Ketone-Modified Polypeptide Conjugation via Oxime Linkage
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Solution Overview
Problem
Current methods for attaching payloads to proteins, such as antibody-drug conjugates, face challenges in achieving consistent and efficient conjugation while maintaining protein stability and activity, particularly when using disulfide linkages, as they often result in heterogeneous products and protein denaturation.
Innovation Solution
The method involves reducing disulfide bonds in proteins to form reactive thiol groups, which are then tethered together with a ketone-containing linkage that allows for efficient oxime formation with aminooxy compounds, enabling precise and stable conjugation of payloads, such as therapeutic agents, while retaining the protein's native conformation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If disulfide bonds are reduced to form reactive thiol groups for payload conjugation, then conjugation efficiency is improved, but protein stability and native conformation are compromised
Solution Approach 1:
The patent segments the conjugation process into two distinct stages: first reducing disulfide bonds to form thiol groups, then tethering these thiol groups with a bifunctional reagent containing a ketone group. This segmentation allows the conjugation to proceed efficiently while preserving the reduced thiol groups for controlled payload attachment, thereby maintaining protein stability.
Solution Approach 2:
The patent introduces a bifunctional reagent as an intermediary between the reduced thiol groups and the payload. This reagent contains a ketone group that forms a stable oxime linkage with the thiol groups, serving as a mediator that enables efficient conjugation while maintaining protein structure and stability.
2Adaptability or versatility
If conventional linker chemistry is used to attach payloads to proteins, then diverse payloads can be attached, but heterogeneous products are formed with inconsistent payload-to-protein ratios
Solution Approach 1:
The patent applies local quality by targeting specific reduced thiol groups generated from disulfide bond reduction. By using a bifunctional reagent that selectively reacts with these localized thiol groups, the method achieves site-specific conjugation, ensuring homogeneous products with consistent payload-to-protein ratios while maintaining the ability to attach diverse payloads.
3Quantity of substance
If multiple thiol groups are reduced for conjugation, then sufficient attachment sites are available, but protein denaturation occurs
Solution Approach 1:
The patent performs preliminary reduction of disulfide bonds to generate thiol groups before conjugation. This preliminary action creates sufficient attachment sites for payload attachment while the subsequent use of a bifunctional reagent with ketone group prevents over-reduction and maintains protein conformational stability throughout the process.
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 homogeneity and stability of protein-payload conjugates, improving the yield and functionality of antibodies and other proteins by maintaining their native conformation and allowing for controlled payload release.
Implementation Method 1
The method involves reducing disulfide bonds in proteins to form reactive thiol groups
Implementation Method 2
ketone-containing linkage that allows for efficient oxime formation with aminooxy compounds
Data Source
AI summary
The invention provides improved methods to prepare protein conjugates from a ketone-modified protein. In one embodiment, the protein is prepared by linking two free cysteines by reaction with a 1,3-dihaloacetone or similar ketone-containing reactant, linking the sulfur atoms of the two cysteines together. The ketone inserted between the sulfur atoms is then used to form an oxime, thus conjugating the protein to a payload. In another embodiment, two cysteine residues are tied together by reaction with a 1,3-dihaloacetone or similar reactant, and the new ketone is used to form an oxime with a suitable payload molecule, thus conjugating the protein to a payload. The method provides improved reaction conditions for the oxime formation, whereby higher yields and improved product homogeneity are achieved.