AAV-Binding Protein Engineering for Alkali-Resistant Vector Purification
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Solution Overview
Problem
Existing AAV-binding proteins lack sufficient alkaline resistance during purification processes, leading to inefficiencies in separating Full AAV vectors from Empty AAV vectors, and existing methods for purifying Full AAV vectors are complex and prone to contamination.
Innovation Solution
Developing an AAV-binding protein with specific amino acid substitutions that enhance alkaline resistance, followed by a two-step purification process involving affinity chromatography and anion-exchange chromatography to achieve high-purity separation of Full AAV vectors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional AAV-binding proteins are used for affinity chromatography, then AAV purification can be performed, but the proteins lack sufficient alkaline resistance during cleaning processes
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues in the AAV-binding protein sequence. The substitution of particular amino acids at defined positions alters the protein's chemical properties to enhance its resistance to alkaline conditions during cleaning processes, while maintaining its AAV binding functionality.
Solution Approach 2:
The patent implements local quality by introducing specific amino acid substitutions at particular positions within the protein sequence rather than uniformly modifying the entire protein. This targeted approach enhances alkaline resistance at critical regions while preserving the overall structure and binding capability of the protein.
2Manufacturing precision
If complex purification methods are used to separate Full AAV vectors from Empty AAV vectors, then separation can be achieved, but the process becomes complex and prone to contamination
Solution Approach 1:
The patent utilizes parameter changes in the form of controlled pH gradients during elution. By adjusting the pH of the elution buffer, the method enables selective elution of Full AAV vectors from the affinity chromatography column, achieving high-purity separation from Empty AAV vectors through a simple single-step process rather than multiple complex steps.
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 modified AAV-binding protein effectively withstands alkali cleaning, allowing for efficient and high-purity purification of Full AAV vectors, reducing the risk of contamination and simplifying the purification process.
Implementation Method 1
an adsorbent including an insoluble support and an AAV-binding protein immobilized on the support is used. In this method, an AAV vector can be recovered and purified from a solution containing the vector in which impurities are also present.
Implementation Method 2
the purification of the AAV is typically followed by alkali cleaning for residual AAV and impurities using a high-concentration (e.g., 0.1 M or more and 0.5 M or less) aqueous solution of sodium hydroxide. Accordingly, there is a need for the creation of a ligand protein that can withstand this alkali cleaning.
Implementation Method 3
a two-step purification process involving affinity chromatography and anion-exchange chromatography to achieve high-purity separation of Full AAV vectors
Data Source
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AI summary
The present disclosure relates to a protein having affinity for an Adeno-Associated Virus (AAV). Provided are: (i) an AAV-binding protein comprising at least the amino acid residues from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO: 2, provided that one or more of particular amino acid substitutions have occurred at the amino acid residues from position 25 to position 213, the protein having AAV-binding activity; (ii) an AAV-binding protein comprising at least the amino acid residues from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO: 2, provided that one or more of the particular amino acid substitutions have occurred at the amino acid residues from position 25 to position 213, and any one or more of substitution, deletion, insertion or addition of one or several amino acid residues at one or several positions, other than the particular amino acid substitutions, have further occurred, the protein having AAV-binding activity; and (iii) an AAV-binding protein comprising an amino acid sequence having a homology of 70% or more to the entire amino acid sequence in which one or more of the particular amino acid substitutions have occurred in the amino acid sequence from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO: 2, wherein the at least any one amino acid substitution is retained, the protein having AAV-binding activity.