Two-Layer Sucrose Gradient for Virus Purification
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Solution Overview
Problem
Current methods for virus purification, such as chromatographic matrices and continuous sucrose gradient centrifugation, suffer from antigen aggregation and reduced yield, making them inefficient and costly for large-scale production, especially when trying to obtain high-purity whole virus antigen fractions.
Innovation Solution
A two-layer sucrose gradient centrifugation method is employed, using a first layer with a sucrose concentration of 34-50% and a second layer with 50-65% in an aqueous buffer with a pH between 6.0 and 9.4, which increases the volume of the peak pool and reduces antigen aggregation by optimizing the concentrations and amounts of the sucrose solutions, and adding salt and a buffer like Tris to achieve physiological conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If continuous sucrose gradient centrifugation is used for virus purification, then the purification process can be performed, but antigen aggregation occurs and yield is reduced
Solution Approach 1:
The patent changes the chemical composition parameters of the centrifugation medium by adding specific salts (NaCl at 4-8 g/kg, KCl at 2-4 g/kg) and buffers (Tris at 10-20 mmol/kg) to the sucrose gradient. This modifies the ionic strength and pH conditions during centrifugation, preventing antigen aggregation while maintaining high purification yield. The optimized salt and buffer concentrations resolve the contradiction between achieving high purity and maintaining productivity.
2Manufacturing precision
If chromatographic matrices are used for virus purification, then specific purification can be achieved, but the process is time and cost intensive and difficult to adapt to large scale production
Solution Approach 1:
The patent replaces the complex chromatographic mechanical system with a simpler centrifugal separation system. Instead of using chromatographic matrices and columns, the invention uses a centrifugal field with chemically optimized sucrose gradient to achieve virus purification. This substitution dramatically reduces device complexity and makes the process more suitable for large-scale production while maintaining effective purification capability.
3Quantity of substance
If conventional sucrose gradient centrifugation is used, then virus separation can be performed, but antigen aggregation inhibits viral inactivation steps
Solution Approach 1:
The patent applies preliminary action by optimizing the chemical environment (adding salts and buffers) before the centrifugation process begins. The Tris buffer maintains physiological pH (6.0-9.4) and the salts provide appropriate ionic strength, preventing antigen aggregation from occurring in the first place during the separation process. This preliminary optimization of conditions ensures that the separated antigen remains in a state suitable for subsequent inactivation steps, thereby ensuring reliability of the overall process.
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 significantly increases the yield of virus particles and reduces antigen aggregation, allowing for more efficient and cost-effective large-scale production of high-purity virus antigen fractions by optimizing the sucrose gradient and adding salt and buffer to the centrifugation process.
Implementation Method 1
centrifugation of said virus preparation in a gradient of a sugar established by a first layer A of the sugar in a concentration of a sucrose equivalent of 34% to 50% (w/w %) and a second layer B of the sugar in a concentration of a sucrose equivalent of 50% to 65% (w/w %) sucrose
Data Source
AI summary
The present invention provides a method for purification of a virus or virus antigen comprising providing a virus preparation and centrifugation of said virus preparation in a gradient of a sugar established by the addition of two or more buffered sugar layers of different concentration. The method leads to higher yields and reduces unwanted aggregation of the virus or virus antigen by increasing the volume of the peak pool.