Antibody Framework Region Mutation Reversion for Aggregation Control
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Solution Overview
Problem
Concentrated immunoglobulin solutions tend to aggregate at high concentrations, posing challenges for downstream processing and formulation, especially for subcutaneous applications requiring concentrations above 100 mg/ml.
Innovation Solution
Reverting one or two mutations in the fourth framework region of the immunoglobulin heavy chain from hydrophobic or non-polar amino acids back to threonine or serine residues, as found in the natural germline sequence, to reduce aggregation in concentrated solutions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Volume of moving object
If immunoglobulin concentration is increased to achieve low volumes for economic handling and storage, then volume is reduced and handling efficiency is improved, but aggregation occurs and solution stability deteriorates
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence parameters of the immunoglobulin heavy chain, specifically in the fourth framework region. By changing the chemical nature of amino acid residues (from hydrophobic to hydrophilic), the patent alters the molecular interaction parameters that govern aggregation behavior, enabling stable high-concentration solutions without aggregation.
2Quantity of substance
If immunoglobulin concentration is increased to more than 100 mg/ml for subcutaneous application, then formulation efficiency is improved, but aggregation occurs and product quality deteriorates
Solution Approach 1:
The patent applies local quality by making targeted modifications only in the fourth framework region of the heavy chain, rather than altering the entire immunoglobulin structure. This localized approach changes the surface properties specifically in the region responsible for aggregation-prone interactions, while preserving the overall structure and function of the immunoglobulin molecule.
Solution Approach 2:
The patent changes the chemical parameter of amino acid residues in the fourth framework region from hydrophobic to hydrophilic character. This parameter change in the local region prevents aggregation while maintaining the ability to achieve high concentrations required for subcutaneous formulation.
3Reliability
If mutations are introduced to improve binding affinity or other functional properties, then functional performance is improved, but aggregation propensity increases due to altered sequence
Solution Approach 1:
The patent segments the immunoglobulin molecule into functional regions (variable domains for binding) and stability regions (framework regions). By focusing modifications on the fourth framework region specifically, the patent separates the functional optimization from the stability optimization, allowing both to be achieved independently.
Solution Approach 2:
The patent applies parameter changes to the fourth framework region amino acid sequence to restore hydrophilic character that was lost through previous mutations. This reverses the aggregation-promoting effects while preserving the functional improvements achieved through CDR mutations.
Data Source
AI summary
Herein is reported a method for reducing the aggregation of an immunoglobulin in solution comprising the steps of i) comparing the amino acid sequence of the fourth framework region of the heavy chain of an antibody with a reference or germline sequence and determining whether one or more threonine residues and/or serine residues have been replaced by a different amino acid residue, and ii) modifying the amino acid sequence of the immunoglobulin by reverting the exchanged threonine residues and/or serine residues back to threonine or serine of the reference or germline sequence and thereby reducing the aggregation of an immunoglobulin in solution.