Manufacturing of Proteins and Antibodies: Chapter Downstream Processing Technologies

  • Richard Turner
  • Adrian Joseph
  • Nigel Titchener-Hooker
  • Jean BenderEmail author
Part of the Advances in Biochemical Engineering/Biotechnology book series (ABE, volume 165)


Cell harvesting is the separation or retention of cells and cellular debris from the supernatant containing the target molecule Selection of harvest method strongly depends on the type of cells, mode of bioreactor operation, process scale, and characteristics of the product and cell culture fluid. Most traditional harvesting methods use some form of filtration, centrifugation, or a combination of both for cell separation and/or retention. Filtration methods include normal flow depth filtration and tangential flow microfiltration. The ability to scale down predictably the selected harvest method helps to ensure successful production and is critical for conducting small-scale characterization studies for confirming parameter targets and ranges. In this chapter we describe centrifugation and depth filtration harvesting methods, share strategies for harvest optimization, present recent developments in centrifugation scale-down models, and review alternative harvesting technologies.


Capillary shear Centrifugation Continuous centrifugation Depth filter Disk-stack centrifuge Filter capacity Harvest Mammalian cell Primary recovery Scale-down Sigma factor Single-use centrifuge Tangential flow filtration 


  1. 1.
    Ambler CM (1959) The theory of scaling up laboratory data for the sedimentation type centrifuge. Biotechnol Bioeng 1(2):185–205CrossRefGoogle Scholar
  2. 2.
    Bender J, Brown A, Winter C (2002) Scale-up of a disk-stack centrifuge for CHO harvest, Downstream Gab '02 abstracts. Amersham Biosciences, pp 10–11Google Scholar
  3. 3.
    Boychyn M, Yim S, Shamlou PA, Bulmer M, More J, Hoare M (2001) Characterization of flow intensity in continuous centrifuges for the development of laboratory mimics. Chem Eng Sci 56(16):4759–4770CrossRefGoogle Scholar
  4. 4.
    Boychyn M, Yim S, Bulmer M, More J, Bracewell D, Hoare M (2004) Performance prediction of industrial centrifuges using scale-down models. Bioprocess Biosyst Eng 26(6):385–391CrossRefGoogle Scholar
  5. 5.
    Chu L, Robinson DK (2001) Industrial choices for protein production by large-scale cell culture. Curr Opin Biotechnol 12(2):180–187CrossRefGoogle Scholar
  6. 6.
    Fiore JV, Olson WP, Holst SL (1980) Depth filtration. In: Curling JM (ed) Methods of plasma protein fractionation. Academic Press, New YorkGoogle Scholar
  7. 7.
    Gerba CP, Hou K (1985) Endotoxin removal by charge-modified filters. Appl Environ Microbiol 50:1375–1377PubMedPubMedCentralGoogle Scholar
  8. 8.
    Glyn J (2009) Process scale precipitation of impurities in mammalian cell culture broth. In: Gottschalk U (ed) Process scale purification of antibodies. Wiley, HobokenGoogle Scholar
  9. 9.
    Han B, Akeprathumchai S, Wickramasinghe SR, Qian X (2003) Flocculation of biological cells: experiment vs. theory. AIChE J 49(7):1687–1701CrossRefGoogle Scholar
  10. 10.
    Hutchison N, Bingham N, Murrell N, Farid S, Hoare M (2006) Shear stress analysis of mammalian cell suspensions for prediction of industrial centrifugations and its verification. Biotechnol Bioeng 95:483–491CrossRefGoogle Scholar
  11. 11.
    Iammarino M, Nti-Gyabaah J, Chandler M, Roush D, Goklen K (2007) Impact of cell density and viability on primary clarification of mammalian cell broth. Bioprocess Int 5:38–50Google Scholar
  12. 12.
    Joseph A, Kenty B, Mollet M, Hwang K, Rose S, Goldrick S, Bender J, Farid SS, Titchener-Hooker N (2016) A scale-down mimic for mapping the process performance of centrifugation, depth and sterile filtration. Biotechnol Bioeng. doi: 10.1002/bit.25967CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Kang Y, Hamzik J, Felo M, Qi B, Lee J, Ng S, Liebisch G, Shanehsaz B, Singh N, Persaud K, Ludwig D, Balderes P (2013) Development of a novel and efficient cell culture flocculation process using a stimulus responsive polymer to streamline antibody purification processes. Biotechnol Bioeng 110(11):2928–2937CrossRefGoogle Scholar
  14. 14.
    Knight RA, Ostreicher EA (1998) Charge-modified filter media. In: Meltzer TH, Jornitz MW (eds) Filtration in the biopharmaceutical industry. Marcel Dekker Inc., New YorkGoogle Scholar
  15. 15.
    Ko H, Bhatia R (2012) Evaluation of single-use fluidized bed centrifuge system for mammalian cell harvesting. BioPharm Int 25(11):34–40Google Scholar
  16. 16.
    Kompala DS, Ozturk SS (2005) Optimization of high cell density perfusion bioreactors. In: Ozturk SS, Hu W-S (eds) Cell culture technology for pharmaceutical and cell-based therapies. Taylor & Francis, New York, pp 387–416Google Scholar
  17. 17.
    Mehta S (2014) Automated single-use centrifugation solution for diverse biomanufacturing process. In: Subramanian G (ed) Continuous processing in pharmaceutical manufacturing. Wiley-VCH Verlag GmbH & Co. KGaA, WeinheimGoogle Scholar
  18. 18.
    Pinheiro H, Cabral JMS (1993) Centrifugation. In: Kennedy JF, Cabral JMS (eds) Recovery processes for biological materials. Wiley, Chichester, p. 145Google Scholar
  19. 19.
    Riske F, Schroeder J, Belliveau J, Kang X, Kutzko J, Menon MK (2007) The use of chitosan as a flocculant in mammalian cell culture dramatically improves clarification throughput without adversely impacting monoclonal antibody recovery. J Biotechnol 128(4):813–823CrossRefGoogle Scholar
  20. 20.
    Roush DJ, Lu Y (2008) Advances in primary recovery: centrifugation and membrane technology. Biotechnol Prog 24(3):488–495CrossRefGoogle Scholar
  21. 21.
    Tait A, Aucamp J, Bugeon A, Hoare M (2009) Ultra scale-down prediction using microwell technology of the industrial scale clarification characteristics by centrifugation of mammalian cell broths. Biotechnol Bioeng 104(2):321–331CrossRefGoogle Scholar
  22. 22.
    Titchener-Hooker N, Dunnill P, Hoare M (2008) Micro biochemical engineering to accelerate the design of industrial-scale downstream processes for biopharmaceutical proteins. Biotechnol Bioeng 100(3):473–487CrossRefGoogle Scholar
  23. 23.
    Trexler-Schmidt M, Sargis S, Chiu J, Sze-Khoo S, Mun M, Kao YH, Laird MW (2010) Identification and prevention of antibody disulfide bond reduction during cell culture manufacturing. Biotechnol Bioeng 106(3):452–461PubMedGoogle Scholar
  24. 24.
    Van der Meer T, Minow B, Lagrange B, Krumbein F, Rolin F (2014) Diatomaceous earth filtration; innovative single-use concepts for clarification of high-density mammalian cell cultures. BioProcess Int 12(8)Google Scholar
  25. 25.
    Van Reis R, Zydney A (2001) Membrane separations in biotechnology. Curr Opin Biotechnol 12:208–211CrossRefGoogle Scholar
  26. 26.
    Voisard D, Meuwly F, Ruffieux PA, Baer G, Kadouri A (2003) Potential of cell retention techniques for large-scale high-density perfusion culture of suspended mammalian cells. Biotechnol Bioeng 82(7):751–765CrossRefGoogle Scholar
  27. 27.
    Westoby M, Rogers JK, Haverstock R, Romero J, Pieracci J (2011) Modeling industrial centrifugation of mammalian cell culture using a capillary based scale-down system. Biotechnol Bioeng 108(5):989–998CrossRefGoogle Scholar
  28. 28.
    Yigzaw Y, Piper R, Tran M, Shukla AA (2006) Exploitation of the adsorptive properties of depth filters for host cell protein removal during monoclonal antibody purification. Biotechnol Prog 22:288–296CrossRefGoogle Scholar

Further Reading

  1. Akeprathumachai S (2004) Murine leukemia virus clearance by flocculation and microfiltration. Biotechnol Bioeng 88:880–889CrossRefGoogle Scholar
  2. Castilho LR, Medronho RA (2002) Cell retention devices for suspended-cell perfusion cultures. Adv Biochem Eng Biotechnol 74:129–169PubMedGoogle Scholar
  3. Dave P, Dizon-Maspat J, Cano T (2009) Evaluation and implementation of a single-stage multimedia harvest depth filter for a large-scale antibody process. BioProcess Int 7:S8–S17CrossRefGoogle Scholar
  4. Hill P, Bender J (2007) Cell harvesting. In: Stacey G, Davis J (eds) Medicines for animal cell cultures. Wiley, Boca RatonGoogle Scholar
  5. Hove S, Cacace B, Felo M, Chefer K (2010) Development of a robust clarification process for high density mammalian cell culture processes. Recovery of Biological Products XIV, Squaw Creek, Lake Tahoe, August 1–5, 2010Google Scholar
  6. Jaber J, Moya W, Hamzik J, Boudif A, Zhang Y, Soice N (2011) Stimulus responsive polymers for the purification of biomolecules. US patent 0313066 A1Google Scholar
  7. Jayapal K (2007) Recombinant protein therapeutics from CHO cells: 20 years and counting. Chem Eng Prog 103:40–47Google Scholar
  8. Joseph A, Kenty B, Mollet M, Hwang K, Rose S, Goldrick S, Bender J, Farid SS, Titchener-Hooker N (2016) A scale-down mimic for mapping the process performance of centrifugation, depth and sterile filtration. Biotechnol Bioeng doi: 10.1002/bit.25967CrossRefPubMedPubMedCentralGoogle Scholar
  9. Kang Y, Ng S, Lee J, Adaelu J, Qi B, Persaud K, Ludwig D, Balderes P (2012) Development of an alternative monoclonal antibody polishing step. Biopharm Int 25(5):34–45Google Scholar
  10. Kelley B, Blank G, Lee A (2009) Downstream processing of monoclonal antibodies: current practices and future opportunities. In: Gottschalk U (ed) Process scale purification of antibodies. Wiley, HobokenGoogle Scholar
  11. Kilander J, Blomström S, Rasmuson A (2007) Scale-up behavior in stirred square flocculation tanks. Chem Eng Sci 62:1606–1618CrossRefGoogle Scholar
  12. Kim J, Akeprathumachai S, Wichramasinghe SR (2001) Flocculation to enhance microfiltration. J Membr Sci 182:161–172CrossRefGoogle Scholar
  13. Laukel M, Rogge P, Dudziak G (2011) Disposable downstream processing for clinical manufacturing. Current capabilities and limitations. BioProcess Int 9(5):14–21Google Scholar
  14. Liu HF, Ma J, Winter C, Bayer R (2010) Recovery and purification process development for monoclonal antibody production. mAbs 2(5):480–499CrossRefGoogle Scholar
  15. Lutz H, Abbott I, Blanchard M, Parampalli A, Setiabudi G, Chiruvolu V, Noguchi M (2009) Considerations for scaling-up depth filtration of harvested cell culture fluid. BioPharm Int 22(3):1–13Google Scholar
  16. McNerney T, Thomas A, Senczuk A, Carvalho J, Chinniah S, Zhao X, Pallitto M, Piper R (2011) PDADMAC flocculation of CHO cells: a centrifuge-less harvest process for mAbs. 241st ACS National Meeting & Exposition, Anaheim, CA. p BIOT-302, February 2011Google Scholar
  17. Mullan B, Dravis B, Lim A, Clarke A, Janes S, Lambooy P, Olson D, O’Riordan T, Ricart B, Tulloch AG (2011) Disulphide bond reduction of a therapeutic monoclonal antibody during cell culture manufacturing operations. BMC Proceedings 5 (Suppl 8):110CrossRefGoogle Scholar
  18. Pailhes M, Lambalot C, Barloga R (2004) Integration of centrifuges with depth filtration for optimized cell culture fluid clarification processes. BioProcessing J 3(3):55–58CrossRefGoogle Scholar
  19. Pegel A, Reiser S, Steurenthaler M, Klein S (2011) Evaluating disposable depth filtration platforms for mAb harvest clarification. BioProcess Int 9(9): 52–56Google Scholar
  20. Przybycien T, Narahari S, Steele L (2004) Alternative bioseparation operations: life beyond packed-bed chromatography. Curr Opin Biotechnol 15:469–478CrossRefGoogle Scholar
  21. Rechtsteiner H (2004) Cell separation from mammalian suspension cultures. BioProcess Int 2:60–62Google Scholar
  22. Rios M (2012) A decade of harvesting methods. BioProcess Int 10:28–31Google Scholar
  23. Romero J, Chrostowski J, De Vilmorin PG, Case JY (2010) Method of isolating biomacromolecules using low pH and divalent cations. USA patent 2010/0145022 A1Google Scholar
  24. Sellick I (2003) Improve product recovery during cell harvesting. BioProcess Int 1:62–65Google Scholar
  25. Shan J, Xia J, Guo Y, Zhang X (1996) Flocculation of cell, cell debris and soluble protein with methacryloyloxyethyl trimethylammonium chloride—acrylonitrile copolymer. J Biotechnol 49:173–178CrossRefGoogle Scholar
  26. Shpritzer R, Vicik S, Orlando S, Acharya H, Coffman JL (2006) Calcium phosphate flocculation of antibody-producing mammalian cells at pilot scale. The 232nd ACS National Meeting; San Francisco, CA. p BIOT-80, September 10–14, 2006Google Scholar
  27. Shukla A, Thömmes J (2010) Recent advances in large-scale production of monoclonal antibodies and related proteins, Trends Biotechnol 28(5):253–261CrossRefGoogle Scholar
  28. Shukla A, Kandula JR, Gottschalk U (2009) Harvest and recovery of monoclonal antibodies: cell removal and clarification. In: Gottschalk U (ed) Process scale purification of antibodies. Wiley, HobokenGoogle Scholar
  29. Suh CW, Kim SE, Lee EK (1997) Effects of filter additives on cake filtration performance. Korean J Chem Eng 14:241–244CrossRefGoogle Scholar
  30. Van Reis R, Leonard LC, Hsu CC, Builder S (1991) Industrial scale harvest of proteins from mammalian cell culture by tangential flow filtration. Biotechnol Bioeng 38:413–422.CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  • Richard Turner
    • 1
  • Adrian Joseph
    • 2
  • Nigel Titchener-Hooker
    • 2
  • Jean Bender
    • 1
    Email author
  1. 1.MedImmune LLC Gaithersburg HeadquartersGaithersburgUSA
  2. 2.The Advanced Centre of Biochemical Engineering, Department of Biochemical EngineeringUniversity College LondonLondonUK

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