I'm transitioning from a fed-batch to a perfusion culture for CHO cells and have observed markedly different performances between my flask and bioreactor systems. In my flask setup, which uses centrifugation for cell retention, the cells double in concentration daily up to a certain point (40M cell/mL) and effectively re-consume lactate. In contrast, the bioreactor—employing a porous membrane filtration system (0.20 µm pore size) with media removal via a peristaltic pump—shows lower cell growth (max 5M cell/mL which is as good as a fedbatch) , high lactate accumulation, despite comparable glucose, nh3 level and same feed/rate strategies.
One key difference is in pH regulation. In flasks, pH is maintained passively via the media’s buffering capacity and CO₂ exchange in incubator, whereas the bioreactor uses active pH control (CO2/NaOH dosing). the bioreactor gave me a lot of troubles for pH regulation as the microsparging of air would remove CO2 in the media. Could these differences in pH regulation be responsible for the discrepancies in cell performance?
Another key difference is that my perfusion is not continuous in flask as it’s done by centrifugation but is continuous in bioreactor after a few days Because of the limiting flux capacity of the filtering probe.
What strategies would you recommend to mitigate these issues? Any insights, suggestions, or relevant literature references would be greatly appreciated.
Feel free to share your experiences and any relevant studies.
Dariouch Darimont
Based on your description, there are several key factors that could be influencing cell performance in the bioreactor compared to the flask setup, and several potential solutions that can be considered to improve performance in the bioreactor. Here's my expert opinion and a few science-based recommendations:
1. pH Regulation and Difference in pH Control
pH regulation is crucial for cell growth and productivity. In your setup, passive pH regulation in flasks, which relies on the buffering capacity of the medium and CO₂ exchange in the incubator, may be less sensitive to pH fluctuations compared to active pH control in the bioreactor. In the bioreactor, micro sparging (air addition) can remove CO₂ from the medium, potentially causing pH shifts that may not be optimal for cell growth. High pH levels can slow cell growth and lead to higher lactate accumulation, which can be toxic to the cells. Recommendation: Try optimizing the micro sparging strategy or use intermittent micro sparging to prevent excessive CO₂ removal. Also, for active pH control, consider using dynamic systems that can quickly respond to changes and maintain a stable pH (in the range of 7.0-7.4 for CHO cells). These systems should be fine-tuned to avoid excessive NaOH additions, which could negatively affect cell growth.
2. Excessive Lactate Accumulation
Your bioreactor shows high lactate accumulation, which indicates that cell metabolism may be more anaerobic, or the capacity to remove lactate or convert it to CO₂ is impaired. This is common in perfusion systems where the metabolite removal capacity is lower due to limited medium flow. Recommendation: Consider improving the medium flow rate and/or introducing strategies for lactate removal (e.g., using higher flow perfusion or continuous metabolite removal). Additionally, optimizing feed and glucose intake strategies can help reduce excessive lactate production.
3. Filtration Capacity (Flux Capacity)
One of the key factors in your bioreactor setup is filtration capacity, especially when using a probe with a porous membrane. Limited flux can affect perfusion efficiency and the capacity for metabolite and cell removal from the medium, which can impact cell growth and health. Recommendation: Consider optimizing filtration parameters. For example, using different pore sizes or adjusting the flow rate could improve cell performance. Improving flow rate could also help with better lactate and other unwanted metabolite removal.
4. Perfusion and Intervals in Flasks
In flasks, perfusion is not continuous, as it is done by centrifugation, while in the bioreactor, it is continuous after a few days due to the limited flux capacity of the filtering probe. This difference can lead to variations in metabolism and cell growth dynamics between the two systems, which may contribute to the observed discrepancies. Recommendation: Consider better alignment between perfusion strategies in flasks and the bioreactor. It might be beneficial to implement continuous perfusion in flasks as well to create a more similar system in both setups. Additionally, carefully manage the duration of perfusion in the bioreactor to avoid overexposure of cells to high medium volumes or stress conditions like high toxin concentrations.
5. Glucose and Nitrogen Feed Strategy
In your bioreactor, it is possible that the glucose and nitrogen feed strategy is not optimized according to the cells' needs. While you mentioned that glucose and NH3 levels are similar in both systems, the dynamic of metabolism might differ, meaning that cells in the bioreactor are not utilizing these substances efficiently. Recommendation: Consider implementing a dynamic glucose and nitrogen feed strategy, adjusting to the cells' needs over time. This could help reduce excessive lactate accumulation and improve metabolic efficiency.
Additional Recommendations:
- Consider using additional media supplements that can improve metabolism in the bioreactor, such as media with a higher CO₂ retention capacity or additives to enhance metabolic performance.
- Implementing real-time monitoring systems (pH, CO₂, glucose, lactate, NH3) could help quickly identify issues and allow for timely corrections.
The primary challenges in your system likely stem from pH regulation, filtration capacity, and metabolite accumulation. Proper management of these parameters, combined with optimizing feed strategies, filtration, and pH regulation, could significantly improve cell performance in the bioreactor.
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