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Dynamic profiling of amino acid transport and metabolism in Chinese hamster ovary cell culture
BMC Proceedings volume 7, Article number: P97 (2013)
Chinese Hamster Ovary (CHO) cells are the most widely used industrial hosts for the production of recombinant DNA technology drugs . In such processes amino acids (a.a.) are vital nutrients for growth, but also building blocks of the recombinant protein (rprotein). Our research aims to establish a better understanding of a.a. transport in and out of cells, since this could have significant impact on increasing productivity and designing feeding strategies during bioprocessing.
There are about 46 a.a. transporter proteins in mammalian cells, the genes of which are presented in Table 1 along with their substrates and all are members of the Solute Carriers (SLC) database . A.a. transporters are subject to different expression profiles among mammalian cells and are grouped into more than 18 systems, based on sequence homology and function.
To our knowledge, there is no comprehensive study of a.a. transporters in industrially relevant CHO cells in the literature. To that direction, a.a. transporter genes were profiled during batch culture of three CHO cell lines with varying levels of productivity. In parallel, the intra- and extracellular levels of a.a. were quantified.
Materials and methods
Three cell lines were kindly donated by Lonza Biologics. GSn8 cell line was transfected with an empty glutamine synthetase (GS) vector. GS35 and GS46 cell lines were both transfected with a GS vector that also carries the heavy and light chains of a chimeric IgG4 antibody. The specific productivity of cell line GS46, quantified by a commercial ELISA kit (Bethyl laboratories, US), is approximately double that of GS35 one.
Batch cultures were performed in triplicate in 1L Erlenmeyer flasks with a working volume of 300mL in CD-CHO medium (Invitrogen, UK) supplemented with 25 μM MSX (Sigma, UK). Viable cell concentration was determined daily using the trypan blue dye exclusion method.
40 a.a. transporters were studied in all cell lines using real time quantitative reverse transcription polymerase chain reaction on samples from different phases of batch culture. Samples were collected at day 4 (exponential phase) and day 6 & day 7 (stationary phase) of the growth curve for all cell lines (samples were also taken at day 3 for IgG4 producers only and day 9 for the null cell line only). Results are reported against the housekeeping gene "actb". Housekeeping genes "vezt" and "hirip3" were also well correlated.
The extracellular and intracellular a.a. profiles were monitored daily using high performance liquid chromatography (PicoTag, Waters, UK). Intracellular samples were quenched with 0.9% w/v NaCl and extracted with a 50% aqueous acetonitrile solution, as described in .
The results (Table 1) reveal that ~30% of transporters are lowly expressed (fractional copies per cell), 9% are below levels of detection, whereas 40% are significantly differentially expressed either during batch cell culture, or between cell lines, or both. The remaining transporters appear to remain stable.
Regulation within culture
The majority of the transporters are found to be upregulated at stationary phase for all cell lines, as also presented in Figure 1, where a mapping of a.a. metabolism and transport has been illustrated for the null cell line. Specifically, five genes encoding for transporters of a.a. relating to the glutathione (GSH) pathway were found to be upregulated significantly higher than 2 fold at stationary phase, when compared to exponential phase for all cell lines. These genes were: slc1a4 (Ala and Cys), slc6a9 (Gly), slc1a2 (Glu and Asp), slc7a11 (Cystine and Glu), and heteromeric transporter slc3a2 which partners with slc7a11. GSH is a well-known marker of oxidative stress , high levels of which have been associated with high productivity .
Regulation between cell lines
In their majority, genes were found to be upregulated for protein producing cell lines at all time points. Genes whose expression is upregulated significantly (two-fold or higher) in the protein-producers at all time points analyzed were: slc43a2 (system L, leucine and branched-chain a.a.) and slc1a2 (system X-AG, glutamate and aspartate). However, no genes, apart from slc6a6 (taurine and b-Ala), were found to be differentially expressed between high (GS46) and low producer (GS35). We find slc6a6 gene differentially expressed early in cell culture (day 3), which makes us hypothesize that the gene could be a candidate for selection purposes. The overexpression of this gene in CHO cells has been found to significantly enhance growth and productivity .
Feeding strategy based on order of feeding
The a.a. transporters gene expression findings correlate well with the extracellular and intracellular concentration profiles of their respective substrates (Figure 1). By analysing the differentially expressed genes for a specific cell line a feeding strategy can be designed. For example, we find transporter slc7a5, of system L, highly upregulated at stationary phase for the null cell line (Figure 1). This transporter exchanges an intracellular neutral a.a. with an extracellular branched chain one (isoleucine, leucine, valine). Branched chain amino acids are associated with the mTor signalling pathway, essential regulator for many physiological roles in mammalian cells . Hence, a feeding strategy can be proposed, where neutral amino acids are fed first and followed by branched chain amino acids, in order for them to be more effectively uptaken. A similar type of pre-conditioning was found to significantly enhance cellular protein production in another type of mammalian cells .
Glutathione pathway associated a.a. transporters (slc1a2, slc1a4, slc6a9, slc7a11/ slc3a2) can be targeted as genetic engineering targets, since are all found highly upregulated at stationary phase of cell culture. Additionally, transporters slc1a2, slc43a2 are associated with rprotein productivity, since all of them are found to be upregulated for producing cell lines vs the null. Gene slc6a6, carrying taurine and β-alanine, can be associated with high productivity (as also suggested in ), as was also found to be differentially expressed in the high vs the low producer early in cell culture. A feeding strategy can be proposed, based on our results that remains to be tested experimentally. Finally, extending this integrative approach to the proteome level would help link regulation at the transcriptomic level to actual differences in transport capability.
Kyriakopoulos S, Kontoravdi C: Analysis of the landscape of biologically-derived pharmaceuticals in Europe: Dominant production systems, molecule types on the rise and approval trends. European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences. 2012, 48: 428-441.
Hediger MA, Romero MF, Peng JB, Rolfs A, Takanaga H, Bruford EA: The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteins - Introduction. Pflug Arch Eur J Phy. 2004, 447: 465-468.
Dietmair S, Timmins NE, Gray PP, Nielsen LK, Kromer JO: Towards quantitative metabolomics of mammalian cells: Development of a metabolite extraction protocol. Anal Biochem. 2010, 404: 155-164.
Selvarasu S, Ho YS, Chong WPK, Wong NSC, Yusufi FNK, Lee YY, Yap MGS, Lee DY: Combined in silico modeling and metabolomics analysis to characterize fed-batch CHO cell culture. Biotechnol Bioeng. 2012, 109: 1415-1429.
Chong WP, Thng SH, Hiu AP, Lee DY, Chan EC, Ho YS: LC-MS-based metabolic characterization of high monoclonal antibody-producing Chinese hamster ovary cells. Biotechnol Bioeng. 2012, 109: 3103-3111.
Tabuchi H, Sugiyama T, Tanaka S, Tainaka S: Overexpression of Taurine Transporter in Chinese Hamster Ovary Cells Can Enhance Cell Viability and Product Yield, While Promoting Glutamine Consumption. Biotechnol Bioeng. 2010, 107: 998-1003.
Nicklin P, Bergman P, Zhang BL, Triantafellow E, Wang H, Nyfeler B, Yang HD, Hild M, Kung C, Wilson C, et al: Bidirectional Transport of Amino Acids Regulates mTOR and Autophagy. Cell. 2009, 136: 521-534.
S.K. would like to thank EPSRC & iChemE for financial support. K.P. would like to thank RCUK and C.K. thanks RCUK & Lonza Biologics for their Fellowships.
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Kyriakopoulos, S., Polizzi, K.M. & Kontoravdi, C. Dynamic profiling of amino acid transport and metabolism in Chinese hamster ovary cell culture. BMC Proc 7, P97 (2013). https://doi.org/10.1186/1753-6561-7-S6-P97
- Glutamine Synthetase
- Chinese Hamster Ovary Cell
- Branch Chain Amino Acid
- Chinese Hamster Ovary Cell Line