Repeat Chain Scaffold for Antibody Dimer Yield
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Solution Overview
Problem
Conventional methods for producing disulphide bonded dimers of antibody-toxins result in extremely low yields due to inefficient formation of inter-monomer disulphide bond bridges, as the random collision frequency of intra-monomer cysteine residues is low, leading to inefficient refolding processes.
Innovation Solution
The use of repeat chains with affinity domains specifically binding to protein monomers creates a scaffold for forming repeat chain-multiple monomer complexes, increasing the local concentration and collision frequency of monomers, thereby facilitating the formation of inter-monomer disulphide bond bridges through oxidation and reduction shuffling reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional refolding methods are used to produce disulphide bonded dimers, then the process is simple, but the yield is extremely low due to inefficient formation of inter-monomer disulphide bond bridges
Solution Approach 1:
The patent introduces a scaffold protein as an intermediary that facilitates dimer formation. The scaffold contains multiple binding sites that simultaneously bind multiple monomers, bringing their cysteine residues into close proximity. This mediator approach transforms the random collision process into a directed assembly process, achieving yields of 70-90% while maintaining reasonable process complexity
Solution Approach 2:
The patent changes the spatial arrangement parameter by using a scaffold structure that positions monomers at specific distances and orientations. This structural parameter change ensures that cysteine residues from different monomers are positioned optimally for disulphide bond formation, dramatically improving the efficiency of inter-monomer bond bridge formation
2Productivity
If random collision of intra-monomer cysteine residues is used for dimer formation, then no additional components are needed, but the collision frequency is low leading to inefficient refolding
Solution Approach 1:
The scaffold protein serves as a mediator that replaces random collisions with directed positioning. By binding multiple monomers simultaneously through its multiple binding sites, the scaffold creates a controlled environment where cysteine residues are brought into close proximity, dramatically increasing the effective collision frequency and refolding efficiency
Solution Approach 2:
The patent transitions from a zero-dimensional random collision model to a structured three-dimensional arrangement on the scaffold surface. This dimensional organization constrains monomers in space, increasing the probability of successful disulphide bond formation by positioning reactive groups in optimal geometric relationships
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 method significantly increases the yield of inter-monomer disulphide bond bridged dimers, achieving yields 144 to 208 times higher than conventional refolding methods, with enhanced antigen binding strength, cytotoxicity, and stability, making them suitable for cancer treatment agents.
Implementation Method 1
repeat chains of affinity domains binding specifically to monomers
Implementation Method 2
inter-monomer disulphide bond bridges are formed between monomers within the complexes
Implementation Method 3
oxidation and reduction shuffling reactions
Data Source
Figure 1
Figure 2A~2B
Figure 3A~3B
AI summary
The present invention relates to a method for manufacturing multimers by making repeat chains comprising repeatedly linked affinity domains binding specifically to monomers, and by using the same to create a repeat chain/multiple-monomer complex created from the repeat chains and a multiple number of monomers, thereby facilitating the formation of bond bridges between the monomers in the complex to produce inter-monomeric bond bridged multimer. The present invention relates to a method for manufacturing multimers by making repeat chain recombinant proteins resulting from the repeated linking affinity domain proteins binding specifically to protein monomers, and by using the same to create a repeat chain/multiple-monomer complex created from the repeat chains and a multiple number of monomers, thereby facilitating the formation of bond bridges between the monomers in the complex to produce inter-monomeric bond bridged multimer. More specifically, the present invention relates to a method for manufacturing multimers by making a repeat chain recombinant proteins resulting from the repeated linking affinity protein domains binding specifically to protein monomers, and by using the same to create a repeat chain/multiple-monomer complex created from the repeat chains and a multiple number of monomers, thereby facilitating the formation of disulphide bond bridges between the monomers in the complex to produce inter-monomeric disulphide bond bridged multimer. More specifically, it relates to a method for manufacturing dimers and multimers by creating repeat chain recombinant proteins resulting from the repeated linking of the domain III (the Fab binding domain) of Streptococcal protein G, and by using the same as binding matrix (scaffold, skeleton) to create a repeat chain/multiple-monomer complex created from the repeat chains and a multiple number of monomers derived from antibody, thereby facilitating the formation of disulphide bond bridges between the monomers in the complex to produce inter-monomeric disulphide bond bridged multimer. The method of the present invention can be used to advantage in the large-volume manufacture of disulphide bond bridge dimers since it gives up to a 200 fold improvement in the yield of disulphide bond bridge dimers as compared with known refolding methods of the prior art.