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Current Pharmaceutical Analysis

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ISSN (Print): 1573-4129
ISSN (Online): 1875-676X

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Research Article

Validation of the T-47D Cell Culture Bioassay for the Potency Assessment of Botulinum Neurotoxin Type A

Author(s): Bruna Xavier, Rafaela Ferreira Perobelli Dumoncel, Clóvis Dervil Appratto Cardoso, Francielle Santos da Silva and Sérgio Luiz Dalmora*

Volume 19, Issue 4, 2023

Published on: 04 April, 2023

Page: [345 - 351] Pages: 7

DOI: 10.2174/1573412919666230320155755

Price: $65

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Abstract

Background: Botulinum neurotoxins (BoNTs) are among the most potent toxins known and are also used for therapeutic and aesthetic applications.

Objective: An alternative in vitro cell culture bioassay based on the induction of apoptosis on T- 47D breast cancer cells, after exposure to BoNTA, was developed and validated.

Methods: The T-47D cells (ATCC HTB-133) were seeded at a density of 3 × 105 cells mL-1, and the bioassay was performed with doses of BoNTA, between 3 and 81 U mL-1. The responses were assessed using 10 μL of Alamar Blue®. The absorbances were read at 570 and 600 nm.

Results: The results were compared with those of the in vivo LD50 mouse bioassay, showing a non-significant 1.08% higher, mean difference of the estimated potencies (p>0.05). Besides, the biopharmaceutics is analyzed by the size exclusion and reversed-phase liquid chromatography methods, showing a significant correlation with values 1.15% higher and 0.85% lower, respectively, related to the cell culture bioassay.

Conclusion: It is concluded that the validated T-47D cell culture assay represents an advancement toward the establishment of an alternative approach for the potency assessment, in the context of the 3 Rs. Besides, the employment of chromatographic methods in conjunction with the bioassays contributes to assessing the quality attributes of the biopharmaceutical formulations of BoNTA.

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[1]
Jankovic, J. Botulinum toxin: State of the art. Mov. Disord., 2017, 32(8), 1131-1138.
[http://dx.doi.org/10.1002/mds.27072] [PMID: 28639368]
[2]
Anandan, C.; Jankovic, J. Botulinum Toxin in Movement Disorders: An Update. Toxins (Basel), 2021, 13(1), 42.
[http://dx.doi.org/10.3390/toxins13010042] [PMID: 33430071]
[3]
Fonfria, E.; Maignel, J.; Lezmi, S.; Martin, V.; Splevins, A.; Shubber, S.; Kalinichev, M.; Foster, K.; Picaut, P.; Krupp, J. The expanding therapeutic utility of botulinum neurotoxins. Toxins (Basel), 2018, 10(5), 208.
[http://dx.doi.org/10.3390/toxins10050208] [PMID: 29783676]
[4]
Oh, H.M.; Park, J.; Song, D.; Chung, M. Efficacy and safety of a new botulinum toxin type A free of complexing proteins. Toxins (Basel), 2015, 8(1), 4.
[http://dx.doi.org/10.3390/toxins8010004] [PMID: 26712786]
[5]
Adler, S.; Bicker, G.; Bigalke, H.; Bishop, C.; Blümel, J.; Dressler, D.; Fitzgerald, J.; Gessler, F.; Heuschen, H.; Kegel, B.; Luch, A.; Milne, C.; Pickett, A.; Ratsch, H.; Ruhdel, I.; Sesardic, D.; Stephens, M.; Stiens, G.; Thornton, P.D.; Thürmer, R.; Vey, M.; Spielmann, H.; Grune, B.; Liebsch, M. The current scientific and legal status of alternative methods to the LD50 test for botulinum neurotoxin potency testing. The report and recommendations of a ZEBET Expert Meeting. Altern. Lab. Anim., 2010, 38(4), 315-330.
[http://dx.doi.org/10.1177/026119291003800401] [PMID: 20822324]
[6]
European Pharmacopoeia. Strasbourg: Council of Europe, 2020.
[7]
Törnqvist, E.; Annas, A.; Granath, B.; Jalkesten, E.; Cotgreave, I.; Öberg, M. Strategic focus on 3R principles reveals major reductions in the use of animals in pharmaceutical toxicity testing. PLoS One, 2014, 9(7), e101638.
[http://dx.doi.org/10.1371/journal.pone.0101638] [PMID: 25054864]
[8]
Jones, R.G.A.; Corbel, M.J.; Sesardic, D. A review of WHO International Standards for botulinum antitoxins. Biologicals, 2006, 34(3), 223-226.
[http://dx.doi.org/10.1016/j.biologicals.2005.11.009] [PMID: 16490362]
[9]
Brin, M.F.; James, C.; Maltman, J. Botulinum toxin type A products are not interchangeable: a review of the evidence. Biologics, 2014, 8, 227-241.
[PMID: 25336912]
[10]
Taylor, K.; Gericke, C.; Alvarez, L.R. Botulinum toxin testing on animals is still a Europe-wide issue. Altern. Anim. Exp., 2019, 36(1), 81-90.
[http://dx.doi.org/10.14573/altex.1807101] [PMID: 30303513]
[11]
Whitemarsh, R.C.M.; Strathman, M.J.; Chase, L.G.; Stankewicz, C.; Tepp, W.H.; Johnson, E.A.; Pellett, S. Novel application of human neurons derived from induced pluripotent stem cells for highly sensitive botulinum neurotoxin detection. Toxicol. Sci., 2012, 126(2), 426-435.
[http://dx.doi.org/10.1093/toxsci/kfr354] [PMID: 22223483]
[12]
Pellett, S.; Schwartz, M.P.; Tepp, W.H.; Josephson, R.; Scherf, J.M.; Pier, C.L.; Thomson, J.A.; Murphy, W.L.; Johnson, E.A. Human induced pluripotent stem cell derived neuronal cells cultured on chemically-defined hydrogels for sensitive in vitro detection of botulinum neurotoxin. Sci. Rep., 2015, 5(1), 14566.
[http://dx.doi.org/10.1038/srep14566] [PMID: 26411797]
[13]
Lamotte, J.D.D.; Roqueviere, S.; Gautier, H.; Raban, E.; Bouré, C.; Fonfria, E.; Krupp, J.; Nicoleau, C. HiPSC-derived neurons provide a robust and physiologically relevant in vitro platform to test botulinum neurotoxins. Front. Pharmacol., 2021, 11, 617867.
[http://dx.doi.org/10.3389/fphar.2020.617867] [PMID: 33519485]
[14]
Fernández-Salas, E.; Wang, J.; Molina, Y.; Nelson, J.B.; Jacky, B.P.S.; Aoki, K.R. Botulinum neurotoxin serotype A specific cell-based potency assay to replace the mouse bioassay. PLoS One, 2012, 7(11), e49516.
[http://dx.doi.org/10.1371/journal.pone.0049516] [PMID: 23185348]
[15]
Pathe-Neuschäfer-Rube, A.; Neuschäfer-Rube, F.; Haas, G.; Langoth-Fehringer, N.; Püschel, G. Cell-based reporter release assay to determine the potency of proteolytic bacterial neurotoxins. Toxins (Basel), 2018, 10(9), 360.
[http://dx.doi.org/10.3390/toxins10090360] [PMID: 30189643]
[16]
Pellett, S.; Tepp, W.H.; Johnson, E.A. Critical analysis of neuronal cell and the mouse bioassay for detection of botulinum neurotoxins. Toxins (Basel), 2019, 11(12), 713.
[http://dx.doi.org/10.3390/toxins11120713] [PMID: 31817843]
[17]
Nepal, M.R.; Jeong, T.C. Alternative methods for testing botulinum toxin: Current status and future perspectives. Biomol. Ther. (Seoul), 2020, 28(4), 302-310.
[http://dx.doi.org/10.4062/biomolther.2019.200] [PMID: 32126735]
[18]
Matak, I.; Lacković, Z. Botulinum neurotoxin type A: Actions beyond SNAP-25? Toxicology, 2015, 335, 79-84.
[http://dx.doi.org/10.1016/j.tox.2015.07.003] [PMID: 26169827]
[19]
Bandala, C.; Perez-Santos, J.L.M.; Lara-Padilla, E.; Delgado Lopez, M.G.; Anaya-Ruiz, M. Effect of botulinum toxin A on proliferation and apoptosis in the T47D breast cancer cell line. Asian Pac. J. Cancer Prev., 2013, 14(2), 891-894.
[http://dx.doi.org/10.7314/APJCP.2013.14.2.891] [PMID: 23621257]
[20]
Karpiński, T.; Adamczak, A. Anticancer activity of bacterial proteins and peptides. Pharmaceutics, 2018, 10(2), 54.
[http://dx.doi.org/10.3390/pharmaceutics10020054] [PMID: 29710857]
[21]
Xavier, B.; Perobelli, R.; Walter, M.; da Silva, F.; Dalmora, S. Content/potency assessment of botulinum neurotoxin type-A by validated liquid chromatography methods and bioassays. Toxins (Basel), 2019, 11(1), 35.
[http://dx.doi.org/10.3390/toxins11010035] [PMID: 30642048]
[22]
ICH- International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use: Guideline on Validation of Analytical Procedure: Text and Methodology Q2 (R1). 2005.
[23]
Sesardic, D.; Das, R.G. Alternatives to the LD50 assay for botulinum toxin potency testing: Strategies and progress towards refinement, reduction and replacement. AATEX, 2007, 14, 581-585.
[24]
FDA-Food and Drug Administration. Guidance for industry: Analytical Procedures and Methods Validation for Drugs and Biologics. Food and Drug Administration: Washington, DC, USA, 2015.
[25]
USP—United States Pharmacopeia. The United States Pharmacopeia Convention: Rockville, 2021.
[26]
Martin Bland, J.; Altman, D. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet, 1986, 327(8476), 307-310.
[http://dx.doi.org/10.1016/S0140-6736(86)90837-8] [PMID: 2868172]
[27]
Wang, X.; An, Z.; Luo, W.; Xia, N.; Zhao, Q. Molecular and functional analysis of monoclonal antibodies in support of biologics development. Protein Cell, 2018, 9(1), 74-85.
[http://dx.doi.org/10.1007/s13238-017-0447-x] [PMID: 28733914]

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