Method for separation of antibodies or antibody fragments being devoid of an Fc region capable of binding to protein A

Patent metadata

Field	Value
Publication number	US-12552857-B2
Application number	US-202017604964-A
Country	US
Kind code	B2
Filing date	Apr 29, 2020
Priority date	Apr 29, 2019
Publication date	Feb 17, 2026
Grant date	Feb 17, 2026

Abstract

The invention discloses a method for separation of antibodies or antibody fragments, comprising the steps of: a) providing a feed comprising antibodies or antibody fragments having a VH3 region and being devoid of an Fc region capable of binding to Protein A; b) contacting the feed with a separation resin having covalently coupled ligands, wherein the ligands comprise a polypeptide as defined by SEQ ID NO 1 and wherein the antibodies or antibody fragments bind to the separation resin; c) optionally washing the separation resin with a washing liquid; d) eluting the antibodies or antibody fragments from the separation resin with an elution liquid and recovering the antibodies or antibody fragments.

First claim

The invention claimed is:

1 . A method for separation of antibodies or antibody fragments, comprising the steps of:
a) providing a feed comprising antibodies or antibody fragments capable of binding to Protein A, where the antibodies or antibody fragments comprise a VH3 region and are devoid of an Fc region capable of binding to Protein A; b) contacting said feed with a separation resin having ligands covalently coupled to a support, wherein said ligands consist essentially of a polypeptide as defined by SEQ ID NO 1:

AQX 1 AFYEILX 2 LPNLTEEQRX 3 AFIQSLKDDPSVSKAILAEAKKLNX 4

AQ

wherein:
X 1 =E, K, Y, T, F, L, W, I, M, V, A, H or R,
X 2 =H or K,
X 3 =N or A, and
X 4 =D, F, Y, W, K or R
and wherein said antibodies or antibody fragments bind to said separation resin;
c) optionally washing said separation resin with a washing liquid; and
d) eluting said antibodies or antibody fragments from said separation resin with an elution liquid and recovering said antibodies or antibody fragments.

2 . The method of claim 1 , wherein X 1 =E.

3 . The method of claim 1 , wherein X 2 =K.

4 . The method of claim 1 , wherein X 3 =N.

5 . The method of claim 1 , wherein X 4 =D.

6 . The method of claim 1 , wherein said ligands comprise multimers of said polypeptide, linked by linker regions comprising 0-15 amino acid residues.

7 . The method of claim 6 , wherein said multimers are tetramers, pentamers or hexamers.

8 . The method of claim 6 , wherein said multimers are coupled to said support via thioether links, derived from a C-terminal cysteine on said multimers.

9 . The method of claim 1 , wherein said support comprises crosslinked agarose beads.

10 . The method of claim 1 , wherein said support comprises crosslinked cellulose nanofibers.

11 . The method of claim 1 , wherein said feed is a clarified cell culture supernatant.

12 . The method of claim 1 , wherein said antibody fragments are selected from the group consisting of Fab, scFv, domain antibodies, nanobodies and BiTe.

13 . The method of claim 1 , wherein the binding strength of said antibody fragments to said separation resin is in the nanomolar range or stronger.

14 . The method of claim 1 , wherein said antibodies or antibody fragments are capable of binding to native Protein A via the VH3 region.

15 . The method of claim 1 , wherein the method comprises step c) and wherein contaminants and/or impurities are removed in step c).

16 . The method of claim 15 , wherein said washing liquid is a buffer of pH 5-7.

17 . The method of claim 1 , wherein said elution liquid is a buffer of pH 2-5.

18 . The method of claim 1 , further comprising, after step d), a step e) of cleaning said separation resin with a cleaning liquid of pH 13 or higher.

19 . The method of claim 18 , wherein said cleaning liquid comprises 0.5-2 M of an alkali metal hydroxide.

20 . The method of claim 18 , wherein steps a)-e) are repeated at least 50 times.

Assignees

• Cytiva Bioprocess R & D Ab

Inventors

• Rodrigo Gustav
• Ander Mats
• Bjorkman Tomas
• Palmgren Ronnie
• Brink Charlotte

Classifications

• C07K2317/55

  Fab or Fab' · CPC title

• C07K16/00 Primary

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

• C07K2317/622

  Single chain antibody (scFv) · CPC title

• C07K2317/569

  Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody® · CPC title

• C07K1/22 Primary

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