Fc binding proteins with cysteine in the c-terminal helical region
Inventors
Fiedler, Erik • Haupts, Ulrich
Assignees
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Publication Number
US-12257530-B2
Publication Date
2025-03-25
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 affinity separation matrix comprising an Fc binding protein. At least one domain of the Fc binding protein includes an amino acid sequence of SEQ ID NO: 3, or an amino acid sequence with at least 91% identity thereto, where at least one amino acid at a position corresponding to position 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 of SEQ ID NO: 3 is cysteine.
The Fc binding protein architecture is intended to provide alkaline (caustic) stability while maintaining IgG binding during repeated harsh NaOH cleaning. The disclosed stability is compared with wild-type Protein A during repeated NaOH treatment, and is supported by remaining binding activity after treatment and by dynamic binding capacity data.
Performance is further characterized by IgG elution at acidic pH with high IgG elution recovery, and by high dynamic binding capacity. The disclosed Fc binding proteins and matrices allow coupling/site-specific immobilization via cysteine-based attachment, and include options for domain and multimer configuration, as well as linker and conjugation variations.
Claims Coverage
The claims cover an affinity separation matrix with Fc binding proteins defined by a specific SEQ ID NO: 3 (or high-identity variant) domain and cysteine substitutions at helix 3 positions, with additional dependent claim refinements covering tighter identity thresholds, specific conjugation constraints, termini deletion options, and the number of linked domains.
Affinity separation matrix with a defined SEQ ID NO: 3 Fc-binding domain
An affinity separation matrix comprising an Fc binding protein comprising one or more domains, wherein at least one domain comprises an amino acid sequence of SEQ ID NO: 3, or an amino acid sequence with at least 91% identity thereto.
Cysteine in helix 3 at specified positions of the SEQ ID NO: 3 domain
At least one amino acid at a position corresponding to position 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 of SEQ ID NO: 3 is cysteine.
Conjugation via cysteine at specified positions relative to SEQ ID NO: 3
The Fc binding protein is conjugated to the matrix through a cysteine located at one of specified positions relative to SEQ ID NO: 3.
Tightened sequence identity threshold for the SEQ ID NO: 3 domain
At least one Fc binding protein domain contains an amino acid sequence with at least 93% identity to SEQ ID NO: 3.
Specified number of linked domains in the Fc binding protein
The Fc binding protein includes 2, 3, 4, 5, 6, 7, or 8 linked domains.
Overall, the claim set defines affinity separation matrices based on an Fc binding protein domain having SEQ ID NO: 3 (or ≥91% identity) together with cysteine at specified helix 3 positions, and narrows embodiments by requiring exact SEQ ID NO: 3, higher identity thresholds, cysteine-based conjugation to the matrix, optional termini deletion constraints, and a discrete range for the number of linked domains.
Stated Advantages
Alkaline (caustic) stability versus wild-type Protein A during repeated harsh NaOH cleaning while maintaining IgG binding.
Improved coupling/site-specific immobilization.
High dynamic binding capacity (DBC).
High IgG elution recovery at acidic pH.
Documented Applications
Affinity purification of Fc-containing immunoglobulins/proteins using affinity chromatography and affinity separation matrix formats.
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