Selective Cysteine Reduction in Antibody Conjugates
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Solution Overview
Problem
Current methods for site-specific conjugation of therapeutic moieties to antibodies, such as ADCs, face challenges including heterogeneous mixtures due to improper disulfide bond reformation and instability of re-oxidation agents, leading to suboptimal in vivo performance and stability.
Innovation Solution
A process using specific phosphine-based reducing agents to selectively reduce engineered cysteines in antibodies, maintaining intact interchain disulfide bonds and preventing the formation of antibody half bodies and scrambled disulfides, thereby achieving a more homogeneous conjugate product.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If complete reduction of all interchain disulfide bonds and capped engineered cysteines is performed using TCEP or DTT, then all cysteine residues are freed for conjugation, but incorrect reformation of disulfide bonds occurs leading to mAb half body and scrambled disulfides
Solution Approach 1:
The patent applies local quality by using a reducing agent that selectively reduces only the capped engineered cysteines while leaving the interchain disulfide bonds intact. This selective reduction allows the engineered cysteines to be freed for conjugation without disrupting the native disulfide bond structure, thereby maintaining both conjugation capability and structural integrity.
Solution Approach 2:
The patent employs an intermediary reducing agent (such as TCEP at controlled conditions or alternative reducing agents like 2-mercaptoethanol) that acts as a selective mediator. This intermediary selectively targets the capped engineered cysteines for reduction while being too mild or specific to reduce the stable interchain disulfide bonds, thus preventing mAb half body and scrambled disulfide formation.
2Temperature
If mild oxidation using DHAA is performed to reform interchain disulfide bonds, then oxidation occurs under mild conditions, but DHAA is unstable in water complicating the process
Solution Approach 1:
The patent applies parameter changes by altering the oxidation method from DHAA-based mild oxidation to alternative oxidation systems. This includes changing the oxidant type, adjusting pH levels, temperature, or using metal-mediated oxidation systems that are more stable in aqueous environments while maintaining mild conditions, thereby resolving the instability issue of DHAA.
3Reliability
If continuous removal of cystine by TFF is performed to prevent re-oxidation, then engineered cysteines remain reduced, but considerable modification of standard equipment is required
Solution Approach 1:
The patent applies the taking out principle by removing the need for continuous TFF filtration equipment. Instead, it uses a chemical approach where the reducing agent is designed to be stable and non-reversing under conjugation conditions, or where the conjugation process itself prevents re-oxidation. This extracts the complex equipment requirement while maintaining reduction stability.
4Ease of operation
If conventional reduction procedure is used to uncapped engineered cysteines, then free cysteine residues are obtained for drug attachment, but heterogeneous ADC mixture is generated due to scrambled disulfides
Solution Approach 1:
The patent applies local quality by implementing a reducing agent that specifically targets only the capped engineered cysteines for reduction while preserving the interchain disulfide bonds. This selective local reduction ensures that only the intended cysteines are freed for conjugation, preventing scrambled disulfide formation and generating homogeneous ADC products with consistent conjugation sites.
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 results in a more homogeneous antibody conjugate with improved stability and in vivo performance by ensuring selective reduction of engineered cysteines without affecting native disulfide bonds, reducing the formation of high molecular weight species and maintaining the integrity of the antibody structure.
Implementation Method 1
A process using specific phosphine-based reducing agents to selectively reduce engineered cysteines in antibodies
Data Source
AI summary
The present invention relates to a process for the selective reduction of cysteine-engineered antibodies comprising reacting an antibody comprising one or more engineered cysteines at positions selected from HC40, HC41,HC42, HC89, HC152, HC153, HC155, HC171, LC40, LC41, LC165, and LC168 with a compound according to formula (I), (II), (III), (IV), (V), (VI) or (VII): (I) (II) (III) (IV) (V) (VI) (VII), and to a process for the preparation of antibody conjugates, including antibody-drug conjugates (ADCs).