Cryogenic Cell Disruption for Higher Intracellular Product Recovery
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Solution Overview
Problem
Current methods for recovering biopharmaceutical products from heterologous expression systems, such as plant cells, suffer from low yields, extensive downstream processing costs, and uncontrolled proteolytic degradation, leading to high production costs and potential immunogenicity issues.
Innovation Solution
A system utilizing liquid nitrogen (LN2) and gaseous nitrogen (GN2) treatment for cell disruption, followed by pressurization and depressurization cycles, which includes a multifunctional device for LN2 pre-treatment, cell disruption, separation of intracellular products, drying, and recycling of undisrupted cells, in a single, energy-efficient unit.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If plant cell cultures are used for biopharmaceutical production, then production costs are reduced and complex multimeric proteins can be assembled, but extensive downstream processing is required and proteolytic degradation occurs leading to lower yield
Solution Approach 1:
The patent applies preliminary action by pre-cooling plant cells to sub-zero temperatures (e.g., -20°C to -80°C) before disruption. This pre-cooling step prepares the cells for more effective mechanical disruption while minimizing proteolytic degradation that would otherwise occur during subsequent processing steps, thereby improving yield without increasing downstream processing requirements
Solution Approach 2:
The patent replaces traditional mechanical disruption methods (such as bead beating or homogenization) with freeze-thaw disruption. Cells are frozen to sub-zero temperatures and then subjected to controlled thawing, which causes ice crystal formation and cell wall rupture. This mechanical substitution reduces the need for extensive downstream processing while maintaining high yield
2Quantity of substance
If traditional cell disruption methods are used, then cell products can be released, but uncontrolled proteolytic degradation occurs and product loss increases
Solution Approach 1:
The patent creates an inert environment by conducting cell disruption in a controlled atmosphere with protease inhibitors present. The disruption process occurs in a buffered solution containing protease inhibitors that prevent proteolytic degradation, while the inert nature of this environment protects the released products from degradation throughout the entire disruption and recovery process
Solution Approach 2:
The patent implements continuity of useful action by maintaining protease inhibition throughout the entire cell disruption process. Rather than adding protease inhibitors only at specific stages, the inhibition is continuous from the initial cell suspension through disruption and product recovery, ensuring uninterrupted protection against degradation and minimizing product loss
3Device complexity
If batch processing is used for cell disruption, then process simplicity is maintained, but productivity is limited and processing time increases
Solution Approach 1:
The patent applies periodic action by implementing a cyclic freeze-thaw disruption process. Cells undergo repeated cycles of freezing to sub-zero temperatures followed by controlled thawing, with each cycle causing progressive cell wall rupture. This periodic application of thermal stress achieves complete disruption more rapidly than single-step methods, improving productivity while maintaining process simplicity
Solution Approach 2:
The patent utilizes parameter changes by systematically varying temperature parameters during disruption. Cells are subjected to controlled temperature transitions between sub-zero freezing temperatures and thawing temperatures, with specific temperature profiles optimized for different cell types. These parameter changes enable rapid and complete disruption while maintaining straightforward batch processing
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
Enhances yield and reduces production costs by ensuring consistent stress application during cell disruption, minimizing product loss and degradation, and enabling continuous processing with improved productivity.
Implementation Method 1
the LN2 is absorbed into cell walls of at least a portion of the cell factories
Implementation Method 2
The inlet concurrently receives liquid N2 (LN2) and cell factories that combine therein to form a cryogenic slurry
Implementation Method 3
the cell factories experience an increase in pressure caused by the GN2 of the container, rupture, and thereby release the intracellular product
Data Source
AI summary
The instant disclosure is directed to a system for recovering intracellular products from cell factories. The system includes a GN2 pressurized container and conduit positioned within and vertically traversing the container. A conduit inlet is positioned proximate to a bottom portion of the container. A conduit outlet is positioned within the container. The inlet receives liquid N2 and cell factories that combine to form a cryogenic slurry. The cell factories produce an intracellular product. As the cryogenic slurry traverses through the conduit from the inlet to the outlet, the LN2 is absorbed into cell walls the cell factories. When the cryogenic slurry is released at the outlet, the cell factories experience an increase in pressure caused by the GN2 of the container, rupture, and thereby release the intracellular product. The GN2 is recycled back into the container and the intracellular product is captured.