Fc binding proteins with cysteine in the C-terminal helical region
Inventors
Fiedler, Erik • Haupts, Ulrich
Assignees
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Publication Number
US-11779860-B2
Publication Date
2023-10-10
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 an Fc binding protein comprising one or more domains derived from Protein A, where the one or more domains comprise a three-helix Fc-binding domain including a C-terminal helical region (helix 3). The Fc binding protein is engineered to include one or more cysteines located in the C-terminal helical region (helix 3), with cysteine(s) required at amino acid positions corresponding to a reference sequence.
The engineered Fc binding protein variants comprise amino acid sequences of SEQ ID NOS: 3-86, 90-99 or amino acid sequences with at least 89.5% identity to one of said sequences, together with the requirement that at least one amino acid in the specified positions corresponding to SEQ ID NO: 3 is cysteine. The approach further covers multimers, including mono-, di-, tri-, tetra-, and hexamer formats, and embodiments in which domains are linked, including direct linking or linking using linkers and conjugation chemistry targets.
The invention provides an affinity separation matrix and an affinity purification method that use the engineered Fc binding protein coupled to a solid support, including epoxy-activated matrices of an agarose/sepharose-type matrix. Proteins comprising an Fc sequence are contacted with the affinity separation matrix under conditions that permit binding, and then the Fc-containing proteins are eluted.
Claims Coverage
The document includes three independent claims: one directed to an Fc binding protein, one directed to an affinity separation matrix, and one directed to a method of affinity purification. Across these independent claims, the inventive coverage is built around specific Fc-binding domain sequence sets defined by SEQ ID NOS and % identity, and a required presence of cysteine at defined amino-acid positions corresponding to SEQ ID NO: 3.
Cys-in-helix-3 Fc-binding protein sequence with defined identity
An Fc binding protein comprising one or more domains, wherein the one or more domains comprises an amino acid sequence of any one of SEQ ID NOS: 3-86, 90-99 or an amino acid sequence with at least 89.5% identity to one of said sequences.
Required cysteine at defined positions corresponding to SEQ ID NO: 3
At least one amino acid in position 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 corresponding to SEQ ID NO: 3 is cysteine.
Affinity separation matrix with SEQ ID-defined Fc binding protein domains
An affinity separation matrix comprising an Fc binding protein comprising one or more domains, wherein the one or more domains comprises an amino acid sequence of any one of SEQ ID NOS: 3-16 or an amino acid sequence with at least 89.5% identity to one of said sequences.
Matrix-defined required cysteine at defined positions corresponding to SEQ ID NO: 3
At least one amino acid in position 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 corresponding to SEQ ID NO: 3 is cysteine.
Affinity purification workflow using coupled Fc-binding matrix and Fc elution
A method of affinity purification of a protein comprising an Fc sequence comprising providing a liquid containing protein comprising an Fc sequence, providing an affinity separation matrix with the Fc binding protein coupled to the matrix, contacting the affinity separation matrix with the liquid under conditions that permit binding, and eluting the protein comprising an Fc sequence from the affinity separation matrix.
Independent claim coverage centers on an Fc binding protein and matrix embodiments where SEQ ID-defined Fc-binding domains with at least 89.5% identity include cysteine at specified positions corresponding to SEQ ID NO: 3, and on a method that binds Fc-containing proteins to the coupled affinity separation matrix and elutes them.
Stated Advantages
Improves caustic/alkaline stability during repeated NaOH cleaning without impairing IgG Fc binding.
Improves site-specific coupling efficiency on matrices.
Improves dynamic binding capacity (DBC).
Provides high IgG elution recovery at ≥pH 3.5.
Documented Applications
Affinity purification of proteins comprising an Fc sequence using an affinity separation matrix comprising an Fc binding protein coupled to a solid support, followed by elution of Fc-containing proteins.
Use of the engineered Fc binding protein in an affinity separation matrix for binding and eluting IgG, including elution recovery reported at ≥pH 3.5.
Matrix use involving repeated NaOH cleaning to assess remaining IgG binding activity and alkaline stability of the coupled affinity separation matrix.
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