Volume 34, Issue 9 (December 2023)
Studies in Medical Sciences 2023, 34(9): 565-575 |
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Mohammad Javad S, Noofeli M, Nazari Shirvan A, Khaki P. OPTIMIZATION OF CULTURE PROCESS AND PURIFICATION OF DIPHTHERIA TOXOID BY STEP REDUCTION. Studies in Medical Sciences 2023; 34 (9) :565-575
URL: http://umj.umsu.ac.ir/article-1-6092-en.html
URL: http://umj.umsu.ac.ir/article-1-6092-en.html
Razi Vaccine & Serum Research Institute , noofeli1234@yahoo.com
Abstract: (1022 Views)
Background & Aim: Diphtheria is an acute vaccine-controlled respiratory disease caused by the gram-positive bacterium Corynebacterium diphtheria. Since the preparation, purification, and detoxification of the vaccine against this disease involves many and complex steps, the present study aimed reduction of time of various stages of toxoid purification.
Materials & Methods: For this purpose, three factors were first optimized for the concentration of iron in the culture medium, incubation temperature and rotation speed to produce more toxins. In each case, the experiment was carried out in triplicate and Lf and Kf values were tested to evaluate toxin production. After toxin inactivation and production of the toxoid, step reductions were performed to purify the toxoid. For this, ultrafiltration, precipitation, dialysis and chromatography with Sephadex G-25 column are used. In step reduction conditions, dialysis, chromatography or both of them were removed in the first, second and third cases, respectively. The results of each method were evaluated by SDS-PAGE and the quantitative data was analyzed using Minitab 18 (p‹0.05).
Results: The results for optimizing the culture medium showed that highest amounts of toxin were produced at an iron concentration of 1 mg/l, a temperature of 35 °C, and a rotation speed of 200 rpm(p‹0.05). In the reduction experiments related to toxoid purification steps, the best results were obtained in a series of purified samples with ultrafiltration, precipitation and column chromatography steps.
Conclusion: Since time and cost are two key factors in industrial production, toxoid production for the diphtheria vaccine can be produced at a lower cost and in less time by eliminating the two-week dialysis phase.
Materials & Methods: For this purpose, three factors were first optimized for the concentration of iron in the culture medium, incubation temperature and rotation speed to produce more toxins. In each case, the experiment was carried out in triplicate and Lf and Kf values were tested to evaluate toxin production. After toxin inactivation and production of the toxoid, step reductions were performed to purify the toxoid. For this, ultrafiltration, precipitation, dialysis and chromatography with Sephadex G-25 column are used. In step reduction conditions, dialysis, chromatography or both of them were removed in the first, second and third cases, respectively. The results of each method were evaluated by SDS-PAGE and the quantitative data was analyzed using Minitab 18 (p‹0.05).
Results: The results for optimizing the culture medium showed that highest amounts of toxin were produced at an iron concentration of 1 mg/l, a temperature of 35 °C, and a rotation speed of 200 rpm(p‹0.05). In the reduction experiments related to toxoid purification steps, the best results were obtained in a series of purified samples with ultrafiltration, precipitation and column chromatography steps.
Conclusion: Since time and cost are two key factors in industrial production, toxoid production for the diphtheria vaccine can be produced at a lower cost and in less time by eliminating the two-week dialysis phase.
Type of Study: Research |
Subject:
میکروبیولوژی
References
1. Donyapoor F, Zeinoddini M, Saeedinia AR. Cloning and expression of recombinant immunotoxin using diphtheria toxin and granulocyte colony stimulating factor (G-CSF). J Arak Un Med Sci 2016;19(5):42-50. [Google Scholar]
2. Eaton MD. The purification and concentration of diphtheria toxin: I. evaluation of previous methods; Description of a new procedure: I. Evaluation of previous methods; Description of a new procedure. J Bacteriol 1936;31(4):347-66. Available from: http://dx.doi.org/10.1128/jb.31.4.347-366.1936 [DOI:10.1128/jb.31.4.347-366.1936] [PMID] []
3. WHO-recommended standards for surveillance of selected vaccine-preventable diseases. World Health Organization Geneva. 1999. [DOI:10.1128/jb.31.4.347-366.1936] [PMID] []
4. Hennart M, Panunzi LG, Rodrigues C, Gaday Q, Baines SL, Barros-Pinkelnig M, et al. Population genomics and antimicrobial resistance in Corynebacterium diphtheriae. Genome Med 2020;12(1):107. Available from: http://dx.doi.org/10.1186/s13073-020-00805-7 [DOI:10.1186/s13073-020-00805-7] [PMID] []
5. . Offit PA, Quarles J, Gerber MA, Hackett CJ, Marcuse EK, Kollman TR, et al. Addressing parents' concerns: do multiple vaccines overwhelm or weaken the infant's immune system? Pediatrics 2002;109(1):124-9. Available from: http://dx.doi.org/10.1542/peds.109.1.124 [DOI:10.1542/peds.109.1.124] [PMID]
6. Organization WH. Diphtheria vaccine: WHO position paper. Vaccine. 2017;36(2):199-201. [PMID]
7. Rappuoli R, Malito E. History of diphtheria vaccine development. Corynebacterium diphtheriae and related toxigenic species. Springer; 2014. [DOI:10.1007/978-94-007-7624-1_11]
8. Iwaki M, Komiya T, Yamamoto A, Ishiwa A, Nagata N, Arakawa Y. Genome organization and pathogenicity of Corynebacterium diphtheriae C7 (−) and PW8 strains. Infection Immunity 2010;78(9):3791-800. [DOI:10.1128/IAI.00049-10] [PMID] []
9. Spiering MM, Ringe D, Murphy JR, Marletta MA. Metal stoichiometry and functional studies of the diphtheria toxin repressor. Proc Natl Acad Sci U S A 2003;100(7):3808-13. Available from: http://dx.doi.org/10.1073/pnas.0737977100 [DOI:10.1073/pnas.0737977100] [PMID] []
10. Sundaran B, Udaya Bhaskara Rao Y, Boopathy R. Process optimization for enhanced production of diphtheria toxin by submerged cultivation. J Biosci Bioeng 2001;91(2):123-8. Available from: http://dx.doi.org/10.1016/s1389-1723(01)80053-0 [DOI:10.1016/S1389-1723(01)80053-0] [PMID]
11. Fratelli F, Abrahão-Neto J, Caricati ATP, Borges MM, Guidolin R, Caricati CP. An alternative method for purifying and detoxifying diphtheria toxin. Toxicon 2011;57(7-8):1093-100. Available from: http://dx.doi.org/10.1016/j.toxicon.2011.04.015 [DOI:10.1016/j.toxicon.2011.04.015] [PMID]
12. Chung Y-J, Lee J-A, Jung M-Y, Lee S-M, Kim T-Y, Choe Y-K, et al. Optimization of diphtheria toxin production by Corynebacterium diphtheriae using a casein-based medium in a fermenter. Biotechnol Bioprocess Eng 2016;21(4):537-43. Available from: http://dx.doi.org/10.1007/s12257-016-0360-9 [DOI:10.1007/s12257-016-0360-9]
13. Suwanpatcharakul M, Pakdeecharoen C, Visuttitewin S, Pesirikan N, Chauvatcharin S, Pongtharangkul T. Process optimization for an industrial-scale production of Diphtheria toxin by Corynebacterium diphtheriae PW8. Biologicals 2016;44(6):534-9. Available from: http://dx.doi.org/10.1016/j.biologicals.2016.08.002 [DOI:10.1016/j.biologicals.2016.08.002] [PMID]
14. Faramarzi A, Noofeli M, Tofighi A, Shahcheraghi F. Comparison of the diphtheria toxin separation methods on standard vaccine strain using separator and filter presses to improve the quality & quantity of the final product. 2019. [URL]
15. Carroll SF, Barbieri JT, Collier RJ. Diphtheria toxin: purification and properties. Methods Enzymol 1988;165:68-76. Available from: http://dx.doi.org/10.1016/s0076-6879(88)65014-2. [DOI:10.1016/S0076-6879(88)65014-2] [PMID]
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