Non-natural Ig-binding proteins for alkaline-stable affinity purification
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Solution Overview
Problem
Current affinity chromatography methods using wild-type Protein A for antibody purification face challenges with alkaline stability, leading to a loss of binding capacity for immunoglobulins when exposed to harsh conditions, necessitating the development of novel Ig binding proteins with improved stability and affinity.
Innovation Solution
Non-natural immunoglobulin binding proteins with specific amino acid sequences, such as SEQ ID NO: 1, are designed to maintain high affinity for immunoglobulins even after alkaline treatment, featuring enhanced stability and binding capabilities compared to naturally occurring Protein A domains.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild-type Protein A is used for affinity purification, then high affinity binding to immunoglobulins is achieved, but binding capacity is lost under alkaline conditions
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of Protein A domains to create variants with altered properties. Specifically, mutations are introduced to enhance alkaline stability while preserving or improving immunoglobulin binding affinity, directly addressing the contradiction between binding capacity and alkaline stability
Solution Approach 2:
The patent creates composite binding proteins by combining multiple Protein A domains (A, B, C, D, E) in various configurations and fusions. These composite structures leverage the complementary properties of different domains to achieve both high binding affinity and improved alkaline stability that single domains cannot provide
2Productivity
If chromatography matrices are reused multiple times, then productivity is improved, but binding capacity deteriorates after alkaline cleaning
Solution Approach 1:
The patent applies preliminary action by pre-modifying the Protein A domains with mutations that confer alkaline resistance before the matrices are subjected to cleaning cycles. This proactive structural modification ensures the matrices can withstand repeated alkaline cleaning without losing binding capacity, enabling multiple reuse cycles
Solution Approach 2:
The patent implements beforehand cushioning by introducing protective amino acid substitutions that act as a buffer against alkaline damage. These modifications cushion the protein structure from the harsh cleaning conditions, preventing denaturation and maintaining binding function throughout the matrix lifecycle
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
These non-natural Ig binding proteins demonstrate increased alkaline stability and high affinity for immunoglobulins, allowing for efficient and repeated use in affinity purification processes without significant loss of binding capacity.
Implementation Method 1
Wild-type Protein A binds to the Fc region of IgG molecules with high affinity and selectivity
Implementation Method 2
An established procedure for capturing and purifying antibodies is affinity chromatography using the bacterial cell surface protein A
Data Source
Figure 1~1A
Figure 1B
Figure 2
AI summary
The present disclosure relates to non-natural binding proteins comprising one or more non-natural immunoglobulin (Ig) binding domains wherein at least one non-natural lg-binding domain comprises the amino acid sequence X1 X2X3XiXsX5X7 XsQQX11AFYX1sX15LX1 sX19PX21 LX23X24X2sQRX28X2gf IQSLKDDPSXio SXi2Xi3Xi4LXi5EAXigKLXs2Xs3Xs4QXs5PX. The idisclosure also relates to compositions such as affinity matrices comprising the non-natural Ig-binding proteins of the invention. Use of these Ig-binding proteins or of the compositions for affinity purification of immunoglobulins and to methods of affinity purification.