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2024
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An advanced acoustic retention system for perfusion culture at high cell densities—BioSep
Overview
Mammalian cell culture is of great significance for protein production in the biotechnology industry. [1] . Approximately in the pharmaceutical industry 70% The recombinant protein is produced using Chinese hamster ovary cells. CHO ) produced by.
In perfusion culture, nutrients are continuously supplied while byproducts are removed. [2] Compared to batch culture and fed-batch techniques, perfusion provides cells with a favorable environment and shorter product residence times. This is particularly important for the quality of unstable products. Another advantage of the perfusion mode is that it allows the use of smaller bioreactors and reduces the need for in-situ cleaning operations. [3] 。
Perfusion requires a device that can keep cells suspended in the culture medium. Most mammalian cell retention systems used in perfusion are based on differences in cell size—for example, by employing filters. However, due to the inevitable contamination caused by filters, conventional filtration membranes cannot achieve true steady-state perfusion culture. Moreover, frequent filter replacements increase both costs and the risk of contamination. [4] 。
An acoustic separator is an alternative cell-retention system that uses forces generated in a standing ultrasonic wave field to separate cells from the supernatant liquid. The cells become trapped in the pressure nodes of the standing wave and are collected as loose aggregates. These cell aggregates then return to the bioreactor via gravitational settling. [4] 。
In this study, a perfusion system designed for high-density cell cultures was used. Applikon Biosep 10 L An advanced version of the acoustic cell separator. In the bioreactor, cell density is... 11~144×10 6 cells/mL between CHO The cell evaluates its performance.
Materials and Methods
01 Cell acoustic trapping device – BioSep
- BioSep The system consists of an acoustic chamber and a controller.
- The controller’s function is to automatically generate the sound field within the acoustic chamber.
- The cell suspension from the bioreactor is pumped into an acoustic chamber installed on the headplate of the bioreactor.
- The standing wave forces the suspended cells into a plane, where they form loose aggregates (Figure). 1 ).
- The clear liquid is harvested by passing upward through the acoustic field, while the concentrated cells are returned to the bioreactor.
- As cell concentration and perfusion rate increase, the power input to the acoustic chamber is adjusted to higher levels to maintain high separation efficiency. [5] 。
- The run time corresponds to the period during which the cells are separated from the supernatant. At the end of the run time, the acoustic field is temporarily turned off, harvesting is paused, and the cells in the chamber are returned to the bioreactor.
- In this study, the power level and operating time were varied to achieve the optimal settings for high density. CHO Cell culture exceeding 125 × 10 6 cells/mL 。
02 Experimental setup
- To evaluate the separation performance under a series of high cell concentrations, we will... CHO The cells are cultured in flasks, concentrated, and then suspended for use. My-Control Operating system Applikon 250 mL MiniBio In a bioreactor.
- BioSep 10 L The power level is 2~7W 。
- The experimental setup is shown in the figure. 2 As shown.
2 |
A ) The experimental setup includes: a feed tank, a waste liquid tank, a harvesting pump, a feed pump, and an acoustic chamber. MiniBio 250 mL 、my-Control
B ) Typical experimental setup [5]
3 | Analytical method
•BioSep The separation efficiency according to the formula. 1 Calculate :
SE (%) = 1 - HX / BX × 100 [1]
Among them HX The live cell concentration corresponding to the harvest pipeline, BX Corresponding to the concentration of living cells in the bioreactor [4] 。
To ensure stable and reproducible acoustic conditions, before sampling from the harvest lines and bioreactors, ultrasonic power input, harvest rate, and operational parameters must be carefully controlled. / The backwash timer should be set to at least a constant value. 30 Minutes.
Based on the duration of the selected operating cycle, harvest samples at specific time points to obtain consistent and comparable data. ( Table 1 ).
Results and Discussion
1| Circulating flow rate
- During perfusion culture at high cell densities, a high circulation rate is required, which can lead to increased turbulence within the acoustic chamber.
- This turbulence-induced effect can influence acoustically induced cell aggregation. [6] 。
- A new one was observed in the current study. BioSep The version allows acoustically induced cell aggregates to settle undisturbed, with a maximum inflow rate of up to 7 mL/min ( ~10 L/ Heaven), allowing retention beyond 100 × 10 6cells/mL The bioreactor concentration.
2| Separation performance
- Collected from the harvest pipeline and bioreactor 70 Determine the separation efficiency in the sample.
- CHO The total cell concentration ranges from 11~144×10 6 cells/mL 。
- Studied 1~15 L/ Different net harvest rates of the sky, 2~7 W the power level and 2 To 10 Running time in minutes (value not shown); results summarized in the figure. 3 Middle.
- From the diagram 3 As can be seen from the middle, when... CHO The total cell concentration is 100 × 10 6cells/mL At that time, it can achieve up to 3L/ The net yield of the sky, while maintaining 98% Typical live cell separation efficiency. Over 4L/ The net yield of the sky affects efficiency at maximum density, but separation still remains. 90% The above cells.
- At a total concentration of 125 × 10 6 cells/mL At the time, with 2L/ The net yield of the day is running, and the cell separation efficiency has reached... 98%。
- Under conditions of increased cell concentration or high harvest yields, it is necessary to use higher power levels and shorter run cycles. [5] 。
- Optimize power ( w ) and runtime ( min ) pairing to achieve high-density cells. The combination of these values yields the highest separation efficiency: 2 W – 10 min; 3 W – 5 min; 5 W – 3 min; 7 W – 2 min 。
These results were expected, as higher power levels allow for increased cell retention under high-concentration or high-flow conditions, while shorter run times prevent excessive accumulation of cell aggregates in the acoustic chamber, thus giving them a chance to settle back into the bioreactor.
Figure 3 Separation efficiency is indicated by black squares, representing the net recovery rate of the inflow pipeline as recorded. CHO A function of the total cell concentration. The power-level matrix represents the maximum applied under this specific net harvest rate. HF Power. The yellow dashed line indicates the cycle rate. 20L/ Tianhe 10L/ The boundary between heaven and earth.
Experimental conclusion
Current research has demonstrated that Biosep As a CHO Cell concentration as high as 125 × 10 6cells/mL The cell retention system enhances the settling efficiency of cells. At this cell concentration, with... 2 L/ Operating at the net yield of the sky, the separation efficiency reaches as high as 98%。
References
[1] S. M. Woodside, B. D. Bowen, and J. M. Piret, “ Mammalian cell retention devices for stirred perfusion bioreactors, ” Cytotechnology, vol. 28, pp. 163 – 175, 1998.
[2] T. Kwon, N. Madziva, J. D. Oliveira, S. K. Chandramohan, L. Yin, H. Prentice, J. Han, ‘ Long-term steady-state perfusion culture of mammalian cells using a robust microfluidic cell retention device ” 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 2015.
[3] M. F. Clincke, C. lleryd, Y. Zhang, E. Lindskog, K. Walsh, and V. Chotteau, “ Very high density of CHO cells in perfusion by ATF or TFF in WAVE bioreactor. Part I: Effect of the cell density on the process, ” Biotechnol. Prog., 2013.
[4] V. M. Gorenflo, J. B. Ritter, D. S. Aeschliman, H. Drouin, B. D. Bowen, and J. M. Piret, “ Characterization and optimization of acoustic filter performance using experimental design methodology, ” Biotechnol. Bioeng., 2005.
[5] Biosep manual, 10 and 50 L per day, Applikon Biotechnology.
[6] I. Z. Shirgaonkar, S. Lanthier & A. Kamen, Acoustic cell filter: A proven cell retention technology for perfusion of animal cell cultures. Biotechnology Advances, 22(6), 433 – 444, 2004.
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