Redox Buffer Re-oxidation for Antibody Fragment Rescue
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Solution Overview
Problem
Current methods for recombinant monoclonal antibody production fail to effectively prevent or mitigate the formation of low molecular weight protein fragments due to disulfide bond reduction, leading to reduced product yield and stability issues during storage.
Innovation Solution
A method involving the use of a redox buffer containing a thiol reducing agent and a thiol oxidizing agent to re-oxidize partial molecules into full molecules, either by admixing the buffer with the starting solution or during chromatography steps, to increase the yield of monomeric proteins and reduce the occurrence of low molecular weight fragments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If disulfide bond reduction occurs during cell culture harvest, then sample contamination with small molecular weight species increases, but current mitigation strategies only prevent HMW aggregation without rescuing LMW fragments
Solution Approach 1:
The patent applies the principle of converting harm into benefit by using the same redox chemistry that causes disulfide bond reduction to now benefit the process. Instead of merely preventing reduction, the invention intentionally introduces a redox buffer system that can reversibly reduce and then re-oxidize disulfide bonds, converting the harmful fragmentation into a recoverable intermediate state that can be rescued to restore full antibodies.
Solution Approach 2:
The patent implements parameter changes by controlling the redox potential of the buffer system throughout the purification process. By adjusting the ratio of reduced to oxidized forms of the redox buffer components, the process dynamically controls disulfide bond formation and breakage, enabling both prevention of aggregation and rescue of fragments at different stages.
2Productivity
If redox buffer is used to re-oxidize partial molecules, then conversion of LMW fragments to full molecules increases, but process complexity increases
Solution Approach 1:
The patent applies universality by designing a redox buffer system that performs multiple functions within a single formulation. The same buffer simultaneously prevents HMW aggregation, rescues LMW fragments, and maintains disulfide bond integrity throughout the chromatography process, eliminating the need for separate treatment steps for each problem.
Solution Approach 2:
The redox buffer system operates autonomously to maintain redox homeostasis throughout the purification process. The buffer automatically adjusts the redox state of protein disulfide bonds based on local conditions, providing self-regulating protection and rescue functions without requiring external intervention or complex process control.
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 effectively converts a significant percentage of partial molecules to full molecules, enhancing the purity and stability of the antibody product, thereby improving the efficiency of the protein purification process and extending the shelf life of therapeutic antibodies.
Implementation Method 1
a redox buffer comprising a redox pair which comprises at least one thiol reducing agent and at least one thiol oxidizing agent, wherein the redox buffer re-oxidizes the partial molecules to full molecules
Data Source
AI summary
Methods for the production of high purity recombinant protein such as monoclonal antibodies (mAb) using disulfide bond re-oxidation are provided. In particular, the present disclosure provides methods for converting partial molecules (e.g., antibody fragments) to full molecules (e.g., full antibodies) comprising admixing a starting solution comprising the partial molecules with a redox buffer comprising a redox pair which comprises at least one thiol reducing agent (e.g., cysteine) and at least one thiol oxidizing agent (e.g., cystine), wherein the redox buffer re-oxidizes the partial molecules to full molecules. The disclosed methods can be used, e.g., to prevent or mitigate the formation of partial molecules during protein purification, or to reprocess or rescue a solution comprising partial molecules (e.g., a partially degraded pharmaceutical formulation).