Factor VII Purification via Multi-Modal Anion Exchange Chromatography
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Solution Overview
Problem
Current methods for purifying Factor VII and/or Factor VIIa from solutions contain drawbacks such as the use of costly monoclonal antibody-based purification, potential contamination from non-Factor VII-related substances, and the need for effective removal of solvent and detergent, which existing technologies fail to address efficiently.
Innovation Solution
A method utilizing mixed-mode anion exchange chromatography with specific buffer conditions, including dilution, binding, washing, and elution steps with reduced calcium ion concentrations, to effectively purify Factor VII and/or Factor VIIa, reducing contaminants and detergent while improving protein recovery and load capacity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If immuno-affinity chromatography using calcium-dependant anti-FVII monoclonal antibody is used, then high purity of Factor VII is achieved, but the cost of production increases considerably and antibodies may leach into the final product affecting safety
Solution Approach 1:
The patent replaces expensive monoclonal antibody-based immuno-affinity matrices with conventional, non-antibody based purification matrices that are cheaper and do not risk leaching into the final product. This substitution maintains purification effectiveness while eliminating the safety and cost issues associated with antibody-based methods.
2Manufacturing precision
If immuno-affinity chromatography is used, then high purity of Factor VII is achieved, but the device complexity and production cost increase due to monoclonal antibody production requirements
Solution Approach 1:
The patent eliminates the need for complex monoclonal antibody production and purification systems by using conventional purification matrices. This simplifies the overall device complexity while maintaining the ability to achieve high purity Factor VII through standard chromatographic techniques.
3Ease of manufacture
If conventional purification methods are used to remove non-Factor VII-related contaminants, then the ability to separate unwanted isoforms is reduced, but the cost and complexity are lowered
Solution Approach 1:
The patent optimizes buffer conditions, particularly calcium ion concentration (maintained at about 35 mM or less throughout the process), to enhance the selective binding of Factor VII to the anion exchange resin. This parameter control enables effective separation of Factor VII isoforms and contaminants using conventional matrices, achieving both cost-effectiveness and high separation precision.
4Productivity
If high load volumes are applied to the purification resin, then productivity increases, but the purification efficiency and protein quality may be adversely affected
Solution Approach 1:
The patent maintains calcium ion concentration at about 35 mM or less during loading, washing, and elution steps to optimize resin binding capacity and selectivity. This parameter control allows high load volumes to be processed while maintaining protein quality and purification efficiency, as the controlled calcium levels prevent non-specific binding and maintain selective Factor VII recovery.
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 enhances the purification efficiency by reducing contaminants and detergent levels, increasing protein recovery, and allowing for greater load volumes without adverse effects on the protein, thereby improving the safety and cost-effectiveness of the final product.
Implementation Method 1
adding the diluted solution from step (i) to a multi-modal anion exchange resin under conditions selected such that Factor VII and/or Factor VIIa is bound to the resin
Implementation Method 2
adding an elution buffer to the resin under conditions selected such that Factor VII and/or Factor VIIa is eluted from the resin
Data Source
AI summary
The present invention relates generally to a method of purifying Factor VII and/or Factor VIIa from a solution containing either or both proteins, the method comprising: diluting the solution containing Factor VII and/or Factor VIIa in a loading buffer; adding the diluted solution from step (i) to a multi-modal anion exchange resinunder conditions selected such that Factor VII and/or Factor VIIa is bound to the resin; optionally washing the resin with a wash buffer; adding an elution buffer to the resin under conditions selected such that Factor VII and/or Factor VIIa is eluted from the resin; and recovering the eluted recombinant Factor VII and/or Factor VIIa; wherein the loading buffer, the optional wash buffer and the elution buffer each comprise about 35 mM or less of calcium ions.