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Current Pharmaceutical Biotechnology

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ISSN (Print): 1389-2010
ISSN (Online): 1873-4316

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Research Article

Multivariate Optimization of the Refolding Process of an Incorrectly Folded Fc-Fusion Protein in a Cell Culture Broth

Author(s): Hossein Behrouz, Behnaz Molavi, Ata Tavakoli, Mansoureh Askari, Shayan Maleknia, Fereidoun Mahboudi and Mehdi Khodadadian*

Volume 21, Issue 3, 2020

Page: [226 - 235] Pages: 10

DOI: 10.2174/1389201020666191002144424

Price: $65

Abstract

Background: Protein misfolding is a common problem in large-scale production of recombinant proteins, which can significantly reduce the yield of the process.

Objective: In this work, we aimed at treating a cell culture broth containing high levels (>45%) of incorrectly folded Fc-fusion proteins by a simple redox buffer system in order to increase the proportion of the protein with correct conformation.

Methods: Multi-variable process optimization was firstly conducted at a small scale (25 mL), employing an experimental design methodology. After identifying the key variables using a resolution IV Fractional Factorial Design (FFD), the process was then optimized by the Central Composite Design (CCD).

Results: The optimal conditions for the refolding reaction were 340 mM Tris-base, 6.0 mM L-cysteine, 0.5 mM L-cystine, a buffer pH of 9.0, a reaction temperature of 8.5ºC and a reaction time of 24 h. Based on the treatment conditions obtained at a small scale, the process was further scaled up to 4500- L. The misfolded content was always less than 20%. The reaction can proceed well in the absence of chemical additives, such as chaotropic agents, aggregation suppressors, stabilizers and chelators.

Conclusion: The refolding process increases the fraction of active protein in the original broth reducing the burden on downstream purification steps markedly.

Keywords: Protein misfolding, protein refolding, fusion protein, recombinant DNA technology, cell culture broth composite design.

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[1]
Glaesner, W.; Vick, A.M.; Millican, R.; Ellis, B.; Tschang, S.H.; Tian, Y.; Bokvist, K.; Brenner, M.; Koester, A.; Porksen, N.; Etgen, G.; Bumol, T. Engineering and characterization of the long-acting glucagon-like peptide-1 analogue LY2189265, an Fc fusion protein. Diabetes Metab. Res. Rev., 2010, 26(4), 287-296.
[http://dx.doi.org/10.1002/dmrr.1080] [PMID: 20503261]
[2]
Chan, A.C.; Carter, P.J. Therapeutic antibodies for autoimmunity and inflammation. Nat. Rev. Immunol., 2010, 10(5), 301-316.
[http://dx.doi.org/10.1038/nri2761] [PMID: 20414204]
[3]
Weiner, L.M.; Surana, R.; Wang, S. Monoclonal antibodies: Versatile platforms for cancer immunotherapy. Nat. Rev. Immunol., 2010, 10(5), 317-327.
[http://dx.doi.org/10.1038/nri2744] [PMID: 20414205]
[4]
Reichert, J.M. Antibodies to watch in 2010. MAbs, 2010, 2(1), 84-100.
[http://dx.doi.org/10.4161/mabs.2.1.10677] [PMID: 20065640]
[5]
Reichert, J.M. Which are the antibodies to watch in 2012? MAbs, 2012, 4(1), 1-3.
[http://dx.doi.org/10.4161/mabs.4.1.18719] [PMID: 22327425]
[6]
Dumont, J.A.; Liu, T.; Low, S.C.; Zhang, X.; Kamphaus, G.; Sakorafas, P.; Fraley, C.; Drager, D.; Reidy, T.; McCue, J.; Franck, H.W.G.; Merricks, E.P.; Nichols, T.C.; Bitonti, A.J.; Pierce, G.F.; Jiang, H. Prolonged activity of a recombinant factor VIII-Fc fusion protein in hemophilia A mice and dogs. Blood, 2012, 119(13), 3024-3030.
[http://dx.doi.org/10.1182/blood-2011-08-367813] [PMID: 22246033]
[7]
Yu, H.K.; Lee, H.J.; Ahn, J.H.; Lim, I.H.; Moon, J.H.; Yoon, Y.; Yi, L.S.H.; Kim, S.J.; Kim, J.S. Immunoglobulin Fc domain fusion to apolipoprotein(a) kringle V significantly prolongs plasma half-life without affecting its anti-angiogenic activity. Protein Eng. Des. Sel., 2013, 26(6), 425-432.
[http://dx.doi.org/10.1093/protein/gzt015] [PMID: 23571426]
[8]
Wang, H.; Davis, J.S.; Wu, X. Immunoglobulin Fc domain fusion to TRAIL significantly prolongs its plasma half-life and enhances its antitumor activity. Mol. Cancer Ther., 2014, 13(3), 643-650.
[http://dx.doi.org/10.1158/1535-7163.MCT-13-0645] [PMID: 24431076]
[9]
Knauf, M.J.; Bell, D.P.; Hirtzer, P.; Luo, Z.P.; Young, J.D.; Katre, N.V. Relationship of effective molecular size to systemic clearance in rats of recombinant interleukin-2 chemically modified with water-soluble polymers. J. Biol. Chem., 1988, 263(29), 15064-15070.
[PMID: 3049599]
[10]
Salahuddin, P.; Siddiqi, M.K.; Khan, S.; Abdelhameed, A.S.; Khan, R.H. Mechanisms of protein misfolding: Novel therapeutic approaches to protein-misfolding diseases. J. Mol. Struct., 2016, 1123, 311-326.
[http://dx.doi.org/10.1016/j.molstruc.2016.06.046]
[11]
Shamsi, T.N.; Athar, T.; Parveen, R.; Fatima, S. A review on protein misfolding, aggregation and strategies to prevent related ailments. Int. J. Biol. Macromol., 2017, 105(Pt 1), 993-1000.
[http://dx.doi.org/10.1016/j.ijbiomac.2017.07.116] [PMID: 28743576]
[12]
Huang, C.J.; Lin, H.; Yang, X. Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements. J. Ind. Microbiol. Biotechnol., 2012, 39(3), 383-399.
[http://dx.doi.org/10.1007/s10295-011-1082-9] [PMID: 22252444]
[13]
Shin, H.C. Protein folding, misfolding, and refolding of therapeutic proteins. Biotechnol. Bioproc. E, 2001, 6, 237-243.
[http://dx.doi.org/10.1007/BF02931984]
[14]
Maas, C.; Hermeling, S.; Bouma, B.; Jiskoot, W.; Gebbink, M.F.B.G. A role for protein misfolding in immunogenicity of biopharmaceuticals. J. Biol. Chem., 2007, 282(4), 2229-2236.
[http://dx.doi.org/10.1074/jbc.M605984200] [PMID: 17135263]
[15]
Preu, M.; Guyot, D.; Petz, M. Development of a gas chromatography-mass spectrometry method for the analysis of aminoglycoside antibiotics using experimental design for the optimisation of the derivatisation reactions. J. Chromatogr. A, 1998, 818(1), 95-108.
[http://dx.doi.org/10.1016/S0021-9673(98)00537-8] [PMID: 9770307]
[16]
Nowak, M.; Seubert, A. Application of experimental design for the characterisation of a novel elution system for high-capacity anion chromatography with suppressed conductivity detection. J. Chromatogr. A, 1999, 855(1), 91-109.
[http://dx.doi.org/10.1016/S0021-9673(99)00683-4] [PMID: 10514975]
[17]
Shah, M.; Pathak, K. Development and statistical optimization of solid lipid nanoparticles of simvastatin by using 2(3) full-factorial design. AAPS PharmSciTech, 2010, 11(2), 489-496.
[http://dx.doi.org/10.1208/s12249-010-9414-z] [PMID: 20309652]
[18]
Dobson, C.M. Principles of protein folding, misfolding and aggregation. Semin. Cell Dev. Biol., 2004, 15(1), 3-16.
[http://dx.doi.org/10.1016/j.semcdb.2003.12.008] [PMID: 15036202]
[19]
Chaturvedi, S.K.; Siddiqi, M.K.; Alam, P.; Khan, R.H. Protein misfolding and aggregation: Mechanism, factors and detection. Process Biochem., 2016, 51, 1183-1192.
[http://dx.doi.org/10.1016/j.procbio.2016.05.015]

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