Nanobody Exchange Chromatography for Conformational Epitope Selection
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Solution Overview
Problem
Conventional methods for selecting polypeptide binders, particularly in recombinant antibody libraries, face challenges such as the need for purified antigens, difficulty in targeting conformational epitopes, and inefficiencies in high-throughput applications, especially when dealing with difficult-to-purify antigens under native conditions.
Innovation Solution
The NANEX technology employs Nanobody-based exchange chromatography, using a pair of Nanobodies (trapper and stripper) to selectively purify and elute target proteins in physiological conditions, allowing for the selection of specific binders from complex samples without the need for purified antigens, and enabling the identification of binders that target conformational epitopes with high affinity and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional selection methods are used for polypeptide binders, then the process is simpler, but purified antigens are required and conformational epitopes cannot be targeted effectively
Solution Approach 1:
The patent uses a first binding agent as an intermediary to capture the target antigen in its native conformation from complex samples. This intermediary enables the preservation of conformational epitopes without requiring antigen purification, allowing subsequent selection of polypeptide binders that recognize these conformational structures.
Solution Approach 2:
The method performs preliminary capture of the target antigen using a first binding agent before the actual selection of polypeptide binders. This preliminary action preserves the native conformation of the antigen and its epitopes, enabling subsequent identification of conformational epitope-specific binders without requiring prior antigen purification.
2Measurement precision
If purified antigens are used in selection methods, then specificity is improved, but the need for antigen purification reduces throughput and increases time
Solution Approach 1:
The patent extracts only the essential function of antigen purification by using a first binding agent to capture and immobilize the target antigen directly from complex samples. This extraction approach eliminates the time-consuming purification steps while maintaining the specificity needed for accurate binder selection, thereby increasing throughput.
Solution Approach 2:
The first binding agent serves as an intermediary that enables direct capture of native antigens from complex samples without purification. This intermediary maintains antigen specificity while eliminating the bottleneck of purification steps, allowing high-throughput selection of specific polypeptide binders.
3Ease of operation
If conventional elution methods are used, then binders can be released, but harsh conditions denature proteins and lose native conformation
Solution Approach 1:
The patent uses a second binding agent that competes with the first binding agent for the same epitope, creating a competitive displacement mechanism. This copying approach allows elution of bound polypeptide binders under physiological conditions by having the second binding agent replicate the epitope-binding function, thereby releasing the antigens and associated binders without denaturation.
Solution Approach 2:
The method changes the binding parameters by introducing a second binding agent with different kinetic properties that competes for the same epitope. This parameter change enables reversible displacement of the first binding agent under physiological conditions, allowing elution of native-conformation proteins without harsh denaturing conditions.
4Productivity
If high-throughput selection is performed, then productivity increases, but selection precision and specificity decrease
Solution Approach 1:
The first binding agent acts as an intermediary that captures native antigens from complex samples, preserving conformational epitopes and enabling specific binder selection. This intermediary maintains high selection accuracy while allowing high-throughput processing by eliminating purification steps and enabling direct selection from crude samples.
Solution Approach 2:
The second binding agent copies the epitope-binding function to competitively displace the first binding agent, enabling specific elution of target-bound polypeptide binders. This copying mechanism maintains selection precision by ensuring only epitope-specific binders are eluted, while supporting high-throughput operations through physiological condition elution.
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 enables efficient and selective identification of target-specific binders, achieving robust enrichment of antigens in 2-3 selection rounds, even from proteome-wide libraries, and allows for the isolation of novel binders that might be overlooked in conventional methods, while maintaining native protein conformation and physiological conditions.
Implementation Method 1
capturing the target of interest on a surface using a first binding agent to form an immobilized antigen complex
Implementation Method 2
eluting the target protein and the selective polypeptide binder, using a second binding agent competing for the target binding site of the first binding agent
Data Source
AI summary
The disclosure relates to a novel method for selection and identification of specific polypeptide binding agents for a target of interest. More specifically, the selection method involves capturing the target of interest on a surface using a first binding agent to form an immobilized antigen complex, selecting for specific antigen-binding polypeptides present in a sample, preferably as a display library, and eluting the target protein and the selective polypeptide binder, using a second binding agent competing for the target binding site of the first binding agent. More specifically, the selection method presented herein provides for an efficient and highly selective medium- to high-throughput technology applicable to recombinant antibody libraries, including immune and unbiased or proteome-wide display libraries, wherein selections can be performed without a need for purified target protein, in physiological conditions.