Fc binding proteins with cysteine in the C-terminal helical region
Inventors
Fiedler, Erik • Haupts, Ulrich
Assignees
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Publication Number
US-12275774-B2
Publication Date
2025-04-15
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 disclosed invention relates to an Fc binding protein comprising one or more domains that include an amino acid sequence derived from Protein A. In particular, the protein is defined by containing an amino acid sequence of SEQ ID NO: 3, or an amino acid sequence with at least 91% identity to SEQ ID NO: 3, with a cysteine at specific positions. The specified cysteine-containing positions include amino acids at position 43, 46, 47, 50, 51, or 53 within the C-terminal helix 3 region.
The invention further defines Fc binding proteins whose cysteine substitutions are designed to increase caustic/alkaline stability during repeated NaOH cleaning, without reducing IgG binding. The disclosed approach is presented as maintaining IgG binding while improving remaining binding capacity under strong alkaline sanitization, including prolonged exposure to NaOH. The document describes this as improved remaining IgG binding after prolonged 0.5 M NaOH exposure, and as enabling repeated use under strong alkaline sanitization conditions.
The disclosed invention also defines affinity separation matrices incorporating the immobilized Fc binders. Using solid support and affinity separation matrix formats, the document describes affinity purification of immunoglobulins (including IgG) with pH/salt-controlled elution and reuse under strong alkaline sanitization. Experimental examples report improved dynamic binding capacity relative to Protein A and near-complete elution behavior from agarose bead matrices using the cysteine variants.
Claims Coverage
The independent claims define three core claim groupings: (i) sequence-defined Fc binding proteins from SEQ ID NO: 3 with cysteine at specified helix 3 positions, (ii) Fc binding proteins that include at least 91% identity to SEQ ID NO: 3 with cysteine at corresponding positions, and (iii) broader Fc binding proteins defined by inclusion of particular SEQ ID NO: 18–20 domains (or ≥91% identity). Across these independent claims, the inventive features focus on cysteine placement and sequence identity/selection, while dependent claims extend coverage to conjugated solid support and affinity separation matrices that incorporate the Fc binders.
Sequence-defined Fc binder with helix-3 cysteine in SEQ ID NO: 3
An Fc binding protein comprising one or more domains, wherein at least one domain comprises an amino acid sequence of SEQ ID NO: 3, wherein the amino acid at position 43, 46, 47, 50, 51, or 53 of SEQ ID NO: 3 is cysteine.
≥91% identity SEQ ID NO: 3 variant with helix-3 cysteine
An Fc binding protein comprising one or more domains, wherein at least one domain comprises an amino acid sequence with at least 91% identity to SEQ ID NO: 3, wherein the amino acid in a position corresponding to position 43, 46, 47, 50, 51, or 53 of SEQ ID NO: 3 is cysteine.
SEQ ID NO: 18–20 domain (or ≥91% identity) as Fc binder core
An Fc binding protein comprising one or more domains, wherein at least one domain comprises an amino acid sequence of any one of SEQ ID NOs: 18-20, or an amino acid sequence with at least 91% identity thereto.
Overall, the independent claims are grounded in Fc binding domain sequence selection/identity (SEQ ID NO: 3 or SEQ ID NO: 18–20, and ≥91% identity alternatives) and cysteine placement at helix 3 positions corresponding to 43, 46, 47, 50, 51, or 53. Dependent coverage further incorporates immobilized formats by requiring conjugation to a solid support and/or inclusion in an affinity separation matrix.
Stated Advantages
Increased caustic/alkaline stability during repeated NaOH cleaning.
Improved remaining IgG binding after prolonged exposure to NaOH.
Improved dynamic binding capacity compared to Protein A.
Near-complete elution from agarose bead matrices (e.g., ~99.8–100% at pH 3.5).
Documented Applications
Affinity purification of immunoglobulins, including IgG, using affinity separation matrices and pH/salt-controlled elution.
Reuse of the affinity separation formats under strong alkaline sanitization conditions involving NaOH cleaning.
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