Establishment of the Carrot-Made LTB-Syn Antigen Cell Line in Shake Flask and Airlift Bioreactor Cultures

Abstract

Carrot (Daucus carota) cells have been used to effectively manufacture recombinant biopharmaceuticals such as cytokines, vaccines, and antibodies. We generated the carrot cell line Z4, genetically modified to produce the LTB-Syn antigen, which is a fusion protein proposed for immunotherapy against synucleinopathies. In this work, the Z4 cell suspension line was cultivated to produce the LTB-Syn protein in a 250 mL shake flask and 2 L airlift bioreactor cultures grown for 45 and 30 days, respectively. Maximum biomass was obtained on day 15 in both the airlift bioreactor (35.00 ± 0.04 g/L DW) and shake flasks (17.00 ± 0.04 g/L DW). In the bioreactor, the highest LTB-Syn protein yield (1.52 ± 0.03 µg/g FW) was obtained on day 15; while the same occurred on day 18 for shake flasks (0.92 ± 0.02 µg/g FW). LTB-Syn protein levels were analyzed by GM1-ELISA and western blot. PCR analysis confirmed the presence of the transgene in the Z4 line. The obtained data demonstrate that the carrot Z4 cell suspension line grown in airlift bioreactors shows promise for a scale-up cultivation producing an oral LTB-Syn antigen.

Key words

Daucus carota - Apiaceae - LTB-Syn antigen - airlift bioreactor - suspension culture - synucleinopathies

Publikationsverlauf

Eingereicht: 15. Juni 2021

Angenommen nach Revision: 14. Oktober 2021

Artikel online veröffentlicht:
16. Dezember 2021

• References

• 1 Shim BS, Hong KJ, Maharjan PM, Choe S. Plant factory: New resource for the productivity and diversity of human and veterinary vaccines. Clin Exp Vaccine Res 2019; 8: 136-139
• 2 Pniewski T, Czyż M, Wyrwa K, Bociąg P, Krajewski P, Kapusta J. Micropropagation of transgenic lettuce containing HBsAg as a method of mass-scale production of standardised plant material for biofarming purposes. Plant Cell Rep 2017; 36: 49-60
• 3 Loh HS, Green BJ, Yusibov V. Using transgenic plants and modified plant viruses for the development of treatments for human diseases. Curr Opin Virol 2017; 26: 81-89
• 4 Kurup VM, Thomas J. Edible vaccines: Promises and challenges. Mol Biotechnol 2020; 62: 79-90
• 5 Rosales-Mendoza S, Tello-Olea MA. Carrot cells: A pioneering platform for biopharmaceuticals production. Mol Biotechnol 2015; 57: 219-232
• 6 Tekoah Y, Shulman A, Kizhner T, Ruderfer I, Fux L, Nataf Y, Bartfeld D, Ariel T, Gingis-Velitski S, Hanania U, Shaaltiel Y. Large-scale production of pharmaceutical proteins in plant cell culture-the Protalix experience. Plant Biotechnol J 2015; 13: 1199-1208
• 7 Rosales-Mendoza S, Nieto-Gómez R. Green therapeutic biocapsules: Using plant cells to orally deliver biopharmaceuticals. Trends Biotechnol 2018; 36: 1054-1067
• 8 Martens EC, Lowe EC, Chiang H, Pudlo NA, Wu M, McNulty NP, Abbott DW, Henrissat B, Gilbert HJ, Bolam DN, Gordon JI. Recognition and degradation of plant cell wall polysaccharides by two human gut symbionts. PLoS Biol 2011; 9: e1001221
• 9 Lawson LB, Norton EB, Clements JD. Defending the mucosa: Adjuvant and carrier formulations for mucosal immunity. Curr Opin Immunol 2011; 23: 414-420
• 10 Palma JA, Kaufmann H. Treatment of autonomic dysfunction in Parkinson disease and other synucleinopathies. Mov Disord 2018; 33: 372-390
• 11 Dorsey ER, Bloem BR. The Parkinson pandemic–A call to action. JAMA Neurol 2018; 75: 9-10
• 12 Jellinger KA. How close are we to revealing the etiology of Parkinsonʼs disease?. Expert Rev Neurother 2015; 15: 1105-1107
• 13 Sanchez-Guajardo V, Barnum CJ, Tansey MG, Romero-Ramos M. Neuroimmunological processes in Parkinsonʼs disease and their relation to α-synuclein: Microglia as the referee between neuronal processes and peripheral immunity. ASN Neuro 2013; 5: 113-139
• 14 Masliah E, Rockenstein E, Adame A, Alford M, Crews L, Hashimoto M, Seubert P, Lee M, Goldstein J, Chilcote T, Games D, Schenk D. Effects of alpha-synuclein immunization in a mouse model of Parkinsonʼs disease. Neuron 2005; 46: 857-868
• 15 Ghochikyan A, Petrushina I, Davtyan H, Hovakimyan A, Saing T, Davtyan A, Cribbs DH, Agadjanyan MG. Immunogenicity of epitope vaccines targeting different B cell antigenic determinants of human α-synuclein: feasibility study. Neurosci Lett 2014; 560: 86-91
• 16 Arevalo-Villalobos JI, Govea Alonso DO, Rosales-Mendoza S. Using carrot cells as biofactories and oral delivery vehicles of LTB-Syn: A low-cost vaccine candidate against synucleinopathies. J Biotechnol 2020; 309: 75-80
• 17 Schneider T, Hahn-Löbmann S, Stephan A, Schulz S, Giritch A, Naumann M, Kleinschmidt M, Tusé D, Gleba Y. Plant-made Salmonella bacteriocins salmocins for control of Salmonella pathovars . Sci Rep 2018; 8: 4078
• 18 Romero-Maldonado A, Monreal-Escalante E, Rosales-Mendoza S. Expression in plants of two new antigens with implications in Alzheimerʼs disease immunotherapy. Plant Cell Tiss Organ Cult 2016; 126: 361-370
• 19 Arevalo-Villalobos JI, Govea-Alonso DO, Bañuelos-Hernández B, González-Ortega O, Zarazúa S, Rosales-Mendoza S. Inducible expression of antigens in plants: A study focused on peptides related to multiple sclerosis immunotherapy. J Biotechnol 2020; 318: 51-56
• 20 Rosales-Mendoza S, Soria-Guerra RE, de Jesús Olivera-Flores MT, López-Revilla R, Argüello-Astorga GR, Jiménez-Bremont JF, García-de la Cruz RF, Loyola-Rodríguez JP, Alpuche-Solís AG. Expression of Escherichia coli heat-labile enterotoxin b subunit (LTB) in carrot (Daucus carota L.). Plant Cell Rep 2007; 26: 969-976
• 21 Macharoen K, Du M, Jung S, McDonald KA, Nandi S. Production of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures in a pilot-scale bioreactor. Biotechnol Bioeng 2020; 118: 1431-1443
• 22 Ramírez-Mosqueda MA, Iglesias-Andreu LG. Vanilla (Vanilla planifolia Jacks.) cell suspension cultures: Establishment, characterization, and applications. 3 Biotech 2017; 7: 242
• 23 Zhang H, Liu M, Li Y, Zhao Y, He H, Yang G, Zheng C. Oral immunogenicity and protective efficacy in mice of a carrot-derived vaccine candidate expressing UreB subunit against Helicobacter pylori . Protein Expr Purif 2010; 69: 127-131
• 24 Son SH, Choi SM, Lee YH, Choi KB, Yun SR, Kim JK, Park HJ, Kwon EW, Noh EW, Seon JH, Park YG. Large-scale growth and taxane production in cell cultures of Taxus cuspidata (Japanese yew) using a novel bioreactor. Plant Cell Rep 2000; 19: 628-633
• 25 Ferri M, Righetti L, Tassoni A. Increasing sucrose concentrations promote phenylpropanoid biosynthesis in grapevine cell cultures. J Plant Physiol 2011; 168: 189-195
• 26 Taticek RA, Moo-Young M, Legge R. The scale-up of plant cell culture: Engineering considerations. Plant Cell Tiss Organ Cult 1991; 24: 139-158
• 27 Saad KR, Parvatam G, Shetty NP. Medium composition potentially regulates the anthocyanin production from suspension culture of Daucus carota . 3 Biotech 2018; 8: 134
• 28 Soderquist RG, Lee JM. Enhanced production of recombinant proteins from plant cells by the application of osmotic stress and protein stabilization. Plant Cell Rep 2005; 24: 127-132
• 29 Suresh B, Bais HP, Raghavarao KSMS, Ravishankar GA, Ghildyal NP. Comparative evaluation of bioreactor design using Tagetes patula L. hairy roots as a model system. Process Biochem 2005; 40: 1509-1515
• 30 Clemente M, Corigliano MG, Pariani SA, Sánchez-López EF, Sander VA, Ramos-Duarte VA. Plant serine protease inhibitors: Biotechnology application in agriculture and molecular farming. Int J Mol Sci 2019; 20: 1345
• 31 Menkhaus TJ, Bai Y, Zhang C, Nikolov ZL, Glatz CE. Considerations for the recovery of recombinant proteins from plants. Biotechnol Prog 2004; 20: 1001-1014
• 32 Huang TK, McDonald KA. Bioreactor systems for in vitro production of foreign proteins using plant cell cultures. Biotechnol Adv 2012; 30: 398-409
• 33 Schillberg S, Raven N, Spiegel H, Rasche S, Buntru M. Critical analysis of the commercial potential of plants for the production of recombinant proteins. Front Plant Sci 2019; 10: 720
• 34 Holland T, Sack M, Rademacher T, Schmale K, Altmann F, Stadlmann J, Fisher R, Hellwig S. Optimal nitrogen supply as a key to increased and sustained production of a monoclonal full-size antibody in BY-2 suspension culture. Biotechnol Bioeng 2010; 107: 278-289
• 35 Nova-López CJ, Muñoz-Pérez JM, Granger-Serrano LF, Arias-Zabala ME, Arango-Isaza RE. Expression of recombinant Cry 1Ac protein in potato plant cell suspension culture: Establishment of culture and optimization of biomass and protein production by nitrogen supply. DYNA 2017; 84: 34-41
• 36 Vasilev N, Grömping U, Lipperts A, Raven N, Fischer R, Schillberg S. Optimization of BY-2 cell suspension culture medium for the production of a human antibody using a combination of fractional factorial designs and the response surface method. Plant Biotechnol J 2013; 11: 867-874
• 37 Maier U, Büchs J. Characterisation of the gas-liquid mass transfer in shaking bioreactors. Biochem Eng J 2001; 7: 99-106
• 38 Arevalo-Villalobos JI, Govea-Alonso DO, Monreal-Escalante E, Zarazúa S, Rosales-Mendoza S. LTB-Syn: a recombinant immunogen for the development of plant-made vaccines against synucleinopathies. Planta 2017; 245: 1231-1239
• 39 Huang TK, McDonald KA. Bioreactor engineering for recombinant protein production in plant cell suspension cultures. Biochem Bioeng J 2009; 45: 168-184
• 40 Xu J, Ge X, Dolan MC. Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures. Biotechnol Adv 2011; 29: 278-299
• 41 Castro-Concha LA, Escobedo RM, Miranda-Ham ML. Measurement of cell viability. Methods Mol Biol 2012; 877: 49-56
• 42 Caspeta L, Nieto I, Zamilpa A, Alvarez L, Quintero R, Villarreal ML. Solanum chrysotrichum hairy root cultures: characterization, scale-up and production of five antifungal saponins for human use. Planta Med 2005; 71: 1084-1087
• 43 Peraza-Luna F, Rodríguez-Mendiola M, Arias-Castro C, Bessiere JM, Calva-Calva G. Sotolone production by hairy root cultures of Trigonella foenum-graecum in airlift with mesh bioreactors. J Agric Food Chem 2001; 49: 6012-6019