Fc-Region Variants for Bispecific Antibody Chain Pairing
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Solution Overview
Problem
The challenge in producing bispecific or multispecific antibodies lies in the chain association issue during expression, leading to complex mixtures and low yields, particularly in achieving correct heavy and light chain pairing, which complicates purification and affects the pharmacokinetic properties of therapeutic monoclonal antibodies.
Innovation Solution
The introduction of specific amino acid mutations in the CH2 and CH3 domains of the Fc-region, such as H310A, H433A, and Y436A, or L251D, L314D, and L432D, along with disulfide bridges, allows for the separation of heterodimeric Fc-regions from homodimeric ones by modulating binding to Staphylococcal protein A and avoiding interaction with human FcRn, facilitating purification and correct chain association.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If specific amino acid mutations (H310A, H433A, Y436A, L251D, L314D, L432D) are introduced in the CH2 and CH3 domains of the Fc-region, then binding to Staphylococcal protein A is modulated enabling separation of heterodimeric Fc-regions from homodimeric ones, but interaction with human FcRn is reduced or eliminated affecting pharmacokinetic properties
Solution Approach 1:
The patent applies local quality by introducing specific amino acid mutations only in the CH2 and CH3 domains of the Fc-region, while leaving other regions unchanged. This localized modification enables differential binding properties: the mutations H310A, H433A, Y436A, L251D, L314D, and L432D specifically alter Protein A binding affinity in these domains, allowing heterodimeric Fc-regions to be separated from homodimeric ones during purification, while the overall pharmacokinetic function is preserved through careful selection of mutation sites that do not disrupt essential FcRn interaction regions
Solution Approach 2:
The patent employs parameter changes by systematically varying amino acid residues at specific positions (H310, H433, Y436, L251, L314, L432) in the Fc-region. These parameter modifications create a gradient of binding affinities that enable purification based on differential Protein A binding. The mutations are designed to modulate rather than eliminate Protein A binding, maintaining enough affinity for purification while creating sufficient differentiation between heterodimeric and homodimeric species for effective separation
2Ease of manufacture
If chain association is enforced during expression to achieve correct heavy and light chain pairing, then purification is simplified, but expression complexity increases leading to complex mixtures and low yields
Solution Approach 1:
The patent applies the taking out principle by extracting the chain association enforcement mechanism from the expression step and relocating it to the purification step. Instead of attempting to control heavy and light chain pairing during expression (which creates complex mixtures and low yields), the invention allows all chain combinations to form during expression, then uses the mutated Fc-region's differential Protein A binding properties to separate correct heterodimeric Fc-regions from incorrect homodimeric ones during purification. This separates the concerns of expression (maximizing yield) from purification (ensuring correctness)
Solution Approach 2:
The patent applies preliminary action by pre-modifying the Fc-region with specific amino acid mutations before expression, which prepares the molecule for subsequent purification based on differential binding. The mutations in the CH2 and CH3 domains are introduced in advance, creating the necessary binding property differences that will enable separation of correct from incorrect chain associations during the purification step, rather than attempting to prevent incorrect associations during expression
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 mutations enable efficient purification and separation of heterodimeric Fc-regions, improving the yield and pharmacokinetic properties of bispecific antibodies by ensuring correct chain pairing and reducing interaction with human FcRn, thus enhancing their therapeutic potential.
Implementation Method 1
The purification of monoclonal antibodies by means of selective Fc-region affinity ligands... The ligands most adopted to bind selectively IgG are Staphylococcal protein A and protein G, which are able to establish highly selective interactions with the Fc-region of most IgGs
Implementation Method 2
These mutations enable efficient purification and separation of heterodimeric Fc-regions, improving the yield and pharmacokinetic properties of bisspecific antibodies by ensuring correct chain pairing and reducing interaction with human FcRn
Data Source
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, whereini) the first and the second polypeptide comprise the mutations H310A, H433A and Y436A, orii) the first and the second polypeptide comprise the mutations L251D, L314D and L432D, oriii) the first and the second polypeptide comprise the mutations L251S, L314S and L432S.