Solid Support Reducing Agent for Trisulfide Bond Conversion
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Solution Overview
Problem
Current methods for eliminating trisulfide bonds in proteins during production and purification procedures, such as those for antibodies, are inefficient and can promote aggregation due to the need for large quantities of reducing agents like cysteine, which also require separate steps for removal and may form undesirable disulfide linkages.
Innovation Solution
A method involving allowing proteins with trisulfide bonds to associate with a solid support and then exposing them to a reducing agent at specific concentrations, followed by replacing the reducing agent solution, effectively converting trisulfide bonds to disulfide bonds, thereby reducing molecular heterogeneity and aggregation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If large quantities of reducing agents like cysteine are used to eliminate trisulfide bonds, then trisulfide bonds are effectively removed, but protein aggregation is promoted and additional removal steps are required
Solution Approach 1:
The patent uses a solid support as an intermediary carrier to bind the reducing agent, enabling controlled delivery to trisulfide bonds. This mediation prevents excessive reducing agent exposure that causes aggregation, while still achieving effective trisulfide bond conversion to disulfide bonds through the controlled interaction on the solid support surface.
Solution Approach 2:
The patent changes the physical state of the reducing agent from free solution to solid support-bound form, and optimizes the concentration to 0.5-3 mM. This parameter change enables precise control of reducing agent availability, achieving sufficient trisulfide bond conversion while minimizing aggregation-promoting effects that occur with higher concentrations in solution.
2Reliability
If large quantities of reducing agents like cysteine are used to eliminate trisulfide bonds, then trisulfide bonds are effectively removed, but additional steps for reducing agent removal are required
Solution Approach 1:
The solid support acts as an intermediary that simplifies the overall process. The reducing agent is delivered and removed with the solid support itself, eliminating the need for separate removal steps. The solid support can be easily separated from the protein solution through standard centrifugation or filtration, streamlining the workflow.
Solution Approach 2:
The patent combines the reducing agent with the solid support into a single reagent system. This merging allows the reducing agent to be introduced and removed as part of a single handling unit, reducing the number of discrete steps compared to adding free reducing agent followed by separate removal procedures.
3Reliability
If reducing agents are used to convert trisulfide bonds to disulfide bonds, then bond conversion is achieved, but undesirable disulfide linkages may form
Solution Approach 1:
The patent optimizes the reducing agent concentration to a specific range (0.5-3 mM) and controls the reaction conditions on the solid support surface. These controlled parameters enable selective conversion of trisulfide bonds to disulfide bonds while preventing over-reduction or scrambling that would create undesirable disulfide linkages, thus maintaining proper disulfide bond patterns.
Solution Approach 2:
The solid support serves as a controlled environment that mediates the reduction reaction. This controlled interface allows selective access to trisulfide bonds for conversion to disulfide bonds, while the solid support structure prevents random disulfide scrambling and maintains the integrity of existing disulfide bond patterns in the protein.
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 efficiently converts trisulfide bonds to disulfide bonds, improving protein stability and reducing aggregation, while minimizing the need for additional steps and reducing agent quantities, thus enhancing the quality and consistency of therapeutic proteins like antibodies.
Implementation Method 1
exposing said proteins bound to the solid support to a solution comprising a reducing agent... converting trisulfide bonds to disulfide bonds
Data Source
Figure 1(A)~1(D)
Figure 2
Figure 3
AI summary
The present invention pertains to methods of preventing and eliminating trisulfide bonds in proteins such as antibodies. In one embodiment, trisulfide bonds in proteins are converted to disulfide bonds as part of chromatographic purification procedures. In another embodiment, the formation of trisulfide bonds in proteins is inhibited by implementation of methods described herein during the cell culture production of such proteins. In another embodiment, monoclonal antibodies are produced by the methods described herein.