Skip to main content
SpringerLink
Log in

Understanding the intracellular effects of yeast extract on the enhancement of Fc-fusion protein production in Chinese hamster ovary cell culture

  • Biotechnological products and process engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Yeast extract (YE), as a non-animal source additive for mammalian cell culture medium, has been widely used for manufacturing of therapeutic proteins. In the present study, one particular YE was found to have significantly improved the specific productivity (q p) of Fc-fusion protein in recombinant Chinese hamster ovary (rCHO) cell culture. In order to elucidate the intracellular effects of YE on protein productivity, steps of the target protein synthesis process were investigated to unveil their variations caused by YE addition. Stepwise analysis on Fc-fusion protein synthesis process showed that YE enhanced Fc-fusion protein gene transcription with cell cycle arrest at G1 phase; mammalian target of rapamycin (mTOR) signaling pathway was activated to enhance the translation of Fc-fusion protein, and the block in post-translational steps of Fc-fusion protein was alleviated by YE addition as well. Our results revealed the responses of multiple protein production steps to the addition of YE and provided a practical guidance for the separation and application of active compounds from hydrolysates.

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
Fig. 7

Similar content being viewed by others

References

  • Aggarwal SR (2014) What’s fueling the biotech engine—2012 to 2013. Nat Biotechnol 32:32–39

    Article  CAS  PubMed  Google Scholar 

  • Allen MJ, Boyce JP, Trentalange MT, Treiber DL, Rasmussen B, Tillotson B, Davis R, Reddy P (2008) Identification of novel small molecule enhancers of protein production by cultured mammalian cells. Biotechnol Bioeng 100(6):1193–1204

    Article  CAS  PubMed  Google Scholar 

  • Becker E, Florin L, Pfizenmaier K, Kaufmann H (2008) An XBP-1 dependent bottle-neck in production of IgG subtype antibodies in chemically defined serum-free Chinese hamster ovary (CHO) fed-batch processes. J Biotechnol 135(2):217–223

    Article  CAS  PubMed  Google Scholar 

  • Buchner J, Pastan I, Brinkmann U (1992) A method for increasing the yield of properly folded recombinant fusion proteins: single-chain immunotoxins from renaturation of bacterial inclusion bodies. Anal Biochem 205(2):263–270

    Article  CAS  PubMed  Google Scholar 

  • Burdakov D, Petersen OH, Verkhratsky A (2005) Intraluminal calcium as a primary regulator of endoplasmic reticulum function. Cell Calcium 38(3):303–310

    Article  CAS  PubMed  Google Scholar 

  • Chen F, Fan L, Wang J, Zhou Y, Ye Z, Zhao L, Tan WS (2012) Insight into the roles of hypoxanthine and thymidine on cultivating antibody-producing CHO cells: cell growth, antibody production and long-term stability. Appl Microbiol Biotechnol 93(1):169–178

    Article  PubMed  Google Scholar 

  • Dreesen IA, 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(4):853–866

    Article  CAS  PubMed  Google Scholar 

  • Du Z, Treiber D, McCarter JD, Fomina-Yadlin D, Saleem RA, McCoy RE, Zhang Y, Tharmalingam T, Leith M, Follstad BD, Dell B, Grisim B, Zupke C, Heath C, Morris AE, Reddy P (2015) Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures. Biotechnol Bioeng 112(1):141–155

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Florin L, Pegel A, Becker E, Hausser A, Olayioye MA, Kaufmann H (2009) Heterologous expression of the lipid transfer protein CERT increases therapeutic protein productivity of mammalian cells. J Biotechnol 141(1):84–90

    Article  CAS  PubMed  Google Scholar 

  • Franěk F, Katinger H (2002) Specific effects of synthetic oligopeptides on cultured animal cells. Biotechnol Prog 18(1):155–158

    Article  PubMed  Google Scholar 

  • Franěk F, Hohenwarter O, Katinger H (2000) Plant protein hydrolysates: preparation of defined peptide fractions promoting growth and production in animal cells cultures. Biotechnol Prog 16(5):688–692

    Article  PubMed  Google Scholar 

  • Gupta AJ, Hageman JA, Wierenga PA, Boots J-W, Gruppen H (2014) Chemometric analysis of soy protein hydrolysates used in animal cell culture for IgG production—an untargeted metabolomics approach. Process Biochem 49(2):309–317

    Article  CAS  Google Scholar 

  • Hendrick V, Winnepenninckx P, Abdelkafi C, Vandeputte O, Cherlet M, Marique T, Renemann G, Loa A, Kretzmer G, Werenne J (2001) Increased productivity of recombinant tissular plasminogen activator (t-PA) by butyrate and shift of temperature: a cell cycle phases analysis. Cytotechnology 36(1-3):71–83

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Jiang Z, Sharfstein ST (2008) Sodium butyrate stimulates monoclonal antibody over-expression in CHO cells by improving gene accessibility. Biotechnol Bioeng 100(1):189–194

    Article  CAS  PubMed  Google Scholar 

  • Kou TC, Fan L, Zhou Y, Ye ZY, Liu XP, Zhao L, Tan WS (2011) Detailed understanding of enhanced specific productivity in Chinese hamster ovary cells at low culture temperature. J Biosci Bioeng 111(3):365–369

    Article  CAS  PubMed  Google Scholar 

  • Kumar N, Gammell P, Clynes M (2007) Proliferation control strategies to improve productivity and survival during CHO based production culture. Cytotechnology 53(1-3):33–46

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Lee CJ, Seth G, Tsukuda J, Hamilton RW (2009) A clone screening method using mRNA levels to determine specific productivity and product quality for monoclonal antibodies. Biotechnol Bioeng 102(4):1107–1118

    Article  CAS  PubMed  Google Scholar 

  • Lee HW, Christie A, Starkey JA, Read EK, Yoon S (2014) Intracellular metabolic flux analysis of CHO cells supplemented with wheat hydrolysates for improved mAb production and cell-growth. J Chem Technol Biotechnol 90(2):291–302

    Article  Google Scholar 

  • Li B, Sirimuthu NMS, Ray BH, Ryder AG (2012) Using surface-enhanced Raman scattering (SERS) and fluorescence spectroscopy for screening yeast extracts, a complex component of cell culture media. J Raman Spectrosc 43(8):1074–1082

    Article  CAS  Google Scholar 

  • Liu C-H, Chu I-M, Hwang S-M (2001) Pentanoic acid, a novel protein synthesis stimulant for Chinese hamster ovary (CHO) cells. J Biosci Bioeng 91(1):71–75

    Article  CAS  PubMed  Google Scholar 

  • Lu C, Gonzalez C, Gleason J, Gangi J, Yang JD (2007) A T-flask based screening platform for evaluating and identifying plant hydrolysates for a fed-batch cell culture process. Cytotechnology 55(1):15–29

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ma XM, Blenis J (2009) Molecular mechanisms of mTOR-mediated translational control. Nat Rev Mol Cell Biol 10(5):307–318

    Article  PubMed  Google Scholar 

  • Mendonca RZ, de Oliveira EC, Pereira CA, Lebrun I (2007) Effect of bioactive peptides isolated from yeastolate, lactalbumin and NZCase in the insect cell growth. Bioprocess Biosyst Eng 30(3):157–164

    Article  CAS  PubMed  Google Scholar 

  • Michiels JF, Barbau J, De Boel S, Dessy S, Agathos SN, Schneider YJ (2011) Characterisation of beneficial and detrimental effects of a soy peptone, as an additive for CHO cell cultivation. Process Biochem 46(3):671–681

    Article  CAS  Google Scholar 

  • Mosser M, Kapel R, Aymes A, Bonanno L-M, Olmos E, Chevalot I, Marc I, Marc A (2012) Chromatographic fractionation of yeast extract: a strategy to identify physicochemical properties of compounds promoting CHO cell culture. Process Biochem 47(7):1178–1185

    Article  CAS  Google Scholar 

  • Mosser M, Chevalot I, Olmos E, Blanchard F, Kapel R, Oriol E, Marc I, Marc A (2013) Combination of yeast hydrolysates to improve CHO cell growth and IgG production. Cytotechnology 65(4):629–641

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Nyberg GB, Balcarcel RR, Follstad BD, Stephanopoulos G, Wang DI (1999) Metabolism of peptide amino acids by Chinese hamster ovary cells grown in a complex medium. Biotechnol Bioeng 62(3):324–335

    Article  CAS  PubMed  Google Scholar 

  • Oslowski CM, Urano F (2011) Measuring ER stress and the unfolded protein response using mammalian tissue culture system. Method Enzymol 490:71–92

    Article  CAS  Google Scholar 

  • Proud CG (2009) mTORC1 signalling and mRNA translation. Biochem Soc Trans 37(1):227–231

    Article  CAS  PubMed  Google Scholar 

  • Richardson J, Shah B, Bondarenko PV, Bhebe P, Zhang Z, Nicklaus M, Kombe MC (2015) Metabolomics analysis of soy hydrolysates for the identification of productivity markers of mammalian cells for manufacturing therapeutic proteins. Biotechnol Prog. doi:10.1002/btpr.2050

    PubMed  Google Scholar 

  • Royle L, Campbell MP, Radcliffe CM, White DM, Harvey DJ, Abrahams JL, Kim Y-G, Henry GW, Shadick NA, Weinblatt ME (2008) HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software. Anal Biochem 376(1):1–12

    Article  CAS  PubMed  Google Scholar 

  • Ruggero D, Sonenberg N (2005) The Akt of translational control. Oncogene 24(50):7426–7434

    Article  CAS  PubMed  Google Scholar 

  • Schlatter S, Senn C, Fussenegger M (2003) Modulation of translation-initiation in CHO-K1 cells by rapamycin-induced heterodimerization of engineered eIF4G fusion proteins. Biotechnol Bioeng 83(2):210–225

    Article  CAS  PubMed  Google Scholar 

  • Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3(6):1101–1108

    Article  CAS  PubMed  Google Scholar 

  • Sengupta S, Peterson TR, Sabatini DM (2010) Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol Cell 40(2):310–322

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Shen AY, Van de Goor J, Zheng L, Reyes AE, Krummen LA, Ozturk S, Hu W (2006) Recombinant DNA technology and cell line development. Biotechnol Biprocess Ser 30:15–40

    Google Scholar 

  • Sommer R (1998) Yeast extracts: production, properties and components. Food Aust 50(4):181–183

    Google Scholar 

  • Sung YH, Lim SW, Chung JY, Lee GM (2004) Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese hamster ovary cells. Appl Microbiol Biotechnol 63(5):527–536

    Article  CAS  PubMed  Google Scholar 

  • Sunley K, Butler M (2010) Strategies for the enhancement of recombinant protein production from mammalian cells by growth arrest. Biotechnol Adv 28(3):385–394

    Article  CAS  PubMed  Google Scholar 

  • Svennerholm L (1957) Quantitive estimation of sialic acids: II. A colorimetric resorcinol-hydrochloric acid method. Biochim Biophys Acta 24:604–611

    Article  CAS  PubMed  Google Scholar 

  • Wilkins J, Shiratori MK, Breece T (2009) Biologically active c-terminal arginine-containing peptides. United States Patent. US 2009/0143248 A1

  • Yang WC, Lu J, Nguyen NB, Zhang A, Healy NV, Kshirsagar R, Ryll T, Huang YM (2014) Addition of valproic acid to CHO cell fed-batch cultures improves monoclonal antibody titers. Mol Biotechnol 56(5):421–428

    Article  CAS  PubMed  Google Scholar 

  • Yoon SK, Song JY, Lee GM (2003) Effect of low culture temperature on specific productivity, transcription level, and heterogeneity of erythropoietin in Chinese hamster ovary cells. Biotechnol Bioeng 82(3):289–298

    Article  CAS  PubMed  Google Scholar 

  • Yu M, Hu Z, Pacis E, Vijayasankaran N, Shen A, Li F (2011) Understanding the intracellular effect of enhanced nutrient feeding toward high titer antibody production process. Biotechnol Bioeng 108(5):1078–1088

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 21206040 and 21406066), the National Science and Technology Major Project (No. 2013ZX10004003-003-003), and the National High Technology Research and Development Program of China (863 Program) (No. 2012AA02A303).

Conflict of interest

The authors declare that they have no competing interests.

Author information

Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, P. O. Box 309, 130 Mei-Long Road, Shanghai, 200237, People’s Republic of China

    Dongdong Hu, Yating Sun, Xuping Liu, Jintao Liu, Xintao Zhang, Liang Zhao, Wen-Song Tan & Li Fan

  2. Zhejiang Hisun Pharmaceutical Co., Ltd., Fuyang, Hangzhou, Zhejiang, 311404, China

    Haibin Wang

Authors
  1. Dongdong Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yating Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xuping Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jintao Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xintao Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Liang Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Haibin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Wen-Song Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Li Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Wen-Song Tan or Li Fan.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 112 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hu, D., Sun, Y., Liu, X. et al. Understanding the intracellular effects of yeast extract on the enhancement of Fc-fusion protein production in Chinese hamster ovary cell culture. Appl Microbiol Biotechnol 99, 8429–8440 (2015). https://doi.org/10.1007/s00253-015-6789-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-015-6789-5

Keywords

Search

Navigation