Fc binding proteins with cysteine in the c-terminal helical region
Inventors
Fiedler, Erik • Haupts, Ulrich
Assignees
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Publication Number
US-11548929-B2
Publication Date
2023-01-10
Expiration Date
Abstract
The present invention relates to Fc binding proteins comprising one or more domains with Cysteine in the C-terminal helical region. The invention further relates to affinity matrices comprising the Fc binding proteins of the invention. The invention also relates to a use of these Fc binding proteins or affinity matrices for affinity purification of immunoglobulins and to methods of affinity purification using the Fc binding proteins of the invention.
Core Innovation
The invention relates to Fc binding proteins comprising one or more domains in which at least one domain comprises an amino acid sequence of SEQ ID NO: 2. In that domain, the amino acid in position 43, 46, 47, 50, 51, or 53 corresponding to SEQ ID NO: 2 is Cysteine. The described Fc binding proteins are based on Protein A-like three-helix Fc-binding domains.
The described approach addresses a need for improved caustic/alkaline stability while maintaining Fc binding. The document describes retention of binding after prolonged contact with caustic/alkaline conditions, indicating increased caustic/alkaline stability for the Fc binding proteins.
The document further links the Cysteine-containing Fc binding proteins to improved site-specific coupling to epoxy/activated matrices on a solid support. It describes affinity separation matrices that immobilize the Fc binding proteins, and affinity purification workflows for Fc-containing immunoglobulins using binding/contacting with subsequent elution.
Claims Coverage
The partial set identifies one independent claim (clm-00001). Across the dependents referenced in the provided claim excerpts, the inventive features cover a cysteine-substituted Protein A-like Fc-binding domain sequence (SEQ ID NO: 2), structural and sequence constraints, binding scope to Fc-containing immunoglobulins, and affinity separation/purification using a coupled matrix and an elution step.
Cysteine substitution at defined SEQ ID NO: 2 positions
An Fc binding protein comprising one or more domains, wherein at least one domain comprises an amino acid sequence of SEQ ID NO: 2 wherein the amino acid in position 43, the amino acid in position 46, the amino acid in position 47, the amino acid in position 50, the amino acid in position 51, or the amino acid in position 53 corresponding to SEQ ID NO: 2 is Cysteine.
Domain sequence variants with specified identity and cysteine positions
The Fc binding protein specifies that at least one domain contains an amino acid sequence matching SEQ ID NOs: 3-16 (or a sequence with at least 89.5% identity), with the residues at positions 43, 46, 47, 50, 51, or 53 being cysteine.
Linked multi-domain architecture
The Fc binding protein includes a linked multi-domain structure comprising 2 to 8 domains.
Cysteine-position conjugation to a solid support
A specified Fc binding protein is conjugated to a solid support through the cysteine at position 43, 46, 47, 50, 51, or 53.
Fc-specific affinity purification using an affinity separation matrix
A method for affinity purification of a protein comprising an Fc sequence by contacting an Fc-binding-protein affinity separation matrix with an Fc-containing protein-containing liquid and then eluting the Fc-sequence protein from the matrix.
Binding multiple Fc-containing immunoglobulin formats
The Fc binding protein is defined by its ability to bind multiple immunoglobulin classes, Fc-containing Ig fragments, Fc-containing fusion proteins, and Fc-containing conjugates.
Overall, the claim set coverage in the provided excerpts centers on Fc binding proteins with a Protein A-like three-helix Fc-binding domain in which defined residues in SEQ ID NO: 2 are cysteine, and on how that cysteine enables site-specific conjugation to a solid support and use in affinity separation and affinity purification for Fc-containing immunoglobulins.
Stated Advantages
Increased caustic/alkaline stability with retention of Fc binding after prolonged exposure to caustic/alkaline conditions.
Improved site-specific coupling to epoxy/activated matrices using the specified cysteines.
Improved dynamic binding capacity (DBC) compared with Protein A, as described in the document.
High recovery of bound IgG upon elution as described in the document.
Documented Applications
Affinity purification of Fc-containing immunoglobulins/fragments using an affinity separation matrix comprising an immobilized Fc binding protein, with binding/contacting and elution steps.
Use of affinity separation matrices/affinity chromatography matrices formed by immobilizing the described Fc binding proteins on a solid support.
Binding and purification for Fc-containing immunoglobulin classes and Fc-containing formats including IgG1, IgG2, IgG4, IgM, IgA, immunoglobulin fragments comprising Fc, fusion proteins comprising an Fc region, and conjugates comprising an Fc region.
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