Supramolecular high affinity protein-binding system for purification of biomacromolecules

Patent metadata

Field	Value
Publication number	US-12466875-B2
Application number	US-202217833941-A
Country	US
Kind code	B2
Filing date	Jun 7, 2022
Priority date	Mar 30, 2017
Publication date	Nov 11, 2025
Grant date	Nov 11, 2025

Abstract

In certain embodiments, the present invention provides novel antibody purification methods and systems using a potentially simple and cost-efficient means. In some embodiments, customized Z-33 derived from Staphylococcus aureus Protein A is used to construct immuno-amphiphile molecules which can assemble into immunofibers in aqueous solution with bioactive epitopes on the surface and have IgG binding ability.

First claim

The invention claimed is:

1 . A method for purifying an antibody or an Fc fusion protein, the method comprising the steps of:
a) dissolving an immuno-amphiphile in an aqueous solution at a physiological pH to provide a dissolved immuno-amphiphile; and aging the dissolved immuno-amphiphile overnight to provide immunofibers (IFs); b) mixing a sample containing an antibody or an Fc fusion protein with the IFs, and allowing the IFs to bind the Fc portion of the antibody or Fc fusion protein to form an immunofiber-antibody complex or immunofiber-Fc fusion protein complex in a solution; c) separating the immunofiber-antibody complex or the immunofiber-Fc fusion protein complex from the solution by adding salt and centrifugation; and d) dissociating the IFs from the antibody or Fc fusion protein and collecting the unbound antibody or Fc fusion protein, wherein the immuno-amphiphile comprises an antibody binding peptide and a hydrocarbon moiety; wherein the hydrocarbon moiety is conjugated to the N-terminus of the antibody binding peptide; wherein the antibody binding peptide consists of the amino acid sequence FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD (SEQ ID NO: 1); and wherein the hydrocarbon moiety is a C12 hydrocarbon chain or two C8 hydrocarbon chains.

2 . The method of claim 1 , wherein the IFs are separated from the antibody or Fc fusion protein by lowering the pH to elution condition.

3 . The method of claim 1 , wherein the antibody binding peptide has an α-helical conformation when in an aqueous solution at physiological pH.

4 . The method of claim 1 , further comprising pretreating the immuno amphiphile in hexafluoroisopropanol prior to dissolving the immuno-amphiphile in the aqueous solution.

5 . The method of claim 4 , further comprising removing the hexafluoroisopropanol.

6 . The method of claim 5 , wherein removing the hexafluoroisopropanol comprises evaporating the hexafluoroisopropanol.

7 . The method of claim 1 , wherein the antibody is an IgG.

8 . The method of claim 1 , wherein the antibody is a monoclonal antibody.

9 . The method of claim 1 , wherein the IFs are separated from the antibody or Fc fusion protein by lowering the pH to elution condition and filtration or microfiltration.

10 . The method of claim 1 , wherein the physiological pH is 7.4.

11 . The method of claim 2 , wherein lowering the pH to elution condition comprises lowering the pH to 2.8.

Assignees

• Univ Johns Hopkins
• Bristol Myers Squibb Co

Inventors

• Cui Honggang
• Li Yi
• Xu Xuankuo
• Lock Lye Lin
• Li Zhengjian

Classifications

• C07K14/31 Primary

  from Staphylococcus (G) · CPC title

• B01J20/28023

  Fibres or filaments (fibres or filaments in the form of membranes B01J20/28038; B01J20/28007 takes precedence) · CPC title

• B01J20/24

  Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives · CPC title

• B01D15/3809

  of the antigen-antibody type, e.g. protein A, G or L chromatography · CPC title

• C07K1/30

  by precipitation · CPC title