Alkaline stable fc-binding proteins for affinity chromatography

Patent metadata

Field	Value
Publication number	US-2019300568-A1
Application number	US-201716324842-A
Country	US
Kind code	A1
Filing date	Aug 7, 2017
Priority date	Aug 11, 2016
Publication date	Oct 3, 2019
Grant date	—

Abstract

The present invention relates to Fc binding proteins comprising one or more Fc binding domains wherein at least one domain comprises of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6 or 21. 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.

First claim

1 - 20 . (canceled)

21 . An Fc binding protein comprising one or more domains, wherein at least one domain comprises an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NOs: 1-6 and 21, provided that the amino acid in position 4 is Q.

22 . The Fc binding protein of claim 21 , wherein the protein comprises 2, 3, 4, 5, or 6 domains linked to each other.

23 . The Fc binding protein of claim 22 , wherein all of the domains comprise sequences selected from any one of SEQ ID NOs: 1-6 and 21.

24 . The Fc binding protein of claim 22 , wherein the domains are derivatives of SEQ ID NOs 1-6 or 21, and further wherein each derivative has an amino acid sequence that is 100% identical to one of SEQ ID NOs: 1-6 and 21 except that it has a deletion of 1, 2, or 3 amino acids within the first 4 amino acids of its N-terminus and/or a deletion of 1 or 2 amino acids within the first 2 amino acids of its C-terminus.

25 . The Fc binding protein of claim 22 , wherein the protein is a homo-multimer.

26 . The Fc binding protein of claim 22 , wherein the protein is a hetero-multimer.

27 . The Fc binding protein of claim 22 , wherein one or more domains are linked to each other directly or with one or more linkers.

28 . The Fc binding protein of claim 27 , wherein the linker is a peptide linker.

29 . The Fc binding protein of claim 21 , wherein the protein has less than a 15% reduction in binding capacity following an incubation in 0.5 M NaOH for at least 5 hours.

30 . The Fc binding protein of claim 21 , wherein the Fc binding protein is conjugated to a solid support.

31 . The Fc binding protein of claim 30 , wherein said Fc binding protein further comprises an attachment site for site-specific covalent coupling of the Fc binding protein to a solid support.

32 . An affinity separation matrix comprising an Fc binding protein, the Fc binding protein comprising an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NOS: 1-6 or 21, provided that the amino acid in position 4 is Q.

33 . The affinity separation matrix of claim 32 , wherein the amino acid sequence has at least 98% sequence identity to SEQ ID NO: 1.

34 . The affinity separation matrix of claim 32 , wherein the amino acid sequence has at least 98% sequence identity to SEQ ID NO: 2.

35 . The affinity separation matrix of claim 32 , wherein the amino acid sequence has at least 98% sequence identity to SEQ ID NO: 3.

36 . The affinity separation matrix of claim 32 , wherein the amino acid sequence has at least 98% sequence identity to SEQ ID NO: 4.

37 . The affinity separation matrix of claim 32 , wherein the amino acid sequence has at least 98% sequence identity to SEQ ID NO: 5.

38 . The affinity separation matrix of claim 32 , wherein the amino acid sequence has at least 98% sequence identity to SEQ ID NO: 6.

39 . The affinity separation matrix of claim 32 , wherein the amino acid sequence has at least 98% sequence identity to SEQ ID NO: 21.

40 . A method for affinity purification of a protein comprising an Fc sequence, the method comprising:
(a) contacting an affinity separation matrix comprising at least one Fc binding protein with a solution containing a protein comprising an Fc sequence under conditions that permit binding of said at least one Fc binding protein to said protein comprising an Fc sequence, wherein the at least one Fc binding protein comprises an amino acid sequence having at least 98% sequence identity to one of SEQ ID NOS: 1-6 or 21, provided that the amino acid in position 4 is Q; and (b) eluting the bound protein comprising an Fc sequence from said affinity purification matrix.

41 . The method of claim 40 , further comprising washing the affinity separation matrix between steps (a) and (b).

42 . The method of claim 40 , wherein there is at least 95% elution of the protein comprising an Fc sequence at a pH of 3.5 or higher.

Assignees

• Repligen Corp

Inventors

• Knick Paul
• Fiedler Erik
• Haupts Ulrich
• Meysing Maren

Classifications

• C07K16/065

  Purification, fragmentation · CPC title

• C07K14/31 Primary

  from Staphylococcus (G) · CPC title

• C07K1/22 Primary

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• C07K17/00

  Carrier-bound or immobilised peptides (carrier-bound or immobilised enzymes C12N11/00); Preparation thereof · CPC title

• C07K16/00 Primary

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title