Factor V Purification via Two-Step Chromatography
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for purifying Factor V (FV) are complex, costly, and often result in protein denaturation, making it difficult to obtain a stable and virus-inactivated FV concentrate suitable for clinical use, especially for treating FV deficiencies like Parahaemophilia, with existing processes being inefficient and unsuitable for industrial-scale production.
Innovation Solution
A two-step chromatography process using weak anion exchangers with DEAE functional groups, where the first step separates FV from coagulation factors like FII, FVII, and FIX through batch incubation, and the second step captures and elutes FV, ensuring protein integrity and viral safety, followed by solvent/detergent treatment and nanofiltration for viral inactivation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If traditional purification methods are used, then FV can be obtained, but the process is complex and costly
Solution Approach 1:
The purification process is divided into distinct sequential steps: initial clarification, chromatography on hydroxylapatite, and chromatography on anion exchanger. Each step targets specific contaminants and progressively purifies FV, transforming a complex single-step process into manageable modular stages that improve manufacturability while controlling overall complexity.
Solution Approach 2:
The process utilizes systematic changes in pH and ionic strength parameters to achieve selective binding and elution of FV at different stages. By adjusting these parameters during chromatography steps, the method achieves high purity FV concentration without requiring complex equipment, thereby improving ease of manufacture through parameter optimization rather than equipment complexity.
2Reliability
If traditional purification methods are used, then FV can be obtained, but protein denaturation occurs
Solution Approach 1:
Protease inhibitors are added to the plasma sample before the purification process begins, and calcium ions are maintained throughout the chromatography steps. These preliminary protective actions prevent protein degradation and denaturation during processing, ensuring FV stability and integrity is preserved from the start of the process through to the final concentrate.
Solution Approach 2:
The chromatography steps are conducted under carefully controlled pH and ionic strength conditions that maintain FV in its native conformational state. By optimizing these parameters to match FV's stability requirements, the process achieves high purity without causing protein denaturation, thereby simultaneously improving reliability and maintaining compositional stability.
3Object-affected harmful factors
If existing processes are used, then FV concentrate can be produced, but viral safety is not ensured
Solution Approach 1:
The chromatography resins (hydroxylapatite and anion exchanger) act as intermediary materials that selectively bind and remove viral particles and contaminants while allowing FV to pass through or be selectively eluted. These intermediary substances provide an additional layer of viral safety by physically separating and removing potential viral contaminants during the purification process.
Solution Approach 2:
The process includes steps that adjust pH and ionic conditions to denature or inactivate viruses while preserving FV activity. By carefully controlling these parameters during chromatography and concentration steps, the method achieves viral inactivation without compromising the therapeutic protein, thereby ensuring viral safety while maintaining reliability.
4Manufacturing precision
If complex purification processes are used, then high purity FV is obtained, but industrial scalability is limited
Solution Approach 1:
The purification process is segmented into standardizable chromatography steps that can be performed in batch mode and easily scaled to industrial size. Each purification stage uses conventional chromatography techniques with well-established scalability, allowing the process to maintain high manufacturing precision while being adaptable to large-scale production requirements.
Solution Approach 2:
The chromatography steps are designed to be universally applicable across different scales of production. The same resin types and general protocol can be used from laboratory to industrial scale, with only parameters like column size and flow rate needing adjustment. This universality enables the process to achieve high purity FV while maintaining industrial scalability and productivity.
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 provides a simple, scalable, and cost-effective process for obtaining a high-yield, virus-inactivated FV concentrate with preserved protein integrity, suitable for clinical use, effectively addressing the limitations of existing purification methods by maintaining FV stability and ensuring viral safety.
Implementation Method 1
A two-step chromatography process using weak anion exchangers with DEAE functional groups, where the first step separates FV from coagulation factors like FII, FVII, and FIX through batch incubation
Implementation Method 2
the second step captures and elutes FV, ensuring protein integrity and viral safety
Implementation Method 3
followed by solvent/detergent treatment and nanofiltration for viral inactivation
Implementation Method 4
followed by solvent/detergent treatment and nanofiltration for viral inactivation
Data Source
Figure 1
Figure 2
Figure 3
AI summary
The present invention provides a process for purifying FV starting from human plasma or a fractionation intermediate thereof, that is simple, scalable to the industrial level and relatively inexpensive compared to the methods described in the literature to date. The invention consists of the use of two anion exchange chromatography steps, the first of which has the purpose of separating the FV from the PTC component factors, while the second has the purpose of isolating the protein of interest from the majority of plasma proteins by means of selective interaction with the weak anion exchange support used. The process developed has also had a viral inactivation step and a viral removal step included, contributing to the safety of the final product obtained, without however significantly altering the process total recovery of FV, and without necessitating the introduction of additional steps for eliminating the inactivating agents used, thanks to the order in which the various steps are conducted. The process described in the present invention also enables an FV concentrate to be obtained that is stable once frozen at -20° C.