Continuous Osmotic Shock Apparatus for Periplasmic Protein Release
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Solution Overview
Problem
Current methods for osmotically shocking bacterial cells to release periplasmic contents are time-consuming, difficult to scale up, and lack precise control, limiting their applicability for large-scale molecule release.
Innovation Solution
A method involving a continuous fluid stream combination of high and low osmolality solutions using a T-joint connected to a static mixer, followed by separation using a device based on size or density, to efficiently release periplasmic proteins from bacterial cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If batch mode osmotic shock is used to selectively release periplasmic contents, then cell disruption is avoided and periplasmic proteins are released, but the process is time-consuming and has low throughput
Solution Approach 1:
The patent transforms the traditional batch-mode osmotic shock process into a continuous flow process. Cells are continuously fed through a flow cell where they undergo osmotic shock, and the released periplasmic contents are continuously collected. This continuous operation eliminates the idle time between batches and significantly increases throughput while maintaining the selective release mechanism.
Solution Approach 2:
The patent introduces dynamic control of flow rates for both the cell suspension and shock buffer through programmable pumps. The flow rates can be adjusted and optimized during the process to control the extent of osmotic shock and maximize periplasmic content release while maintaining cell integrity where needed. This dynamic adjustment capability allows the system to adapt to different production requirements.
2Reliability
If batch mode osmotic shock is used to release periplasmic contents, then selective release is achieved, but scaling up is difficult
Solution Approach 1:
The patent divides the osmotic shock process into distinct functional modules: a flow cell for osmotic shock, programmable pumps for fluid delivery, and separation devices for product recovery. Each module can be independently optimized and scaled. The flow cell design with controlled geometry allows for scalable production by increasing flow rates or adding parallel units without changing the fundamental process mechanism.
Solution Approach 2:
The continuous flow osmotic shock apparatus is designed to handle different cell types and periplasmic products using the same basic configuration. By adjusting flow rates, shock buffer composition, and separation parameters, the system can be adapted to various bioproduction applications, making it a universal platform for periplasmic content recovery at different scales.
3Reliability
If batch mode osmotic shock is used, then periplasmic contents are released, but exposure time control is inaccurate
Solution Approach 1:
The patent incorporates flow rate monitoring and control systems that provide feedback to maintain precise exposure times. The programmable pumps deliver the shock buffer at controlled rates, and the residence time of cells in the flow cell is determined by the flow rate and cell volume, which can be precisely calculated and controlled. This feedback control ensures consistent and accurate exposure times for optimal periplasmic content release.
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 a more efficient and scalable release of recombinant polypeptides with improved control over the osmotic shock process, increasing throughput and facilitating large-scale production.
Implementation Method 1
osmotic shock procedure has been used to selectively release the periplasmic contents
Implementation Method 2
equilibrating fermentation broth with high molarity salt or sugar solution (soak buffer) to build high osmotic pressure within the cells
Data Source
AI summary
A method of preparing a recombinant polypeptide of interest includes fermenting a host cell being transformed with a recombinant expression system capable of bringing about secretion of a polypeptide of interest into the periplasm of said host cell. The polypeptide of interest is extracted from the periplasm by applying a continuous osmotic shock to the host cells contained in a fermentation medium. An apparatus for osmotically shocking cells includes a first reservoir containing cells in a first solution and a second reservoir containing a second solution, the first solution having a higher osmolality than the second solution. A method for osmotically shocking cells using the first and second soluctions is also disclosed. Also disclosed is a method of isolating a recombinant polypeptide of interest from a cell.