FER Format Antibody Mutations for Low pH Stability
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Solution Overview
Problem
Current antibody engineering to eliminate Fc-mediated effector functions can lead to stability issues under low pH conditions and increased glycosylation heterogeneity, complicating manufacturing and clinical use.
Innovation Solution
The introduction of specific mutations, such as L234F, L235E, and G236R in the Fc region of IgG1 antibodies, known as the FER format, which suppresses residual complement-dependent cytotoxicity and provides a wild-type-like glycosylation profile while improving tolerance to low pH conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If non-activating mutations are introduced to eliminate Fc-mediated effector functions, then unwanted immune activation is reduced, but protein stability under low pH conditions deteriorates
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions (L234F, L235E, G236R) in the Fc region to modify the protein's chemical structure. These substitutions change the local physicochemical properties of the Fc region, enabling it to resist low pH-induced denaturation while maintaining suppression of Fc-mediated effector functions. The mutations alter the conformational stability and charge distribution in the Fc region, allowing the antibody to maintain structural integrity under low pH conditions without activating unwanted immune responses.
2Object-affected harmful factors
If non-activating mutations are introduced to eliminate Fc-mediated effector functions, then unwanted immune activation is reduced, but glycosylation heterogeneity increases
Solution Approach 1:
The patent uses parameter changes through specific amino acid substitutions (L234F, L235E, G236R) that modify the local environment around the N297 glycosylation site. These substitutions stabilize the Fc region conformation, creating a more uniform microenvironment for glycan attachment. The mutations reduce conformational flexibility and stabilize the glycosylation pocket, leading to more homogeneous glycosylation patterns while maintaining suppression of Fc-mediated effector functions.
3Reliability
If standard viral inactivation protocols are used, then viral contamination is controlled, but protein stability under low pH conditions deteriorates
Solution Approach 1:
The patent applies preliminary action by pre-engineering the Fc region with stabilizing mutations (L234F, L235E, G236R) before subjecting the antibody to low pH viral inactivation protocols. These mutations are introduced in advance to preemptively strengthen the protein's resistance to low pH stress. The pre-modified Fc region maintains structural integrity during the viral inactivation process, allowing standard low pH protocols to be used effectively without causing protein denaturation or aggregation.
Data Source
AI summary
Described herein are proteins comprising an Fc region or the like, such as monoclonal, bispecific and multispecific antibodies, wherein the Fc region has been modified to eliminate or strongly reduce Fc-mediated effector functions, while at the same time allow for good developability, for therapeutic purposes and where such effector functions are undesired.