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Disposable polymeric cryogel bioreactor matrix for therapeutic protein production

Nature Protocols volume 8, pages 821–835 (2013)Cite this article

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Abstract

Low cost and high efficiency make disposable bioreactors feasible for small-scale therapeutic development and initial clinical trials. We have developed a cryogel-based disposable bioreactor matrix, which has been used for production of protein therapeutics such as urokinase and monoclonal antibodies (mAbs). The protocol discusses the application of a cryogel bioreactor for mAb production. Cryogels composed of either polyacrylamide (PAAm) coupled to gelatin or semi-interpenetrating PAAm-chitosan are synthesized by free-radical polymerization at −12 °C. Hybridoma cells are immobilized over the cryogel bioreactor and incubated for 48 h. Medium is circulated thereafter at 0.2 ml min−1 and bioreactors can be run continuously for 60 d. The cryogel-based packed-bed bioreactor can be formulated as a monolith or as beads; it also has an efficiency four times what can be obtained using a tissue-culture flask, a high surface-to-volume ratio and effective nutrient transport. After incubation, the bioreactor setup will take about 60 min using a pre-prepared sterilized cryogel.

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Figure 1: Integrated setup for the production and capture of urokinase.
Figure 2: Urokinase production using a cryogel bioreactor.
Figure 3: Diagrammatic representation of cryogel bioreactor setup.
Figure 4: Digital image of a cryogel bioreactor setup.
Figure 5: Production of mAbs in a PAAC cryogel matrix.
Figure 6: Growth and attachment of hybridoma cells in a PAAm-gelatin cryogel bioreactor.
Figure 7: mAb production in PAAm-gelatin cryogel.

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References

  1. Chu, L. & Robinson, D.K. Industrial choices for protein production by large-scale cell culture. Curr. Opin. Biotechnol. 12, 180–187 (2001).

    Article  CAS  Google Scholar 

  2. Jain, E. & Kumar, A. Upstream processes in antibody production: evaluation of critical parameters. Biotechnol. Adv. 26, 46–72 (2008).

    Article  CAS  Google Scholar 

  3. Warnock, J.N. & Al-Rubeai, M. Bioreactor systems for the production of biopharmaceuticals from animal cells. Biotechnol. Appl. Biochem. 45, 1–12 (2006).

    Article  CAS  Google Scholar 

  4. Eibl, R., Kaiser, S., Lombriser, R. & Eibl, D. Disposable bioreactors: the current state-of-the-art and recommended applications in biotechnology. Appl. Microbiol. Biotechnol. 86, 41–49 (2010).

    Article  CAS  Google Scholar 

  5. Poles-Lahille, A. et al. Disposable bioreactors: from process development to production. BMC Proc. 5 (suppl. 8), P2 (2011).

    Article  Google Scholar 

  6. Langer, E.S. Trends in perfusion bioreactors: next revolution in bioprocessing. BioProcess Int. 9, 18–22 (2011).

    Google Scholar 

  7. Meuwly, F., Ruffieux, P.-A., Kadouri, A. & von Stockar, U. Packed-bed bioreactors for mammalian cell culture: bioprocess and biomedical applications. Biotechnol. Adv. 25, 45–56 (2007).

    Article  CAS  Google Scholar 

  8. Cadwell, J.J.S. New developments in hollow-fiber cell culture. Curr. Pharm. Biotechnol. 6, 397–403 (2005).

    Article  Google Scholar 

  9. Bansal, V., Roychoudhury, P.K., Mattiasson, B. & Kumar, A. Recovery of urokinase from integrated mammalian cell culture cryogel bioreactor and purification of the enzyme using p-aminobenzamidine affinity chromatography. J. Mol. Recognit. 19, 332–339 (2006).

    Article  CAS  Google Scholar 

  10. Jain, E., Karande, A.A. & Kumar, A. Supermacroporous polymer-based cryogel bioreactor for monoclonal antibody production in continuous culture using hybridoma cells. Biotechnol. Prog. 27, 170–180 (2011).

    Article  CAS  Google Scholar 

  11. Kumar, A. et al. Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices. Biotechnol. Bioeng. 93, 636–646 (2006).

    Article  CAS  Google Scholar 

  12. Nilsang, S. et al. Monoclonal antibody production using a new supermacroporous cryogel bioreactor. Biotechnol. Prog. 23, 932–939 (2007).

    Article  CAS  Google Scholar 

  13. Nilsang, S. et al. Three-dimensional culture for monoclonal antibody production by hybridoma cells immobilized in macroporous gel particles. Biotechnol. Prog. 24, 1122–1131 (2008).

    Article  CAS  Google Scholar 

  14. Lozinsky, V.I. et al. Polymeric cryogels as promising materials of biotechnological interest. Trends Biotechnol. 21, 445–451 (2003).

    Article  CAS  Google Scholar 

  15. Dainiak, M.B., Kumar, A., Galaev, I.Y. & Mattiasson, B. Detachment of affinity-captured bioparticles by elastic deformation of a macroporous hydrogel. Proc. Natl. Acad. Sci. USA 103, 849–854 (2006).

    Article  CAS  Google Scholar 

  16. Plieva, F.M., Galaev, I.Y., Noppe, W. & Mattiasson, B. Cryogel applications in microbiology. Trends Microbiol. 16, 543–551 (2008).

    Article  CAS  Google Scholar 

  17. Plieva, F.M. et al. Pore structure in supermacroporous polyacrylamide based cryogels. Soft Matter 1, 303–309 (2005).

    Article  CAS  Google Scholar 

  18. Persson, P. et al. Characterization of a continuous supermacroporous monolithic matrix for chromatographic separation of large bioparticles. Biotechnol. Bioeng. 88, 224–236 (2004).

    Article  CAS  Google Scholar 

  19. Jain, E. & Kumar, A. Designing supermacroporous cryogels based on polyacrylonitrile and a polyacrylamide-chitosan semi-interpenetrating network. J. Biomater. Sci. Polym. Ed. 20, 877–902 (2009).

    Article  CAS  Google Scholar 

  20. Kumar, A., Plieva, F.M., Galaev, I.Y. & Mattiasson, B. Affinity fractionation of lymphocytes using a monolithic cryogel. J. Immunol. Methods 283, 185–194 (2003).

    Article  CAS  Google Scholar 

  21. Kumar, A., Bansal, V., Andersson, J., Roychoudhury, P.K. & Mattiasson, B. Supermacroporous cryogel matrix for integrated protein isolation—immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line. J. Chromatogr. 1103, 35–42 (2006).

    Article  CAS  Google Scholar 

  22. Roychoudhury, P.K., Khaparde, S.S., Mattiasson, B. & Kumar, A. Synthesis, regulation and production of urokinase using mammalian cell culture: a comprehensive review. Biotechnol. Adv. 24, 514–528 (2006).

    Article  CAS  Google Scholar 

  23. Babac, C. et al. Binding of antibodies to concanavalin A–modified monolithic cryogel. React. Funct. Polym. 66, 1263–1271 (2006).

    Article  CAS  Google Scholar 

  24. Deraz, S., Plieva, F.M., Galaev, I.Y., Karlsson, E.N. & Mattiasson, B. Capture of bacteriocins directly from non-clarified fermentation broth using macroporous monolithic cryogels with phenyl ligands. Enzyme Microb. Technol. 40, 786–793 (2007).

    Article  CAS  Google Scholar 

  25. Williams, S.L., Eccleston, M.E. & Slater, N.K.H. Affinity capture of a biotinylated retrovirus on macroporous monolithic adsorbents: Towards a rapid single-step purification process. Biotechnol. Bioeng. 89, 783–787 (2005).

    Article  CAS  Google Scholar 

  26. Srivastava, A., Shakya, A.K. & Kumar, A. Boronate affinity chromatography of cells and biomacromolecules using cryogel matrices. Enzyme Microb. Technol. 51, 373–381 (2012).

    Article  CAS  Google Scholar 

  27. Kumar, A. et al. Affinity binding of cells to cryogel adsorbents with immobilized specific ligands: effect of ligand coupling and matrix architecture. J. Mol. Recognit. 18, 84–93 (2005).

    Article  CAS  Google Scholar 

  28. Kumar, A. & Srivastava, A. Cell separation using cryogel-based affinity chromatography. Nat. Protoc. 5, 1737–1747 (2010).

    Article  CAS  Google Scholar 

  29. Lozinsky, V.I. & Plieva, F.M. Poly(vinyl alcohol) cryogels employed as matrices for cell immobilization. 3. Overview of recent research and developments. Enzyme Microb. Technol. 23, 227–242 (1998).

    Article  CAS  Google Scholar 

  30. Lozinsky, V.I., Plieva, F.M., Galaev, I.Y. & Mattiasson, B. The potential of polymeric cryogels in bioseparation. Bioseparation 10, 163–188 (2001).

    Article  CAS  Google Scholar 

  31. Plieva, F.M. & Mattiasson, B. Macroporous gel particles as novel sorbent materials: rational design. Ind. Eng. Chem. Res. 47, 4131–4141 (2008).

    Article  CAS  Google Scholar 

  32. Bhat, S., Tripathi, A. & Kumar, A. Supermacroprous chitosan-agarose-gelatin cryogels: in vitro characterization and in vivo assessment for cartilage tissue engineering. J. R. Soc. Interface 8, 540–554 (2011).

    Article  CAS  Google Scholar 

  33. Bolgen, N. et al. Three-dimensional ingrowth of bone cells within biodegradable cryogel scaffolds in bioreactors at different regimes. Tissue Eng. Part A 14, 1743–1750 (2008).

    Article  Google Scholar 

  34. Dainiak, M.B. et al. Gelatin-fibrinogen cryogel dermal matrices for wound repair: preparation, optimisation and in vitro study. Biomaterials 31, 67–76 (2010).

    Article  CAS  Google Scholar 

  35. Tripathi, A., Kathuria, N. & Kumar, A. Elastic and macroporous agarose-gelatin cryogels with isotropic and anisotropic porosity for tissue engineering. J. Biomed. Mater. Res. A 90, 680–694 (2009).

    Article  Google Scholar 

  36. Reddy, B.M., Karande, A.A. & Adiga, P.R. A common epitope of β-lactoglobulin and serum retinol-binding proteins: elucidation of its core sequence using synthetic peptides. Mol. Immunol. 29, 511–516 (1992).

    Article  CAS  Google Scholar 

  37. Mo, J.A. & Holmdahl, R. The B cell response to autologous type II collagen: biased V gene repertoire with V gene sharing and epitope shift. J. Immunol. 157, 2440–2448 (1996).

    CAS  PubMed  Google Scholar 

  38. Nandakumar, K.S. et al. Induction of arthritis by single monoclonal IgG anti-collagen type II antibodies and enhancement of arthritis in mice lacking inhibitory FcγRIIB. Eur. J. Immunol. 33, 2269–2277 (2003).

    Article  CAS  Google Scholar 

  39. Favre, E., Pugeaud, P. & Peringer, P. Automated HPLC monitoring of glucose, glutamine, lactate and alanine on suspended mammalian cell reactors. Biotechnol. Tech. 4, 315–320 (1990).

    CAS  Google Scholar 

  40. McCullough, H. The determination of ammonia in whole blood by a direct colorimetric method. Clin. Chim. Acta 17, 297–304 (1967).

    Article  CAS  Google Scholar 

  41. Plieva, F., Xiao, H.T., Galaev, I.Y., Bergenstahl, B. & Mattiasson, B. Macroporous elastic polyacrylamide gels prepared at subzero temperatures: control of porous structure. J. Mater. Chem. 16, 4065–4073 (2006).

    Article  CAS  Google Scholar 

  42. Chisti, Y. & MooYoung, M. Bioprocess intensification through bioreactor engineering. Chem. Eng. Res. Des. 74, 575–583 (1996).

    CAS  Google Scholar 

  43. Stump, D.C., Thienpont, M. & Collen, D. Urokinase-related proteins in human urine—isolation and characterization of single-chain urokinase (prourokinase) and urokinase-inhibitor complex. J. Biol. Chem. 261, 1267–1273 (1986).

    CAS  PubMed  Google Scholar 

  44. Lewis, L.J. Plasminogen activator (urokinase) from cultured cells. Thromb. Haemost. 42, 895–900 (1979).

    Article  CAS  Google Scholar 

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Acknowledgements

We acknowledge the financial support from the Department of Biotechnology, Ministry of Science and Technology, Government of India; Protista Biotechnology Sweden; and the Swedish Research Council/SIDA Research Link Project. We are also thankful to all other co-workers, especially V. Bansal, S. Nilsang and B. Mattiasson who have contributed to the development of this work in one way or another. We also thank A. Karande for her kind help in providing the hybridoma cell lines and for mAb analysis.

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  1. Era Jain

    Present address: Present address: Department of Chemical Engineering, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA.,

Authors and Affiliations

  1. Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur, India

    Era Jain & Ashok Kumar

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Contributions

Both the authors (E.J. and A.K.) conceived, planned and conducted experiments on different aspects of this study. A.K. was responsible for supervising all the work described in this protocol.

Corresponding author

Correspondence to Ashok Kumar.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

Determination of Monoclonal Antibody; Analysis of Glucose in spent media samples; Determination of Ammonia; Simultaneous Determination of Glucose, Lactic acid, and Glutamine by HPLC; and Determination of Urokinase Activity (PDF 369 kb)

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Jain, E., Kumar, A. Disposable polymeric cryogel bioreactor matrix for therapeutic protein production. Nat Protoc 8, 821–835 (2013). https://doi.org/10.1038/nprot.2013.027

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