AAV Purification Using Fatty Acid Mobile Phase
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Solution Overview
Problem
Existing AAV purification systems fail to effectively remove non-functional capsids, leading to impurities in AAV products and insufficient vector genomes for gene therapy applications.
Innovation Solution
The development of an AAV product with fewer non-functional capsids, achieved by separating functional capsids containing a transgene from non-functional capsids based on hydrophobicity and charge, using techniques such as hydrophobic interaction chromatography (HIC) and anion exchange chromatography (AEX) with a fatty acid mobile phase.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional purification systems (ultracentrifugation or AEX without fatty acid mobile phase) are used, then the purification process is simple, but non-functional capsids cannot be effectively removed leading to impure AAV products
Solution Approach 1:
The patent introduces fatty acid mobile phase as an intermediary substance in the AEX chromatography process. This fatty acid mobile phase acts as a mediator that enables selective binding and separation of non-functional capsids from functional capsids, achieving high purity AAV product without requiring complex multi-step purification systems
Solution Approach 2:
The patent changes the chemical parameters of the mobile phase by incorporating fatty acids, which alters the binding characteristics of the anion exchange resin. This parameter change enables the system to differentiate between functional and non-functional capsids based on their hydrophobicity and charge properties, achieving effective separation while maintaining system simplicity
2Productivity
If conventional purification systems are used, then the process is straightforward, but insufficient quantities of vector genomes are produced for robust gene therapy applications
Solution Approach 1:
The patent extracts and removes non-functional capsids (empty capsids, partial capsids, and deamidated capsids) from the AAV product using AEX chromatography with fatty acid mobile phase. By selectively removing these impurities in a single step, the system achieves high productivity with sufficient vector genome quantities while maintaining ease of manufacture
Solution Approach 2:
The AEX chromatography system with fatty acid mobile phase performs multiple functions simultaneously: it removes empty capsids, partial capsids, and deamidated capsids in a single purification step. This multi-functional approach increases vector genome quantity and purity without complicating the manufacturing process
3Object-affected harmful factors
If non-functional capsids are not removed, then the capsid protein amount in a single effective dose is reduced, but immunoreactivity in administered subjects increases
Solution Approach 1:
The patent exploits the hydrophobicity and charge properties of non-functional capsids (which are otherwise harmful due to causing immunoreactivity) to enable their selective binding and removal in the AEX chromatography process. By converting these harmful properties into separation characteristics, the system effectively removes immunogenic impurities while preserving functional capsids
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
The resulting AAV product has improved transgene expression and reduced immunoreactivity, with a higher ratio of functional capsids to non-functional capsids, leading to more effective gene therapy applications.
Implementation Method 1
separating functional capsids containing a transgene from non-functional capsids based on hydrophobicity
Implementation Method 2
anion exchange chromatography (AEX) with a fatty acid mobile phase
Data Source
AI summary
Provided is an adenovirus-associated virus (AAV) product containing fewer non-functional capsids, such as deamidated empty AAV capsids, deamidated AAV capsids comprising a portion of the transgene, and deamidated full capsids, than AAV products currently in use. The AAV product contains a higher ratio of functional capsids to non-functional capsids (deamidated, hydrophobic capsids), compared to a control AAV product purified solely by ultracentrifugation, by anion-exchange chromatography (AEX) without a fatty acid mobile phase. Methods of making and using the AAV product are also provided, including separating functional capsids from non-functional capsids with anion-exchange chromatography (with fatty acid mobile phase), HIC, or chromatography with a mixed mode resin. Such methods also include maintaining transfected cells in a bioreactor about two days post-transfection, optionally at about 30-38° C., and subsequently separating the functional capsids from the non-functional capsids.