Alternating Tangential Flow Perfusion for High Cell Density
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Solution Overview
Problem
Conventional perfusion culturing methods fail to achieve extremely high viable cell densities and cell viability, often leading to cell aggregation, which complicates process control and metabolic heterogeneity.
Innovation Solution
The process involves perfusion culturing using a filter module with hollow fibers and alternating tangential flow, which maintains a lower cell density outflow, achieving high cell retention and minimizing aggregation, thereby allowing for extremely high viable cell densities and high cell viability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional perfusion culturing is used, then cell retention is achieved, but cell aggregation occurs and cell density is limited
Solution Approach 1:
The patent applies periodic action by alternating the flow direction through the hollow fiber membrane every 15-60 seconds. This periodic reversal creates alternating tangential flow that prevents cells from adhering to the membrane surface, thereby eliminating cell aggregation while maintaining high cell retention and enabling extremely high viable cell densities to be achieved
2Reliability
If perfusion culturing is used, then cell retention is achieved, but process control becomes difficult due to metabolic heterogeneity
Solution Approach 1:
The periodic reversal of flow direction ensures that all cells in the bioreactor are continuously exposed to fresh medium and experience uniform shear conditions. This eliminates the formation of stagnant zones and metabolic heterogeneity, resulting in a homogeneous cell population with consistent metabolic profiles and improved process control
3Reliability
If low shear conditions are used in perfusion culturing, then cell damage is reduced, but cell disaggregation does not occur
Solution Approach 1:
The periodic alternation of flow direction creates transient tangential flow that provides sufficient shear force to prevent cell aggregation and disaggregate cell clusters without causing cell damage. The brief duration of each flow reversal pulse ensures that shear forces remain below damaging thresholds while still being effective at preventing aggregation
Solution Approach 2:
The patent changes the flow parameters by periodically reversing flow direction, which dynamically alters the shear stress distribution in the bioreactor. This parameter change enables the system to maintain low average shear conditions for cell viability while creating periodic high-shear pulses that prevent aggregation
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method achieves cell densities of at least 80×10^6 cells per mL with viability exceeding 90%, significantly reducing cell aggregation and maintaining a culture as a single cell suspension, even for aggregating cell types.
Implementation Method 1
the cell culture is circulated over a filter module comprising hollow fibers resulting in an outflow of liquid having a lower cell density than the cell culture
Implementation Method 2
the flow within the filter module is an alternating tangential flow
Data Source
AI summary
The invention relates to a process for the culturing of cells by continuous perfusion culturing of a cell culture comprising cell culture medium and cells, wherein cell culture medium is added to the cell culture, the cell culture is circulated over a filter module comprising hollow fibers resulting in an outflow of liquid having a lower cell density than the cell culture and the flow within the filter module is an alternating tangential flow. Preferably, culture medium is added at a particular perfusion rate and/or biomass is removed form the culture at least once. The method is especially suitable for the culturing of aggregating cells. The invention also relates to such a process wherein a biological substance, preferably an antibody, is produced by the cells, which biological substance may be further purified in downstream processing.