pp 1-24 | Cite as

Innovation in Cell Banking, Expansion, and Production Culture

  • Rashmi Kshirsagar
  • Thomas Ryll
Part of the Advances in Biochemical Engineering/Biotechnology book series


Cell culture-based production processes enable the development and commercial supply of recombinant protein products. Such processes consist of the following elements: thaw and initiation of culture, seed expansion, and production culture. A robust cell source storage system in the form of a cell bank is developed and cells are thawed to initiate the cell culture process. Seed culture expansion generates sufficient cell mass to initiate the production culture. The production culture provides an environment where the cells can synthesize the product and is optimized to deliver the highest possible product concentration with acceptable product quality. This chapter describes the significant innovations made in these process elements and the resulting improvements in the overall efficiency, robustness, and safety of the processes and products.


Cell banking, Fed-batch, Innovation, Mammalian cell culture, Productivity 


  1. 1.
    De Jesus M, Wurm FM (2011) Manufacturing recombinant proteins in kg-ton quantities using animal cells in bioreactors. Eur J Pharm Biopharm 78(2):184–188Google Scholar
  2. 2.
    Huang YM, Hu W, Rustandi E, Chang K, Yusuf-Makagiansar H, Ryll T (2010) Maximizing productivity of CHO cell-based fed-batch culture using chemically defined media conditions and typical manufacturing equipment. Biotechnol Prog 26(5):1400–1410Google Scholar
  3. 3.
    Li J, Gu W, Edmondson DG, Lu C, Vijayasankaran N, Figueroa B, Stevenson D, Ryll T, Li F (2012) Generation of a cholesterol-independent, non-GS NS0 cell line through chemical treatment and application for high titer antibody production. Biotechnol Bioeng 109(7):1685–1692Google Scholar
  4. 4.
    Lu F, Toh PC, Burnett I, Li F, Hudson T, Amanullah A, Li J (2013) Automated dynamic fed-batch process and media optimization for high productivity cell culture process development. Biotechnol Bioeng 110(1):191–205Google Scholar
  5. 5.
    Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22(11):1393–1398Google Scholar
  6. 6.
    Yang WC, Lu J, Kwiatkowski C, Yuan H, Kshirsagar R, Ryll T, Huang Y-M (2014) Perfusion seed cultures improve biopharmaceutical fed-batch production capacity and product quality. Biotechnol Prog 30(3):616–625Google Scholar
  7. 7.
    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):1078–1088Google Scholar
  8. 8.
    Costa AR, Rodrigues ME, Henriques M, Oliveira R, Azeredo J (2014) Glycosylation: impact, control and improvement during therapeutic protein production. Crit Rev Biotechnol 34(4):281–299Google Scholar
  9. 9.
    Higel F, Seidl A, Sorgel F, Friess W (2016) N-Glycosylation heterogeneity and the influence on structure, function and pharmacokinetics of monoclonal antibodies and Fc fusion proteins. Eur J Pharm Biopharm 100:94–100Google Scholar
  10. 10.
    Jefferis R (2012) Isotype and glycoform selection for antibody therapeutics. Arch Biochem Biophys 526(2):159–166Google Scholar
  11. 11.
    van Beers MM, Bardor M (2012) Minimizing immunogenicity of biopharmaceuticals by controlling critical quality attributes of proteins. Biotechnol J 7(12):1473–1484Google Scholar
  12. 12.
    Patil R, Walther J (2016) Continuous manufacturing of recombinant therapeutic proteins: upstream and downstream technologies. Adv Biochem Eng Biotechnol. No. 10 in Volume Overview. Scholar
  13. 13.
    Turner R, Joseph A, Titchener-Hooker N, Bender J (2016) Harvest operations. Adv Biochem Eng Biotechnol. No. 5 in Volume Overview.
  14. 14.
    Gramer MJ, Eckblad JJ, Donahue R, Brown J, Shultz C, Vickerman K, Priem P, van den Bremer ET, Gerritsen J, van Berkel PH (2011) Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose. Biotechnol Bioeng 108(7):1591–1602Google Scholar
  15. 15.
    Hayflick L, Plotkin S, Stevenson RE (1987) History of the acceptance of human diploid cell strains as substrates for human virus vaccine manufacture. Dev Biol Stand 68:9–17Google Scholar
  16. 16.
    Hesse F, Wagner R (2000) Developments and improvements in the manufacturing of human therapeutics with mammalian cell cultures. Trends Biotechnol 18(4):173–180Google Scholar
  17. 17.
    Groth J, Steinmann J, Eckstein V, Muller-Ruchholtz W (1991) Freezing of cells--replacement of serum by oxypolygelatine. J Immunol Methods 141(1):105–109Google Scholar
  18. 18.
    Merten OW, Petres S, Couve E (1995) A simple serum-free freezing medium for serum-free cultured cells. Biologicals 23(2):185–189Google Scholar
  19. 19.
    Muller P, Aurich H, Wenkel R, Schaffner I, Wolff I, Walldorf J, Fleig WE, Christ B (2004) Serum-free cryopreservation of porcine hepatocytes. Cell Tissue Res 317(1):45–56Google Scholar
  20. 20.
    Schoenherr I, Stapp T, Ryll T (2000) A comparison of different methods to determine the end of exponential growth in CHO cell cultures for optimization of scale-up. Biotechnol Prog 16(5):815–821Google Scholar
  21. 21.
    Tsang VL, Wang AX, Yusuf-Makagiansar H, Ryll T (2014) Development of a scale down cell culture model using multivariate analysis as a qualification tool. Biotechnol Prog 30(1):152–160Google Scholar
  22. 22.
    Heidemann R, Mered M, Wang DQ, Gardner B, Zhang C, Michaels J, Henzler HJ, Abbas N, Konstantinov K (2002) A new seed-train expansion method for recombinant mammalian cell lines. Cytotechnology 38(1–2):99–108Google Scholar
  23. 23.
    Seth G (2012) Freezing mammalian cells for production of biopharmaceuticals. Methods 56(3):424–431Google Scholar
  24. 24.
    Seth G, Hamilton RW, Stapp TR, Zheng LS, Meier A, Petty K, Leung S, Chary S (2013) Development of a new bioprocess scheme using frozen seed train intermediates to initiate CHO cell culture manufacturing campaigns. Biotechnol Bioeng 110(5):1376–1385Google Scholar
  25. 25.
    Tao Y, Shih J, Sinacore M, Ryll T, Yusuf-Makagiansar H (2011) Development and implementation of a perfusion-based high cell density cell banking process. Biotechnol Prog 27(3):824–829Google Scholar
  26. 26.
    Clincke MF, Molleryd C, Samani PK, Lindskog E, Faldt E, Walsh K, Chotteau V (2013) Very high density of Chinese hamster ovary cells in perfusion by alternating tangential flow or tangential flow filtration in WAVE Bioreactor - Part II: Applications for antibody production and cryopreservation. Biotechnol Prog 29(3):768–777Google Scholar
  27. 27.
    Heidemann R, Lunse S, Tran D, Zhang C (2010) Characterization of cell-banking parameters for the cryopreservation of mammalian cell lines in 100-ML cryobags. Biotechnol Prog 26(4):1154–1163Google Scholar
  28. 28.
    Horvath B, Tsang VL, Lin W, Dai X-P, Kunas K, Frank G (2013) A generic growth test method for improving quality control of disposables in industrial cell culture. Biopharm Int 26(6):34–41Google Scholar
  29. 29.
    Tao Y, Yusuf-Makagiansar H, Shih J, Ryll T, Sinacore M (2012) Novel cholesterol feeding strategy enables a high-density cultivation of cholesterol-dependent NS0 cells in linear low-density polyethylene-based disposable bioreactors. Biotechnol Lett 34(8):1453–1458Google Scholar
  30. 30.
    Hecht V, Duvar S, Ziehr H, Burg J, Jockwer A (2014) Efficiency improvement of an antibody production process by increasing the inoculum density. Biotechnol Prog 30(3):607–615Google Scholar
  31. 31.
    Pohlscheidt M, Jacobs M, Wolf S, Thiele J, Jockwer A, Gabelsberger J, Jenzsch M, Tebbe H, Burg J (2013) Optimizing capacity utilization by large scale 3000 L perfusion in seed train bioreactors. Biotechnol Prog 29(1):222–229Google Scholar
  32. 32.
    Eibl R, Kaiser S, Lombriser R, Eibl D (2010) Disposable bioreactors: the current state-of-the-art and recommended applications in biotechnology. Appl Microbiol Biotechnol 86(1):41–49Google Scholar
  33. 33.
    Eibl R, Werner S, Eibl D (2009) Bag bioreactor based on wave-induced motion: characteristics and applications. Adv Biochem Eng Biotechnol 115:55–87Google Scholar
  34. 34.
    Shukla AA, Gottschalk U (2013) Single-use disposable technologies for biopharmaceutical manufacturing. Trends Biotechnol 31(3):147–154Google Scholar
  35. 35.
    Stettler M, Zhang X, Hacker DL, De Jesus M, Wurm FM (2007) Novel orbital shake bioreactors for transient production of CHO derived IgGs. Biotechnol Prog 23(6):1340–1346Google Scholar
  36. 36.
    Minow B, Tschoepe S, Regner A, Populin M, Reiser S, Noack C, Neubauer P (2014) Biological performance of two different 1000 L single-use bioreactors applying a simple transfer approach. Eng Life Sci 14(3):283–291Google Scholar
  37. 37.
    Smelko JP, Wiltberger KR, Hickman EF, Morris BJ, Blackburn TJ, Ryll T (2011) Performance of high intensity fed-batch mammalian cell cultures in disposable bioreactor systems. Biotechnol Prog 27(5):1358–1364Google Scholar
  38. 38.
    Griffiths B (1990) Perfusion systems for cell cultivation. Bioprocess Technol 10:217–250Google Scholar
  39. 39.
    Konstantinov K, Goudar C, Ng M, Meneses R, Thrift J, Chuppa S, Matanguihan C, Michaels J, Naveh D (2006) The “push-to-low” approach for optimization of high-density perfusion cultures of animal cells. Adv Biochem Eng Biotechnol 101:75–98Google Scholar
  40. 40.
    Ryll T, Dutina G, Reyes A, Gunson J, Krummen L, Etcheverry T (2000) Performance of small-scale CHO perfusion cultures using an acoustic cell filtration device for cell retention: characterization of separation efficiency and impact of perfusion on product quality. Biotechnol Bioeng 69(4):440–449Google Scholar
  41. 41.
    Woodside SM, Bowen BD, Piret JM (1998) Mammalian cell retention devices for stirred perfusion bioreactors. Cytotechnology 28(1–3):163–175Google Scholar
  42. 42.
    Lehmann J, Buntemeyer H, Jager V (1990) Bulk culture of animal cells for antibody production: a comparison of reactors. Food Biotechnol 4(1):423–431Google Scholar
  43. 43.
    Ryll T, Lucki-Lange M, Jager V, Wagner R (1990) Production of recombinant human interleukin-2 with BHK cells in a hollow fibre and a stirred tank reactor with protein-free medium. J Biotechnol 14(3–4):377–392Google Scholar
  44. 44.
    Ecker DM, Ransohoff TC (2014) Mammalian cell culture capacity for biopharmaceutical manufacturing. Adv Biochem Eng Biotechnol 139:185–225Google Scholar
  45. 45.
    Kantardjieff A, Zhou W (2014) Mammalian cell cultures for biologics manufacturing. Adv Biochem Eng Biotechnol 139:1–9Google Scholar
  46. 46.
    Langer ES (2009) Trends in capacity utilization for therapeutic monoclonal antibody production. MAbs 1(2):151–156Google Scholar
  47. 47.
    Gilbert A, McElearney K, Kshirsagar R, Sinacore MS, Ryll T (2013) Investigation of metabolic variability observed in extended fed batch cell culture. Biotechnol Prog 29(6):1519–1527Google Scholar
  48. 48.
    Mulukutla BC, Gramer M, Hu WS (2012) On metabolic shift to lactate consumption in fed-batch culture of mammalian cells. Metab Eng 14(2):138–149Google Scholar
  49. 49.
    Mulukutla BC, Yongky A, Grimm S, Daoutidis P, Hu WS (2015) Multiplicity of steady states in glycolysis and shift of metabolic state in cultured mammalian cells. PLoS One 10(3):e0121561Google Scholar
  50. 50.
    Toussaint C, Henry O, Durocher Y (2016) Metabolic engineering of CHO cells to alter lactate metabolism during fed-batch cultures. J Biotechnol 217:122–131Google Scholar
  51. 51.
    Zagari F, Jordan M, Stettler M, Broly H, Wurm FM (2013) Lactate metabolism shift in CHO cell culture: the role of mitochondrial oxidative activity. N Biotechnol 30(2):238–245Google Scholar
  52. 52.
    Gawlitzek M, Ryll T, Lofgren J, Sliwkowski MB (2000) Ammonium alters N-glycan structures of recombinant TNFR-IgG: degradative versus biosynthetic mechanisms. Biotechnol Bioeng 68(6):637–646Google Scholar
  53. 53.
    Khetan A, Huang YM, Dolnikova J, Pederson NE, Wen D, Yusuf-Makagiansar H, Chen P, Ryll T (2010) Control of misincorporation of serine for asparagine during antibody production using CHO cells. Biotechnol Bioeng 107(1):116–123Google Scholar
  54. 54.
    Kshirsagar R, McElearney K, Gilbert A, Sinacore M, Ryll T (2012) Controlling trisulfide modification in recombinant monoclonal antibody produced in fed-batch cell culture. Biotechnol Bioeng 109(10):2523–2532Google Scholar
  55. 55.
    Ryll T, Valley U, Wagner R (1994) Biochemistry of growth inhibition by ammonium ions in mammalian cells. Biotechnol Bioeng 44(2):184–193Google Scholar
  56. 56.
    Gagnon M, Hiller G, Luan YT, Kittredge A, DeFelice J, Drapeau D (2011) High-end pH-controlled delivery of glucose effectively suppresses lactate accumulation in CHO fed-batch cultures. Biotechnol Bioeng 108(6):1328–1337Google Scholar
  57. 57.
    Zhang A, Tsang VL, Moore B, Shen V, Huang YM, Kshirsagar R, Ryll T (2015) Advanced process monitoring and feedback control to enhance cell culture process production and robustness. Biotechnol Bioeng 112(12):2495–2504Google Scholar
  58. 58.
    Zhu J, Hatton D (2016) New mammalian expression systems. Adv Biochem Eng Biotechnol. No. 2 in Volume Overview. Scholar
  59. 59.
    Backliwal G, Hildinger M, Kuettel I, Delegrange F, Hacker DL, Wurm FM (2008) Valproic acid: a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures. Biotechnol Bioeng 101(1):182–189Google Scholar
  60. 60.
    Etcheverry T, Ryll T (1997) Mammalian cell culture process. Genentech Inc.Google Scholar
  61. 61.
    Lee MS, Lee GM (2000) Hyperosmotic pressure enhances immunoglobulin transcription rates and secretion rates of KR12H-2 transfectoma. Biotechnol Bioeng 68(3):260–268Google Scholar
  62. 62.
    Moore A, Mercer J, Dutina G, Donahue CJ, Bauer KD, Mather JP, Etcheverry T, Ryll T (1997) Effects of temperature shift on cell cycle, apoptosis and nucleotide pools in CHO cell batch cultures. Cytotechnology 23(1–3):47–54Google Scholar
  63. 63.
    Fan Y, Jimenez Del Val I, Muller C, Wagtberg Sen J, Rasmussen SK, Kontoravdi C, Weilguny D, Andersen MR (2015) Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation. Biotechnol Bioeng 112(3):521–535Google Scholar
  64. 64.
    Kang S, Mullen J, Miranda LP, Deshpande R (2012) Utilization of tyrosine- and histidine-containing dipeptides to enhance productivity and culture viability. Biotechnol Bioeng 109(9):2286–2294Google Scholar
  65. 65.
    Alves CS, Gilbert A, Dalvi S, Germain BS, Xie W, Estes S, Kshirsagar R, Ryll T (2015) Integration of cell line and process development to overcome the challenge of a difficult to express protein. Biotechnol Prog 31(5):1201–1211Google Scholar
  66. 66.
    Estes S, Melville M (2014) Mammalian cell line developments in speed and efficiency. Adv Biochem Eng Biotechnol 139:11–33Google Scholar
  67. 67.
    Johari YB, Estes SD, Alves CS, Sinacore MS, James DC (2015) Integrated cell and process engineering for improved transient production of a “difficult-to-express” fusion protein by CHO cells. Biotechnol Bioeng 112(12):2527–2542Google Scholar
  68. 68.
    Konitzer JD, Muller MM, Leparc G, Pauers M, Bechmann J, Schulz P, Schaub J, Enenkel B, Hildebrandt T, Hampel M, Tolstrup AB (2015) A global RNA-seq-driven analysis of CHO host and production cell lines reveals distinct differential expression patterns of genes contributing to recombinant antibody glycosylation. Biotechnol J 10(9):1412–1423Google Scholar
  69. 69.
    Croughan MS, Konstantinov KB, Cooney C (2015) The future of industrial bioprocessing: batch or continuous? Biotechnol Bioeng 112(4):648–651Google Scholar
  70. 70.
    Pollock J, Ho SV, Farid SS (2013) Fed-batch and perfusion culture processes: economic, environmental, and operational feasibility under uncertainty. Biotechnol Bioeng 110(1):206–219Google Scholar
  71. 71.
    Chon JH, Zarbis-Papastoitsis G (2011) Advances in the production and downstream processing of antibodies. N Biotechnol 28(5):458–463Google Scholar
  72. 72.
    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):141–155Google Scholar
  73. 73.
    Yang WC, Minkler DF, Kshirsagar R, Ryll T, Huang YM (2015) Concentrated fed-batch cell culture increases manufacturing capacity without additional volumetric capacity. J Biotechnol 217:1–11Google Scholar
  74. 74.
    Schiestl M, Stangler T, Torella C, Cepeljnik T, Toll H, Grau R (2011) Acceptable changes in quality attributes of glycosylated biopharmaceuticals. Nat Biotechnol 29(4):310–312Google Scholar
  75. 75.
    Schneider CK (2013) Biosimilars in rheumatology: the wind of change. Ann Rheum Dis 72(3):315–318Google Scholar
  76. 76.
    Chung CH, Mirakhur B, Chan E, Le QT, Berlin J, Morse M, Murphy BA, Satinover SM, Hosen J, Mauro D, Slebos RJ, Zhou Q, Gold D, Hatley T, Hicklin DJ, Platts-Mills TA (2008) Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose. N Engl J Med 358(11):1109–1117Google Scholar
  77. 77.
    Janakiraman V, Kwiatkowski C et al (2015) Application of high-throughput minibioreactor system for systematic scale-down modeling, process characterization, and control strategy development. Biotechnol Prog 31:1623–1632Google Scholar
  78. 78.
    Bruhlmann D, Jordan M, Hemberger J, Sauer M, Stettler M, Broly H (2015) Tailoring recombinant protein quality by rational media design. Biotechnol Prog 31(3):615–629Google Scholar
  79. 79.
    Rouiller Y, Bielser JM, Bruhlmann D, Jordan M, Broly H, Stettler M (2016) Screening and assessment of performance and molecule quality attributes of industrial cell lines across different fed-batch systems. Biotechnol Prog 32(1):160–170Google Scholar
  80. 80.
    Kildegaard HF, Fan Y, Sen JW, Larsen B, Andersen MR (2016) Glycoprofiling effects of media additives on IgG produced by CHO cells in fed-batch bioreactors. Biotechnol Bioeng 113(2):359–366Google Scholar
  81. 81.
    Wong NS, Wati L, Nissom PM, Feng HT, Lee MM, Yap MG (2010) An investigation of intracellular glycosylation activities in CHO cells: effects of nucleotide sugar precursor feeding. Biotechnol Bioeng 107(2):321–336Google Scholar
  82. 82.
    Dicker M, Strasser R (2015) Using glyco-engineering to produce therapeutic proteins. Expert Opin Biol Ther 15(10):1501–1516Google Scholar
  83. 83.
    Jacobs PP, Callewaert N (2009) N-Glycosylation engineering of biopharmaceutical expression systems. Curr Mol Med 9(7):774–800Google Scholar
  84. 84.
    von Horsten HH, Ogorek C, Blanchard V, Demmler C, Giese C, Winkler K, Kaup M, Berger M, Jordan I, Sandig V (2010) Production of non-fucosylated antibodies by co-expression of heterologous GDP-6-deoxy-D-lyxo-4-hexulose reductase. Glycobiology 20(12):1607–1618Google Scholar
  85. 85.
    Weikert S, Papac D, Briggs J, Cowfer D, Tom S, Gawlitzek M, Lofgren J, Mehta S, Chisholm V, Modi N, Eppler S, Carroll K, Chamow S, Peers D, Berman P, Krummen L (1999) Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins. Nat Biotechnol 17(11):1116–1121Google Scholar
  86. 86.
    Berry B, Dobrowsky T, Timson R, Wiltberger K, Kshirsagar R, Ryll T (2015) Quick generation of Raman spectroscopy based in-process glucose control to influence biopharmaceutical protein product quality during mammalian cell culture. Biotechnol Prog 32(1):224–234Google Scholar
  87. 87.
    Quan C, Alcala E, Petkovska I, Matthews D, Canova-Davis E, Taticek R, Ma S (2008) A study in glycation of a therapeutic recombinant humanized monoclonal antibody: where it is, how it got there, and how it affects charge-based behavior. Anal Biochem 373(2):179–191Google Scholar
  88. 88.
    Abu-Absi NR, Kenty BM, Cuellar ME, Borys MC, Sakhamuri S, Strachan DJ, Hausladen MC, Li ZJ (2011) Real time monitoring of multiple parameters in mammalian cell culture bioreactors using an in-line Raman spectroscopy probe. Biotechnol Bioeng 108(5):1215–1221Google Scholar
  89. 89.
    Andre S, Cristau LS, Gaillard S, Devos O, Calvosa E, Duponchel L (2015) In-line and real-time prediction of recombinant antibody titer by in situ Raman spectroscopy. Anal Chim Acta 892:148–152Google Scholar
  90. 90.
    Berry B, Moretto J, Matthews T, Smelko J, Wiltberger K (2015) Cross-scale predictive modeling of CHO cell culture growth and metabolites using Raman spectroscopy and multivariate analysis. Biotechnol Prog 31(2):566–577Google Scholar

Copyright information

© Springer International Publishing AG 2016

Open Access This chapter is distributed under the terms of the Creative Commons Attribution Noncommercial License, which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Authors and Affiliations

  1. 1.Technical Development, BiogenCambridgeUSA
  2. 2.Technical Operations, ImmunoGen, Inc.WalthamUSA

Personalised recommendations