Skip to main content

Advertisement

SpringerLink
Log in

Clonal variations in CHO IGF signaling investigated by SILAC-based phosphoproteomics and LFQ-MS

  • Genomics, transcriptomics, proteomics
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Chinese hamster ovary (CHO) cells are commonly used for the production of monoclonal antibodies. Omics technologies have been used to elucidate cellular switch points which result in higher monoclonal antibody (mAb) productivity and process yields in CHO and other biopharmaceutical production cell lines such as human or mouse. Currently, investigations of the phosphoproteome in CHO cell lines are rare yet could provide further insights into cellular mechanisms related to target product expression. Therefore, we investigated CHO IGF–signaling events using a comparative expression and phosphoproteomic approach in recombinant mAb-producing XL99 cell lines and corresponding parental strain. Differences were found on the level of protein expression between producer and parental cells in the exponential growth phase, mainly in proteins related to the lysosome, oligosaccharide metabolic processes, stress response, and cellular homeostasis. Within a stable isotope labeling by amino acids in cell culture (SILAC)–based phosphoproteomic investigation of IGF signaling, expected general regulation of phosphorylation sites and cell line–specific responses were observed. Detected early phosphorylation events can be associated to observed effects of IGF on cellular growth, metabolism, and cell cycle distribution. Producer cell line–specific signaling exhibited differences to parental cells in intracellular trafficking and transcriptional processes, along with an overall lower amount of observable cross talk to other signaling pathways. By combining label-free and SILAC-based expression for phosphoproteomic analyses, cellular differences in the highly interactive levels of signaling and protein expression were detected, indicating alterations in metabolism and growth following treatment with an exogenous growth factor. The characterization of cell lines and effects of IGF addition resulted in identification of metabolic switch points. With this data, it will be possible to modulate pathways towards increased CHO process yield by targeted application of small-molecule inhibitors.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Baillat D, Hakimi M-A, Näär AM, Shilatifard A, Cooch N, Shiekhattar R (2005) Integrator, a multiprotein mediator of small nuclear RNA processing, associates with the C-terminal repeat of RNA polymerase II. Cell 123:265–276

  • Barnes LM, Bentley CM, Dickson AJ (2003) Stability of protein production from recombinant mammalian cells. Biotechnol Bioeng 81:631–639

    Article  CAS  PubMed  Google Scholar 

  • Baycin-Hizal D, Tabb DL, Chaerkady R, Chen L, Lewis NE, Nagarajan H, Sarkaria V, Kumar A, Wolozny D, Colao J, Jacobson E, Tian Y, O’Meally RN, Krag SS, Cole RN, Palsson BO, Zhang H, Betenbaugh M (2012) Proteomic analysis of Chinese hamster ovary cells. J Proteome Res 11:5265–5276

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Blue RE, Curry EG, Engels NM, Lee EY, Giudice J (2018) How alternative splicing affects membrane-trafficking dynamics. J Cell Sci 131:1–13

    Article  CAS  Google Scholar 

  • Brink BG, Seidel A, Kleinbölting N, Nattkemper TW, Albaum SP (2016) Omics fusion - a platform for integrative analysis of omics data. J Integr Bioinform 13:296

    Article  PubMed  Google Scholar 

  • Cairns RA, Harris IS, Mak TW (2011) Regulation of cancer cell metabolism. Nat Rev Cancer 11:85–95

    Article  CAS  PubMed  Google Scholar 

  • Carlage T, Kshirsagar R, Zang L, Janakiraman V, Hincapie M, Lyubarskaya Y, Weiskopf A, Hancock WS (2012) Analysis of dynamic changes in the proteome of a Bcl-XL overexpressing Chinese hamster ovary cell culture during exponential and stationary phases. Biotechnol Prog 28:814–823

    Article  CAS  PubMed  Google Scholar 

  • Clippinger AJ, Alwine JC (2012) Dynein mediates the localization and activation of mTOR in normal and human cytomegalovirus-infected cells. Genes Dev 26:2015–2026

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dahodwala H, Nowey M, Mitina T, Sharfstein ST (2012) Effects of clonal variation on growth, metabolism, and productivity in response to trophic factor stimulation. A study of Chinese hamster ovary cells producing a recombinant monoclonal antibody. Cytotechnology 64:27–41

    Article  CAS  PubMed  Google Scholar 

  • Davies SL, Lovelady CS, Grainger RK, Racher AJ, Young RJ, James DC (2013) Functional heterogeneity and heritability in CHO cell populations. Biotechnol Bioeng 110:260–274

    Article  CAS  PubMed  Google Scholar 

  • Dinnis DM, James DC (2005) Engineering mammalian cell factories for improved recombinant monoclonal antibody production. Lessons from nature? Biotechnol Bioeng 91:180–189

    Article  CAS  PubMed  Google Scholar 

  • Dorai H (2013) Proteomic analysis of bioreactor cultures of an antibody expressing CHOGS cell line that promotes high productivity. J Proteomics Bioinform 06

  • Dorai H, Kyung YS, Ellis D, Kinney C, Lin C, Jan D, Moore G, Betenbaugh MJ (2009) Expression of anti-apoptosis genes alters lactate metabolism of Chinese hamster ovary cells in culture. Biotechnol Bioeng 103:592–608

    Article  CAS  PubMed  Google Scholar 

  • Dreesen IAJ, Fussenegger M (2011) Ectopic expression of human mTOR increases viability, robustness, cell size, proliferation, and antibody production of Chinese hamster ovary cells. Biotechnol Bioeng 108:853–866

    Article  CAS  PubMed  Google Scholar 

  • Fan L, Frye CC, Racher AJ (2013) The use of glutamine synthetase as a selection marker. Recent advances in Chinese hamster ovary cell line generation processes. Pharm Bioprocess 1:487–502

    Article  Google Scholar 

  • Feichtinger J, Hernández I, Fischer C, Hanscho M, Auer N, Hackl M, Jadhav V, Baumann M, Krempl PM, Schmidl C, Farlik M, Schuster M, Merkel A, Sommer A, Heath S, Rico D, Bock C, Thallinger GG, Borth N (2016) Comprehensive genome and epigenome characterization of CHO cells in response to evolutionary pressures and over time. Biotechnol Bioeng 113:2241–2253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Funahashi A, Morohashi M, Kitano H, Tanimura N (2003) CellDesigner. A process diagram editor for gene-regulatory and biochemical networks. BIOSILICO 1:159–162

    Article  Google Scholar 

  • Geetha T, Langlais P, Caruso M, Yi Z (2012) Protein phosphatase 1 regulatory subunit 12A and catalytic subunit δ, new members in the phosphatidylinositide 3 kinase insulin-signaling pathway. J Endocrinol 214:437–443

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gullerova M, Barta A, Lorkovic ZJ (2006) AtCyp59 is a multidomain cyclophilin from Arabidopsis thaliana that interacts with SR proteins and the C-terminal domain of the RNA polymerase II. RNA 12:631–643

  • Hartl FU (2016) Cellular homeostasis and aging. Annu Rev Biochem 85:1–4

    Article  CAS  PubMed  Google Scholar 

  • Hartley F, Walker T, Chung V, Morten K (2018) Mechanisms driving the lactate switch in Chinese hamster ovary cells. Biotechnol Bioeng 115:1890–1903

    Article  CAS  PubMed  Google Scholar 

  • Heffner KM, Hizal DB, Yerganian GS, Kumar A, Can Ö, O’Meally R, Cole R, Chaerkady R, Wu H, Bowen MA, Betenbaugh MJ (2017) Lessons from the hamster. Cricetulus griseus tissue and CHO cell line proteome comparison. J Proteome Res 16:3672–3687

    Article  CAS  PubMed  Google Scholar 

  • Henry M, Coleman O, Prashant CM, Meleady P (2017a) Phosphopeptide enrichment and LC-MS/MS analysis to study the phosphoproteome of recombinant Chinese hamster ovary cells. Methods Mol Biol 1603:195–208

    Article  CAS  PubMed  Google Scholar 

  • Henry M, Power M, Kaushik P, Coleman O, Clynes M, Meleady P (2017b) Differential phosphoproteomic analysis of recombinant Chinese hamster ovary cells following temperature shift. J Proteome Res 16:2339–2358

    Article  CAS  PubMed  Google Scholar 

  • Huang DW, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44–57

    Article  CAS  Google Scholar 

  • Humphrey SJ, Azimifar SB, Mann M (2015) High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics. Nat Biotechnol 33:990–995

    Article  CAS  PubMed  Google Scholar 

  • Jardon MA, Sattha B, Braasch K, Leung AO, Côté HCF, Butler M, Gorski SM, Piret JM (2012) Inhibition of glutamine-dependent autophagy increases t-PA production in CHO cell fed-batch processes. Biotechnol Bioeng 109:1228–1238

    Article  CAS  PubMed  Google Scholar 

  • Kanehisa M, Sato Y, Morishima K (2016) BlastKOALA and GhostKOALA. KEGG tools for functional characterization of genome and metagenome sequences. J Mol Biol 428:726–731

    Article  CAS  PubMed  Google Scholar 

  • Kaushik P, Henry M, Clynes M, Meleady P (2018) The expression pattern of the phosphoproteome is significantly changed during the growth phases of recombinant CHO cell culture. Biotechnol J:e1700221

  • Kim YJ, Baek E, Lee JS, Lee GM (2013) Autophagy and its implication in Chinese hamster ovary cell culture. Biotechnol Lett 35:1753–1763

    Article  CAS  PubMed  Google Scholar 

  • Korrodi-Gregório L, Ferreira M, Vintém AP, Wu W, Muller T, Marcus K, Vijayaraghavan S, Brautigan DL, da Cruz E Silva OAB, Fardilha M, da Cruz E Silva EF (2013) Identification and characterization of two distinct PPP1R2 isoforms in human spermatozoa. BMC Cell Biol 14:15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kumar N, Gammell P, Meleady P, Henry M, Clynes M (2008) Differential protein expression following low temperature culture of suspension CHO-K1 cells. BMC Biotechnol 8:42

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee JS, Lee GM (2012) Rapamycin treatment inhibits CHO cell death in a serum-free suspension culture by autophagy induction. Biotechnol Bioeng 109:3093–3102

    Article  CAS  PubMed  Google Scholar 

  • Lee D-F, Kuo H-P, Chen C-T, Hsu J-M, Chou C-K, Wei Y, Sun H-L, Li L-Y, Ping B, Huang W-C, He X, Hung J-Y, Lai C-C, Ding Q, Su J-L, Yang J-Y, Sahin AA, Hortobagyi GN, Tsai F-J, Tsai C-H, Hung M-C (2007) IKK beta suppression of TSC1 links inflammation and tumor angiogenesis via the mTOR pathway. Cell 130:440–455

    Article  CAS  PubMed  Google Scholar 

  • Li X, Jiang Y, Meisenhelder J, Yang W, Hawke DH, Zheng Y, Xia Y, Aldape K, He J, Hunter T, Wang L, Lu Z (2016) Mitochondria-translocated PGK1 functions as a protein kinase to coordinate glycolysis and the TCA cycle in tumorigenesis. Mol Cell 61:705–719

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu Z, Dai S, Bones J, Ray S, Cha S, Karger BL, Li JJ, Wilson L, Hinckle G, Rossomando A (2015) A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells. Biotechnol Prog 31:1026–1038

    Article  CAS  PubMed  Google Scholar 

  • Maldonado-Agurto R, Dickson AJ (2018) Multiplexed digital mRNA expression analysis profiles system-wide changes in mRNA abundance and responsiveness of UPR-specific gene expression changes during batch culture of recombinant Chinese hamster ovary cells. Biotechnol J 13:e1700429

    Article  CAS  PubMed  Google Scholar 

  • McIlwain CC, Townsend DM, Tew KD (2006) Glutathione S-transferase polymorphisms. Cancer incidence and therapy. Oncogene 25:1639–1648

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Meleady P, Gallagher M, Clarke C, Henry M, Sanchez N, Barron N, Clynes M (2012a) Impact of miR-7 over-expression on the proteome of Chinese hamster ovary cells. J Biotechnol 160:251–262

    Article  CAS  PubMed  Google Scholar 

  • Meleady P, Hoffrogge R, Henry M, Rupp O, Bort JH, Clarke C, Brinkrolf K, Kelly S, Müller B, Doolan P, Hackl M, Beckmann TF, Noll T, Grillari J, Barron N, Pühler A, Clynes M, Borth N (2012b) Utilization and evaluation of CHO-specific sequence databases for mass spectrometry based proteomics. Biotechnol Bioeng 109:1386–1394

    Article  CAS  PubMed  Google Scholar 

  • Müller B, Heinrich C, Jabs W, Kaspar-Schönefeld S, Schmidt A, Rodrigues de Carvalho N, Albaum SP, Baessmann C, Noll T, Hoffrogge R (2017) Label-free protein quantification of sodium butyrate treated CHO cells by ESI-UHR-TOF-MS. J Biotechnol 257:87–98

    Article  CAS  PubMed  Google Scholar 

  • Niewiadomski P, Kong JH, Ahrends R, Ma Y, Humke EW, Khan S, Teruel MN, Novitch BG, Rohatgi R (2014) Gli protein activity is controlled by multisite phosphorylation in vertebrate Hedgehog signaling. Cell Rep 6:168–181

  • Orellana CA, Marcellin E, Schulz BL, Nouwens AS, Gray PP, Nielsen LK (2015) High-antibody-producing Chinese hamster ovary cells up-regulate intracellular protein transport and glutathione synthesis. J Proteome Res 14:609–618

    Article  CAS  PubMed  Google Scholar 

  • Palsgaard J, Emanuelli B, Winnay JN, Sumara G, Karsenty G, Kahn CR (2012) Cross-talk between insulin and Wnt signaling in preadipocytes. Role of Wnt co-receptor low density lipoprotein receptor-related protein-5 (LRP5). J Biol Chem 287:12016–12026

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Park JH, Noh SM, Woo JR, Kim JW, Lee GM (2016) Valeric acid induces cell cycle arrest at G1 phase in CHO cell cultures and improves recombinant antibody productivity. Biotechnol J 11:487–496

    Article  CAS  PubMed  Google Scholar 

  • Pereira S, Kildegaard HF, Andersen MR (2018) Impact of CHO metabolism on cell growth and protein production. An overview of toxic and inhibiting metabolites and nutrients. Biotechnol J 13:e1700499

    Article  CAS  PubMed  Google Scholar 

  • Pilbrough W, Munro TP, Gray P (2009) Intraclonal protein expression heterogeneity in recombinant CHO cells. PLoS ONE 4:e8432

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rossignol M, Keriel A, Staub A, Egly JM (1999) Kinase activity and phosphorylation of the largest subunit of TFIIF transcription factor. J Biol Chem 274:22387–22392

  • Schönbrunner ER, Schmid FX (1992) Peptidyl-prolyl cis-trans isomerase improves the efficiency of protein disulfide isomerase as a catalyst of protein folding. Proc Natl Acad Sci U S A 89:4510–4513

    Article  PubMed  PubMed Central  Google Scholar 

  • Shaw PE (2002) Peptidyl-prolyl isomerases. A new twist to transcription. EMBO Rep 3:521–526

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sommeregger W, Mayrhofer P, Steinfellner W, Reinhart D, Henry M, Clynes M, Meleady P, Kunert R (2016) Proteomic differences in recombinant CHO cells producing two similar antibody fragments. Biotechnol Bioeng 113:1902–1912

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Takagi Y, Kikuchi T, Wada R, Omasa T (2017) The enhancement of antibody concentration and achievement of high cell density CHO cell cultivation by adding nucleoside. Cytotechnology 69:511–521

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tang X, Hui Z-G, Cui X-L, Garg R, Kastan MB, Xu B (2008) A novel ATM-dependent pathway regulates protein phosphatase 1 in response to DNA damage. Mol Cell Biol 28:2559–2566

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Thapar R (2015) Roles of prolyl isomerases in RNA-mediated gene expression. Biomolecules 5:974–999

  • Trummer E, Fauland K, Seidinger S, Schriebl K, Lattenmayer C, Kunert R, Vorauer-Uhl K, Weik R, Borth N, Katinger H, Müller D (2006) Process parameter shifting. Part I. Effect of DOT, pH, and temperature on the performance of Epo-Fc expressing CHO cells cultivated in controlled batch bioreactors. Biotechnol Bioeng 94:1033–1044

    Article  CAS  PubMed  Google Scholar 

  • Tyanova S, Temu T, Cox J (2016a) The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 11:2301–2319

    Article  CAS  PubMed  Google Scholar 

  • Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, Mann M, Cox J (2016b) The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods 13:731–740

    Article  CAS  Google Scholar 

  • Vizcaíno JA, Csordas A, del-Toro N, Dianes JA, Griss J, Lavidas I, Mayer G, Perez-Riverol Y, Reisinger F, Ternent T, Xu Q-W, Wang R, Hermjakob H (2016) 2016 update of the PRIDE database and its related tools. Nucleic Acids Res 44:D447–D456

    Article  CAS  PubMed  Google Scholar 

  • Walsh G (2018) Biopharmaceutical benchmarks 2018. Nat Biotechnol 36:1136–1145

    Article  CAS  PubMed  Google Scholar 

  • Wang Y, Ding Q, Yen C-J, Xia W, Izzo JG, Lang J-Y, Li C-W, Hsu JL, Miller SA, Wang X, Lee D-F, Hsu J-M, Huo L, Labaff AM, Liu D, Huang T-H, Lai C-C, Tsai F-J, Chang W-C, Chen C-H, Wu T-T, Buttar NS, Wang KK, Wu Y, Wang H, Ajani J, Hung M-C (2012) The crosstalk of mTOR/S6K1 and Hedgehog pathways. Cancer Cell 21:374–387

  • Wingens M, Gätgens J, Schmidt A, Albaum SP, Büntemeyer H, Noll T, Hoffrogge R (2015) 2D-DIGE screening of high-productive CHO cells under glucose limitation--basic changes in the proteome equipment and hints for epigenetic effects. J Biotechnol 201:86–97

    Article  CAS  PubMed  Google Scholar 

  • Xu S, Liu C-X, Xu W, Huang L, Zhao J-Y, Zhao S-M (2017) Butyrate induces apoptosis by activating PDC and inhibiting complex I through SIRT3 inactivation. Signal. Transduct. Target. Ther. 2:16035

  • Xu X, Nagarajan H, Lewis NE, Pan S, Cai Z, Liu X, Chen W, Xie M, Wang W, Hammond S, Andersen MR, Neff N, Passarelli B, Koh W, Fan HC, Wang J, Gui Y, Lee KH, Betenbaugh MJ, Quake SR, Famili I, Palsson BO, Wang J (2011) The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line. Nat Biotechnol 29:735–741

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yi J-M, Huan X-J, Song S-S, Zhou H, Wang Y-Q, Miao Z-H (2016) Triptolide induces cell killing in multidrugresistant tumor cells via CDK7/RPB1 rather than XPB or p44. Mol. Cancer Ther. 15:1495–1503

Download references

Acknowledgements

We would like to thank the Australian Institute for Bioengineering and Nanotechnology, University of Queensland-Brisbane, Australia (AIBN), for providing the CHO clones. In addition, we would like to honor the refinement of graphical illustrations by our bachelor student Marina Simunovic and our lab staff Larissa Leßmann for performing the western blots.

Author information

Authors and Affiliations

  1. Cell Culture Technology, Technical Department, Bielefeld University, Bielefeld, Germany

    Louise Schelletter, Thomas Noll & Raimund Hoffrogge

  2. Center for Biotechnology, CeBiTec, Bioinformatics Resource Facility, Bielefeld University, Bielefeld, Germany

    Stefan Albaum

  3. Biological Department, Osnabrück University, Osnabrück, Germany

    Stefan Walter

Authors
  1. Louise Schelletter
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stefan Albaum
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stefan Walter
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas Noll
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Raimund Hoffrogge
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Louise Schelletter.

Ethics declarations

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

All authors declare that he/she has no conflict of interest.

Additional information

Publisher’s note

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

Electronic supplementary material

ESM 1

(XLSX 29 kb)

ESM 2

(XLSX 643 kb)

ESM 3

(XLSX 1370 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Schelletter, L., Albaum, S., Walter, S. et al. Clonal variations in CHO IGF signaling investigated by SILAC-based phosphoproteomics and LFQ-MS. Appl Microbiol Biotechnol 103, 8127–8143 (2019). https://doi.org/10.1007/s00253-019-10020-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-019-10020-z

Keywords

Search

Navigation