Perfusion-fed-batch cell culture for high-density protein production
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Solution Overview
Problem
Mammalian cell culture methods for protein production, such as those used in biopharmaceutical industries, face challenges in achieving high cell density and protein yield due to limitations from waste products like lactate and ammonium, resulting in lower protein expression levels compared to microbial systems.
Innovation Solution
The method involves growing cells in perfusion culture to achieve super high densities (above 40×10^6 cells/mL) without being restricted by waste products, then switching to fed-batch culture to enter a high protein production phase, allowing for significantly higher protein production and improved protein quality.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If cells are grown to high density in conventional cell culture, then cell density increases, but waste products (lactate, ammonium) accumulate and become toxic to cell growth
Solution Approach 1:
The cell culture process is divided into two distinct phases: a perfusion phase for rapid cell growth to high density, and a fed-batch phase for protein production. This segmentation allows the system to achieve high cell density without prolonged exposure to toxic waste products, as the perfusion phase efficiently removes metabolites while the fed-batch phase focuses on productivity.
Solution Approach 2:
The perfusion phase is used preliminarily to grow cells to super high density before transitioning to the production phase. By establishing a high cell density population first, the system ensures sufficient biomass is available for subsequent high-level protein production during the fed-batch phase, without the constraints of waste accumulation.
2Quantity of substance
If cells are grown in perfusion culture to super high density, then cell density increases significantly, but the process complexity increases
Solution Approach 1:
The invention merges the advantages of perfusion culture (efficient waste removal enabling high density) and fed-batch culture (simplified operation and high productivity) into a sequential two-phase process. This combination allows the system to achieve super high cell density while maintaining operational simplicity during the production phase, as fed-batch requires less complex monitoring and control compared to continuous perfusion.
3Productivity
If cells are grown to high density and then switched to fed-batch, then protein production increases, but the transition timing must be precisely controlled
Solution Approach 1:
The system uses feedback control to monitor cell density and physiological parameters during the perfusion phase, enabling precise determination of the optimal transition point to fed-batch culture. By continuously monitoring cell growth and metabolic state, the system can identify when super high density has been achieved and when switching to production mode will maximize protein yield while maintaining cell health.
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 approach enables the production of higher quantities of high-quality protein in a shorter time, overcoming traditional limitations and enhancing protein yield and stability compared to conventional methods.
Implementation Method 1
adjusting the pH of the cell culture to below neutral pH (i.e., below a pH of 7) and settling the cell culture, such that the cell culture separates to form a supernatant layer and a cell-bed layer, wherein the protein is present in (and can be isolated from) the supernatant layer
Implementation Method 2
settling the cell culture, such that the cell culture separates to form a supernatant layer and a cell-bed layer
Data Source
AI summary
The present invention provides a method of increasing protein production in a cell culture by growing cells that produce the protein (e.g., the growth phase) in a perfusion cell culture to a high cell density (i.e., at least above about 40×106 cells/mL) and then switching to a protein production phase, wherein the cells are cultured in a fed-batch cell culture. The present invention further provides a method for clarifying a protein from a cell culture by adjusting the pH of the cell culture to below neutral pH (i.e., below a pH of 7) and settling the cell culture, such that the cell culture separates to form a supernatant layer and a cell-bed layer, wherein the protein is in the supernatant layer.