Mutant Protein A Ligands Alkaline Stability
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current affinity chromatography matrices containing protein A ligands face challenges in withstanding high alkaline pH conditions during cleaning-in-place procedures, leading to reduced capacity and stability, despite previous mutations that only offer limited improvement.
Innovation Solution
Development of a mutant Fc-binding polypeptide and multimer with enhanced alkaline stability by mutating specific asparagine residues to amino acids like glutamic acid, lysine, or tyrosine, which maintains high selectivity for immunoglobulins and improves resistance to alkaline cleaning agents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If native or recombinant protein A ligands are used in affinity chromatography matrices, then high affinity and selectivity for immunoglobulins are achieved, but the matrices show decreased stability and capacity under high alkaline pH conditions during cleaning-in-place procedures
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the protein A sequence (particularly asparagine residues at positions 3, 6, and 40 in the B-domain) to alter the chemical stability parameters of the ligand. These mutations change the protein's resistance to alkaline conditions without significantly affecting its immunoglobulin-binding affinity, thereby resolving the contradiction between maintaining high selectivity and improving alkaline stability.
Solution Approach 2:
The invention applies local quality by introducing specific mutations at particular positions within the protein A sequence rather than globally modifying the entire protein. The mutations are localized to specific residues (e.g., N3A, N6A, N40A combinations) that are critical for alkaline stability, while leaving the immunoglobulin-binding regions intact, thus achieving improved alkaline resistance without compromising binding affinity.
2Reliability
If multiple amino acid mutations are introduced to improve alkaline stability, then resistance to cleaning agents is enhanced, but the complexity of protein production and characterization increases
Solution Approach 1:
The patent applies segmentation by dividing the protein A sequence into distinct domains (B-domain, C-domain, etc.) and focusing mutations specifically on the B-domain where they provide maximum alkaline stability improvement. This segmented approach allows for systematic evaluation of different mutation combinations (N3A, N6A, N40A) and simplifies the engineering process compared to random mutagenesis throughout the entire 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
The mutant polypeptides and multimers exhibit significantly improved alkaline stability, allowing for more effective cleaning and extended use in bioprocesses without significant loss of immunoglobulin-binding capacity, even at higher NaOH concentrations.
Implementation Method 1
proteins capable of specific binding to invariable parts of an immunoglobulin molecule, such interaction being independent on the antigen-binding specificity of the antibody
Implementation Method 2
the mutant polypeptides and multimers exhibit significantly improved alkaline stability, allowing for more effective cleaning and extended use in bioprocesses without significant loss of immunoglobulin-binding capacity, even at higher NaOH concentrations
Data Source
AI summary
An Fc-binding polypeptide of improved alkali stability, comprising a mutant of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 22, SEQ ID NO: 51 or SEQ ID NO: 52, wherein at least the asparagine or serine residue at the position corresponding to position 11 in SEQ ID NO: 4-7 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.