observed following Zn supplemented conditions (Dineley et al. 2005; Dakubo et al. 2006), which might indicate possible tissue-specific effects. While the role of zinc in the regulation of energy metabolism in suspension CHO cells is still unclear, the results presented here suggest that zinc supplementation strategies at stationary phases on the cultures might be suitable for enhancing CHO final titers due to the increased OCR levels and titers displayed.

Conclusion

We have found that supplementation of protein-free media with zinc at 25 mg/l (86.93 µM) resulted in a significant increase of both recombinant EPO and IgG titers in two CHO cell lines; SK15 and DP12. Although lower peak VCD was also displayed following supplementation, Viabilities were maintained above 80% throughout. Increased oxidative respiration was also observed to correlate with the increased titer profiles in both cell lines. Together, these data indicate that zinc supplementation strategies may be a viable mechanism for increasing specific productivity in CHO cell lines.

Electronic supplementary material

Below is the link to the electronic supplementary material.

10616_2019_334_MOESM1_ESM.docx (42.5KB, docx)

Supplementary material 1 (DOCX 42 kb). Supplementary Fig. 1. Titer (a, b), VCD (c, d) and Viability (e, f) of SK15 (a, c, e) and DP12 (b, d, f) cells grown in suspension in in-house chemically-defined medium CDM + A supplemented with copper at: 1 mg/l (Cu-1), 7.5 mg/l (Cu-7.5), 13.7 mg/l (Cu-13.7) and 20 mg/l (Cu-20). Statistical differences in titer data compared to the control (CDM + A) are represented as: p-value < 0.05 (*) and < 0.01 (**)

Acknowledgements

This work was conducted with the financial support of Scientific Foundation of Ireland (SFI) Grant No. [13/IA/1841] and Science Foundation Ireland co-funded by ERDF, Grant No. [12/RC/2275_P2].

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Padraig Doolan and Martin Clynes contributed equally to this work.

Contributor Information

Berta Capella Roca, Phone: +353-1-7005700/5624, Email: berta.capellaroca2@mail.dcu.ie.

Antonio Alarcón Miguez, Email: antonio.alarconmiguez2@mail.dcu.ie.

Joanne Keenan, Email: joanne.keenan@dcu.ie.

Srinivas Suda, Email: srinivas.suda@nibrt.ie.

Niall Barron, Email: niall.barron@nibrt.ie.

Donal O’Gorman, Email: donal.ogorman@dcu.ie.

Padraig Doolan, Email: padraig.doolan@dcu.ie.

Martin Clynes, Email: martin.clynes@dcu.ie.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

10616_2019_334_MOESM1_ESM.docx (42.5KB, docx)

Supplementary material 1 (DOCX 42 kb). Supplementary Fig. 1. Titer (a, b), VCD (c, d) and Viability (e, f) of SK15 (a, c, e) and DP12 (b, d, f) cells grown in suspension in in-house chemically-defined medium CDM + A supplemented with copper at: 1 mg/l (Cu-1), 7.5 mg/l (Cu-7.5), 13.7 mg/l (Cu-13.7) and 20 mg/l (Cu-20). Statistical differences in titer data compared to the control (CDM + A) are represented as: p-value < 0.05 (*) and < 0.01 (**)

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