Fc-Region Variants for Bispecific Antibody Purification
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Solution Overview
Problem
The production of bispecific antibodies is challenging due to issues with chain association and purification, particularly in large-scale production, where efficient separation of heterodimeric Fc-regions from homodimeric forms is difficult, and the use of existing Fc-receptor binding ligands like Staphylococcal protein A and FcRn complicates purification processes.
Innovation Solution
Introduction of specific amino acid mutations in the CH2- and CH3-domains of the Fc-region, such as I253A, H310A, H433A, and Y436A, to create bispecific antibodies that bind to Staphylococcal protein A but not human FcRn, allowing for efficient separation of heterodimeric Fc-regions using protein A affinity chromatography.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If FcRn binding is used for purification, then purification selectivity is improved, but FcRn ligand availability and cost are worsened
Solution Approach 1:
The invention segments the purification approach by creating two distinct Fc-region variants: one optimized for FcRn binding (for purification) and one with native FcRn binding properties (for therapeutic function). This segmentation allows each variant to be optimized for its specific purpose without compromise
Solution Approach 2:
The invention introduces FcRn binding as an intermediary mechanism for purification. By engineering enhanced FcRn binding in the first Fc-region variant, the patent creates a intermediate purification step that separates heterodimers from homodimers before final product formulation, avoiding direct reliance on scarce FcRn ligands throughout the process
2Adaptability or versatility
If heterodimeric Fc-regions are produced, then bispecific antibody functionality is improved, but separation from homodimers is worsened
Solution Approach 1:
The invention introduces asymmetry into the Fc-region structure by creating two non-identical Fc-region variants (first and second variants) with different amino acid sequences. This asymmetric design ensures that only heterodimeric combinations can form the functional bispecific antibody, while homodimers are structurally distinct and can be separated through affinity chromatography or other purification methods
Solution Approach 2:
The invention applies local quality changes by introducing specific amino acid mutations in the CH2- and CH3-domains of the Fc-region variants. These localized modifications (e.g., I253A, H310A, H433A, Y436A) alter binding properties in specific regions without affecting the overall antibody structure or function, enabling selective purification while maintaining bispecific functionality
3Productivity
If Fc-region mutations are introduced, then purification properties are improved, but Fc-receptor binding is worsened
Solution Approach 1:
The invention segments the Fc-region into two distinct variants with differentiated functions: the first Fc-region variant contains mutations optimized for purification properties (enhanced FcRn binding), while the second Fc-region variant maintains native Fc-receptor binding properties. This segmentation allows each variant to fulfill its specific role without compromise
Solution Approach 2:
The invention introduces localized amino acid mutations specifically in the CH2- and CH3-domains of the Fc-region variants. These targeted mutations (e.g., I253A, H310A, H433A, Y436A) modify local binding properties to enhance purification efficiency while leaving other critical Fc-receptor interaction sites intact, thereby maintaining therapeutic functionality
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
Enables high-yield purification of bispecific antibodies by ensuring correct chain association and separation from homodimers, maintaining functional properties and enabling efficient production processes.
Implementation Method 1
The protein A-IgG interaction can be described using two main binding sites: the first is positioned in the heavy chain CH2 domain and is characterized by hydrophobic interactions between Phe 132, Leu 136, Ile 150 (of protein A) and the IgG hydrophobic knob constituted by Ile 253 and Ser 254
Implementation Method 2
The second site is located in the heavy chain CH3 domain and is dominated by electrostatic interactions between Gln 129 and Tyr 133 (protein A) and His 433, Asn 434, and His 435 (IgG)
Implementation Method 3
The FcRn functions to salvage IgG from the lysosomal degradation pathway, resulting in reduced clearance and increased half-life. The interaction between an antibody of the class IgG and the FcRn is pH dependent
Data Source
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AI summary
Herein is reported a polypeptide comprising a first polypeptide and a second polypeptide each comprising in N-terminal to C-terminal direction at least a portion of an immunoglobulin hinge region, which comprises one or more cysteine residues, an immunoglobulin CH2-domain and an immunoglobulin CH3-domain, wherein i) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations H310A, H433A and Y436A, or ii) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251D, L314D and L432D, or iii) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251S, L314S and L432S.