Dual Affinity Polypeptide Purification
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Solution Overview
Problem
Conventional affinity chromatography is costly and limited in its ability to handle proteins with dissociation constants outside the range of 10^-5 to 10^-7 M, leading to high costs and inefficiencies in the purification of therapeutic proteins, as it often results in protein denaturation during dissociation.
Innovation Solution
The use of target binding polypeptides (TBP) with dual binding functionalities, capable of forming higher-order complexes with target molecules, allowing for increased binding efficiency and purification through specific elution while the TBP remains immobilized on a support.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional affinity chromatography is used with dissociation constants in the range of 10^-5 to 10^-7 M, then the purification process is efficient and specific, but the cost is very substantial and prohibitive
Solution Approach 1:
The patent introduces a dual affinity polypeptide (DAP) as an intermediary molecule that mediates between the target protein and the solid phase matrix. The DAP has two distinct binding domains: one that binds to the target protein with moderate affinity and another that binds to the solid phase matrix with high affinity. This intermediary approach allows the system to achieve both efficient purification and reduced costs by enabling the use of cheaper solid phase matrices while maintaining high purification efficiency through the specific binding properties of the DAP.
2Adaptability or versatility
If conventional affinity chromatography is used for proteins with dissociation constants outside the range of 10^-5 to 10^-7 M, then all proteins can be purified, but protein denaturation occurs during dissociation
Solution Approach 1:
The patent utilizes parameter changes in binding affinity by designing the DAP with two different binding domains having distinct dissociation constants. The first domain binds to the target protein with moderate affinity (suitable for various protein types), while the second domain binds to the solid phase matrix with high affinity. This parameter differentiation allows the system to purify proteins with various dissociation constants without causing denaturation, as the moderate affinity interaction facilitates gentle elution conditions that preserve protein integrity.
3Ease of manufacture
If a generic capturing ligand immobilized to a matrix is used with a dual affinity polypeptide, then the system is cost-effective and versatile, but the binding capacity of the DAP molecule is limited
Solution Approach 1:
The patent employs composite materials by fusing two different affinity domains into a single dual affinity polypeptide molecule. The DAP comprises a first domain with affinity for the target protein and a second domain with affinity for the capturing ligand on the solid phase matrix. This composite structure allows the system to maintain cost-effectiveness through the use of generic capturing ligands while significantly enhancing the binding capacity through the synergistic effect of multiple affinity domains within the same polypeptide chain.
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 binding capacity and efficiency of protein purification, reducing costs and maintaining protein integrity by forming higher-order complexes that can be easily isolated and eluted, thus overcoming the limitations of conventional affinity chromatography.
Implementation Method 1
contacting a target molecule, and a population of target binding polypeptides (TBP), in solution for a sufficient time to allow complex formation
Implementation Method 2
The dual affinity polypeptide (DAP) reacts with the target biomolecule to form a 1:1 complex of medium binding affinity in which one DAP binds to one target, and this complex subsequently binds non-covalently to a generic affinity matrix with a strong binding affinity
Data Source
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AI summary
The present invention relates to a process for purification of a target molecule, comprising the steps: (a) contacting a target molecule, and a population of target binding polypeptides (TBP), in solution for a sufficient time to allow complex formation; and (b) isolating the target from the complex from (a) by subsequent purification steps, wherein (i) the target binding polypeptides have at least two binding functionalities; a first binding functionality towards the target and a second binding functionality towards a catching ligand comprised in a solid support; and (ii) the first binding functionality comprises at least two binding sites for the target, and the target com¬ prises at least two binding sites for the TBP.