Engineered Protein A Ligand Alkali Resistance Antibody Dissociation
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Solution Overview
Problem
Current engineered Protein A ligands face challenges in achieving optimal alkali resistance and acid-induced antibody dissociation, with existing mutations primarily focusing on replacing Gly residues at position 29 with Ala, limiting the exploration of amino acids with larger side chains that may offer better performance.
Innovation Solution
Development of recombinant Protein A mutants where Gly residues in the E, D, A, B, and C domains are replaced with amino acids like Val, Leu, Ile, Phe, Tyr, Trp, Glu, Arg, His, Met, or Cys, enhancing alkali resistance and antibody dissociation properties compared to conventional mutants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Gly residues at position 29 are replaced with Ala in Protein A domains, then alkali resistance is improved, but antibody dissociation in acidic environments is limited
Solution Approach 1:
The patent systematically varies the amino acid substitution at position 29 across all five domains (E, D, A, B, C), testing multiple amino acids (Ala, Val, Leu, Ile, Phe, Tyr, Trp, Glu, Arg, His, Met, Cys) to optimize the balance between alkali resistance and antibody dissociation properties
Solution Approach 2:
The patent introduces different amino acid substitutions at position 29 in each of the five domains independently, allowing each domain to have optimized properties for its specific function while contributing to overall protein performance
2Object-generated harmful factors
If recombinant Protein A without XM region is used, then non-specific adsorption is reduced, but binding capacity and reuse efficiency are limited
Solution Approach 1:
The patent creates a composite structure by combining five mutated domains (each with optimized position 29 substitution) into a multi-domain Protein A molecule, achieving synergistic effects that enhance both specific binding capacity and alkali resistance while maintaining low non-specific adsorption
Solution Approach 2:
The patent designs a multi-domain Protein A variant that simultaneously achieves multiple functions: high specific binding capacity, reduced non-specific adsorption, improved alkali resistance, and enhanced antibody dissociation, making it universally applicable for antibody purification
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
The new Protein A mutants demonstrate improved chemical stability in alkaline conditions and enhanced antibody dissociation in acidic environments, leading to more effective separation and purification of antibodies using affinity chromatography.
Implementation Method 1
a protein having an affinity for an immunoglobulin... specifically binds to an antibody
Implementation Method 2
a method for separating and purifying, or adsorbing and removing an antibody with the use of this matrix
Data Source
AI summary
An object of the present invention is to create a novel engineered Protein A ligand having better antibody dissociation properties in the presence of an acid than conventional engineered Protein A ligands and a further object of the present invention is to create a novel engineered Protein A ligand having higher alkali resistance. The present invention is to provide a protein having an affinity for an immunoglobulin, including an amino acid sequence derived from any of E, D, A, B and C domains of Protein A, wherein at least one Gly residue in the amino acid sequence is replaced with an amino acid other than Ala, and the protein has a lower affinity for an Fab region of an immunoglobulin than a protein including an amino acid sequence in which the Gly residue is replaced with Ala. Also, the present invention is to provide the protein having an affinity for an immunoglobulin, which has improved chemical stability in an alkaline condition compared to the corresponding domain.