Adenovirus Purification via Direct Anion Exchange Chromatography
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for adenovirus purification, such as those used in vaccine production, are complex, costly, and inefficient, particularly due to the need for large volumes and multiple steps like tangential flow filtration (TFF) before ion exchange chromatography, which complicates large-scale process development and technology transfer.
Innovation Solution
A method that omits the pre-anion exchange chromatography TFF step, directly applying the clarified adenovirus sample to an anion exchange column using membrane anion exchange chromatography with specific salt concentrations and a Sartobind Q membrane, exploiting the charge characteristics of the viral capsid for efficient purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a multi-step downstream process including TFF concentration/diafiltration, depth filter clarification, AEX, and formulation is used, then adequate product recovery and host cell protein reduction are achieved, but the process complexity and manufacturing cost increase
Solution Approach 1:
The invention extracts and removes the TFF concentration/diafiltration step from the traditional multi-step downstream process, retaining only depth filter clarification followed by AEX chromatography. This extraction eliminates the need for high MWCO membranes and large buffer volumes while maintaining adequate product recovery and host cell protein reduction through optimized AEX conditions
Solution Approach 2:
The AEX chromatography step is designed to perform multiple functions simultaneously: it serves as both the primary purification step and the host cell protein removal step that traditionally required separate TFF and chromatography operations. This multi-functionality simplifies the overall process while maintaining effectiveness
2Object-generated harmful factors
If TFF concentration/diafiltration with high MWCO membrane is used before AEX, then lower MW protein and nucleic acid impurities are cleared, but large volumes of buffers and equipment are required
Solution Approach 1:
The invention extracts the TFF concentration/diafiltration step entirely from the process, eliminating the need for high MWCO membranes and the associated large buffer volumes. Impurity removal is achieved through optimized AEX chromatography conditions instead
Solution Approach 2:
The invention changes the operational parameters of AEX chromatography, specifically using load and wash salt concentrations in the range 24 to 31 mS/cm (higher than conventional conditions), which enables effective impurity removal without requiring prior TFF concentration and without needing large buffer volumes
3Reliability
If conventional downstream process with multiple steps is used, then purification is achieved, but yield per liter of culture is limited
Solution Approach 1:
By removing the TFF concentration/diafiltration step that traditionally preceded AEX, the invention eliminates the volume reduction bottleneck that limited yield per liter. The optimized direct AEX process from clarified lysate preserves higher virus concentrations while maintaining purification quality
Solution Approach 2:
The invention changes the AEX operational parameters (higher salt concentrations in load and wash steps at 24-31 mS/cm) to improve virus binding efficiency and recovery, directly increasing yield per liter of culture while maintaining adequate purification quality
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 simplifies the process, reduces costs, and significantly increases adenovirus yield per liter of culture, achieving up to 8000 doses per liter with lower host cell protein levels, making it more efficient and scalable for clinical use.
Implementation Method 1
performing anion exchange chromatography comprising directly applying the clarified sample to an anion exchange column and eluting adenovirus from the anion exchange column
Implementation Method 2
exploiting the charge characteristics of the viral capsid for efficient purification
Data Source
AI summary
The invention relates to a method for purifying an adenovirus comprising (a) providing a liquid sample comprising adenovirus, (b) clarifying said sample by depth filtration, (c) performing anion exchange chromatography comprising the steps of (i) directly applying the clarified sample of (b) to an anion exchange column, (ii) eluting adenovirus from the anion exchange column to provide an eluate. The invention also relates to adenovirus produced from said methods, and to compositions comprising same.