Bio24350 Vol. 24 No. 1 (2025)

Vol. 24, No. 1 (2025), Bio24350 https://doi.org/10.24275/rmiq/Bio24350

Scaling E. coli disruption for a continuous mill design for plasmid recovery

Authors

R. Herrera-Imperial, P. Guerrero-Germán, C. A. Gutiérrez-Valenzuela, A. R. Mártin-García, J. Ortega-Lopez, A. Tejeda-Mansir

Abstract

The production of plasmid DNA (pDNA) is fundamental for creating DNA and RNA vaccines, which have proven to be a novel and effective alternative to traditional vaccines. The pDNA production process includes the stages of fermentation, primary recovery, intermediate recovery, and purification. Primary recovery involves cell harvest and rupture, which is a critical step in the bioprocess. In this research, a bead milling operation was employed as an alternative to the conventional alkaline lysis technique to enhance the efficiency of the primary recovery stage. Batch kinetic studies were performed with milling chambers with L/D of 0.51, 0.93 and 1.67. By fitting a kinetic model to the experimental data, the specific kinetic constants of 0.46, 0.44 and 0.21 min-1, respectively, were determined. Electrophoretic analysis shows that more than 60% of the supercoiled plasmid was recovered in less than 5 min, with no RNA content. The results of the milling kinetics in batch mode were used to analyze the behavior of a continuous mill using the Perfectly Agitated Tanks in series model. The results indicate that the highest overall efficiency is achieved with a chamber having an L/D ratio of 0.51 in 4 stages.

Keywords

mechanical lysis, plasmid, E. coli, bead milling.

References
Abdulrahman, A., & Ghanem, A. (2018). Recent advances in chromatographic purification of plasmid DNA for gene therapy and DNA vaccines: A review. Analytica Chimica Acta, 1025, 41-57. https://doi.org/10.1016/j.aca.2018.04.001 Aires-Barros, M. R., & Azevedo, A. M. (2017). 7 - Fundamentals of biological separation processes. In A. Pandey & J. A. C. Teixeira (Eds.), Current Developments in Biotechnology and Bioengineering (pp. 187-237). Elsevier. https://doi.org/10.1016/B978-0-444-63668-3.00007-X Carlson, A., Signs, M., Liermann, L., Boor, R., & Jem, K. J. (1995). Mechanical disruption of Escherichia coli for plasmid recovery. Biotechnol Bioeng, 48(4), 303-315. https://doi.org/10.1002/bit.260480403 Delran, P., Barthe, L., Peydecastaing, J., Pontalier, P. Y., Guihéneuf, F., & Frances, C. (2024). Integrating wet stirred-bead milling for Tetraselmis suecica biorefinery: operating parameters influence and specific energy efficiency. Bioresource Technology, 394, 130181. https://doi.org/10.1016/j.biortech.2023.130181 Diogo, M., Queiroz, J., & Prazeres, D. (2003). Assessment of purity and quantification of plasmid DNA in process solutions using high-performance hydrophobic interaction chromatography. Journal of Chromatography A, 998(1-2), 109-117. https://doi.org/10.1016/s0021-9673(03)00618-6 Estrada-Martínez, R.J., Favela-Torres, E., Soto-Cruz, N.O., Saucedo-Castañeda, G., & Martínez-Valdez, F.J. (2023). Respiro-fermentative metabolism in yeast cultivated in solid-state culture: The Crabtree effect and ethanol production. Revista Mexicana de ingeniería Química, (22)1, Bio3025 - Bio3025. https://doi.org/10.24275/rmiq/Bio3025 Günerken, E., D'Hondt, E., Eppink, M., Elst, K., & Wijffels, R. (2017). Flow cytometry to estimate the cell disruption yield and biomass release of Chlorella sp. during bead milling. Algal Research, 25, 25-31. https://doi.org/10.1016/j.algal.2017.04.033 Haque, S., Khan, S., Wahid, M., Mandal, R. K., Tiwari, D., Dar, S. A., . . . Jawed, A. (2016). Modeling and optimization of a continuous bead milling process for bacterial cell lysis using response surface methodology. RSC advances, 6(20), 16348-16357. https://doi.org/10.1039/C5RA26893A Jaén, K. E., Velazquez, D., Sigala, J.-C., & Lara, A. R. (2021). Enhancing microaerobic plasmid DNA production by chromosomal expression of Vitreoscilla hemoglobin in E. coli. Biochemical Engineering Journal, 166, 107862. https://doi.org/10.1016/j.bej.2020.107862 Juárez, M., de la Rosa, C. G., Alanis, J. S., & Lara-Rodríguez, A. (2021). Effect of Vitreoscilla hemoglobin on recombinant protein expression and energy and redox state of CHO cells. Revista Mexicana de Ingeniería Química, 20(1), 281-288. https://doi.org/10.24275/rmiq/Bio1886 Ohlson, J. (2020). Plasmid manufacture is the bottleneck of the genetic medicine revolution. Drug discovery today, 25(11), 1891. https://doi.org/10.1016/j.drudis.2020.09.040 Padilla-Zamudio, A., Lucero-Acuña, J. A., Guerrero-Germán, P., Ortega-López, J., & Tejeda-Mansir, A. (2018). Efficient disruption of Escherichia coli for plasmid DNA recovery in a bead mill. Applied Sciences, 8(1), 30. https://doi.org/10.3390/app8010030 Prazeres, D. M. F. (2011). Plasmid biopharmaceuticals: basics, applications, and manufacturing. John Wiley & Sons. https://doi.org/10.1002/9780470939918 Roush, D. J., & Lu, Y. (2008). Advances in primary recovery: centrifugation and membrane technology. Biotechnology progress, 24(3), 488-495. https://doi.org/10.1021/bp070414x Sanchez-Casco, M., Dumonteil, E., & Ortega-Lopez, J. (2013). Production optimisation of a DNA vaccine candidate against leishmaniasis in flask culture. African Journal of Biotechnology, 12(31). https://doi.org/10.5897/AJB12.1360 Selvaraj, S., & Vytla, R. M. (2017). Evaluation of model parameters for growth, tannic acid utilization and tannase production in Bacillus gottheilii M2S2 using polyurethane foam blocks as support. 3 Biotech, 7, 1-9. https://doi.org/10.1007/s13205-017-0909-0 Shuler, M. L., & Kargi, F. (2017). Bioprocess Engineering. Prentice Hall. Tejeda Mansir, A., Montesinos Cisneros, R. M., & & Guzmán Zamudio, R. (2011). Bioseparaciones. Pearson.

References

Abdulrahman, A., & Ghanem, A. (2018). Recent advances in chromatographic purification of plasmid DNA for gene therapy and DNA vaccines: A review. Analytica Chimica Acta, 1025, 41-57. https://doi.org/10.1016/j.aca.2018.04.001
Aires-Barros, M. R., & Azevedo, A. M. (2017). 7 - Fundamentals of biological separation processes. In A. Pandey & J. A. C. Teixeira (Eds.), Current Developments in Biotechnology and Bioengineering (pp. 187-237). Elsevier. https://doi.org/10.1016/B978-0-444-63668-3.00007-X
Carlson, A., Signs, M., Liermann, L., Boor, R., & Jem, K. J. (1995). Mechanical disruption of Escherichia coli for plasmid recovery. Biotechnol Bioeng, 48(4), 303-315. https://doi.org/10.1002/bit.260480403
Delran, P., Barthe, L., Peydecastaing, J., Pontalier, P. Y., Guihéneuf, F., & Frances, C. (2024). Integrating wet stirred-bead milling for Tetraselmis suecica biorefinery: operating parameters influence and specific energy efficiency. Bioresource Technology, 394, 130181. https://doi.org/10.1016/j.biortech.2023.130181
Diogo, M., Queiroz, J., & Prazeres, D. (2003). Assessment of purity and quantification of plasmid DNA in process solutions using high-performance hydrophobic interaction chromatography. Journal of Chromatography A, 998(1-2), 109-117. https://doi.org/10.1016/s0021-9673(03)00618-6
Estrada-Martínez, R.J., Favela-Torres, E., Soto-Cruz, N.O., Saucedo-Castañeda, G., & Martínez-Valdez, F.J. (2023). Respiro-fermentative metabolism in yeast cultivated in solid-state culture: The Crabtree effect and ethanol production. Revista Mexicana de ingeniería Química, (22)1, Bio3025 - Bio3025.
https://doi.org/10.24275/rmiq/Bio3025
Günerken, E., D'Hondt, E., Eppink, M., Elst, K., & Wijffels, R. (2017). Flow cytometry to estimate the cell disruption yield and biomass release of Chlorella sp. during bead milling. Algal Research, 25, 25-31. https://doi.org/10.1016/j.algal.2017.04.033
Haque, S., Khan, S., Wahid, M., Mandal, R. K., Tiwari, D., Dar, S. A., . . . Jawed, A. (2016). Modeling and optimization of a continuous bead milling process for bacterial cell lysis using response surface methodology. RSC advances, 6(20), 16348-16357. https://doi.org/10.1039/C5RA26893A
Jaén, K. E., Velazquez, D., Sigala, J.-C., & Lara, A. R. (2021). Enhancing microaerobic plasmid DNA production by chromosomal expression of Vitreoscilla hemoglobin in E. coli. Biochemical Engineering Journal, 166, 107862. https://doi.org/10.1016/j.bej.2020.107862
Juárez, M., de la Rosa, C. G., Alanis, J. S., & Lara-Rodríguez, A. (2021). Effect of Vitreoscilla hemoglobin on recombinant protein expression and energy and redox state of CHO cells. Revista Mexicana de Ingeniería Química, 20(1), 281-288. https://doi.org/10.24275/rmiq/Bio1886
Ohlson, J. (2020). Plasmid manufacture is the bottleneck of the genetic medicine revolution. Drug discovery today, 25(11), 1891. https://doi.org/10.1016/j.drudis.2020.09.040
Padilla-Zamudio, A., Lucero-Acuña, J. A., Guerrero-Germán, P., Ortega-López, J., & Tejeda-Mansir, A. (2018). Efficient disruption of Escherichia coli for plasmid DNA recovery in a bead mill. Applied Sciences, 8(1), 30. https://doi.org/10.3390/app8010030
Prazeres, D. M. F. (2011). Plasmid biopharmaceuticals: basics, applications, and manufacturing. John Wiley & Sons. https://doi.org/10.1002/9780470939918
Roush, D. J., & Lu, Y. (2008). Advances in primary recovery: centrifugation and membrane technology. Biotechnology progress, 24(3), 488-495. https://doi.org/10.1021/bp070414x
Sanchez-Casco, M., Dumonteil, E., & Ortega-Lopez, J. (2013). Production optimisation of a DNA vaccine candidate against leishmaniasis in flask culture. African Journal of Biotechnology, 12(31). https://doi.org/10.5897/AJB12.1360
Selvaraj, S., & Vytla, R. M. (2017). Evaluation of model parameters for growth, tannic acid utilization and tannase production in Bacillus gottheilii M2S2 using polyurethane foam blocks as support. 3 Biotech, 7, 1-9. https://doi.org/10.1007/s13205-017-0909-0
Shuler, M. L., & Kargi, F. (2017). Bioprocess Engineering. Prentice Hall.
Tejeda Mansir, A., Montesinos Cisneros, R. M., & & Guzmán Zamudio, R. (2011). Bioseparaciones. Pearson.