Chromatography Ligand Comprising Domain C From Staphylococcus Aureus Protein A For Antibody Isolation

Patent metadata

Field	Value
Publication number	US-2016200797-A1
Application number	US-201615063471-A
Country	US
Kind code	A1
Filing date	Mar 7, 2016
Priority date	Sep 29, 2006
Publication date	Jul 14, 2016
Grant date	—

Abstract

The present invention relates to a chromatography ligand, which comprises Domain C from Staphylococcus protein A (SpA), or a functional fragment or variant thereof. The chromatography ligand presents an advantageous capability of withstanding harsh cleaning in place (CIP) conditions, and is capable of binding Fab fragments of antibodies. The ligand may be provided with a terminal coupling group, such as arginine or cysteine, to facilitate its coupling to an insoluble carrier such as beads or a membrane. The invention also relates to a process of using the ligand in isolation of antibodies, and to a purification protocol which may include washing steps and/or regeneration with alkali.

First claim

1 - 18 . (canceled)

19 . A process of isolating one or more target compounds, the process comprising,
contacting a liquid comprising said compound(s) with a chromatography matrix; allowing said compound(s) to adsorb to ligands present on the matrix, wherein said ligands consist of one or more Staphylococcus protein A (SpA) Domain C, or functional fragments or variant thereof; and, optionally, eluting said compound(s) by the passing across said matrix of a liquid that releases them from ligands.

20 . A process of isolating one or more target compounds, the process comprising,
contacting a liquid comprising said compound(s) with a chromatography matrix; allowing said compound(s) to adsorb to ligands present on the matrix, wherein said ligands are multimers comprising at least two Staphylococcus protein A(SpA) Domain C units, or functional fragments or variant thereof; and, optionally, eluting said compound(s) by the passing across said matrix of a liquid that releases them from ligands.

21 . A process of claim 19 , wherein the target compound is a protein an antibody, or a Fab fragment.

22 - 26 . (canceled)

27 . The process of claim 19 , wherein the ligands present on the matrix has retained at least 95% of its original binding capacity after 5 hours incubation in 0.5 M NaOH.

28 . The process of claim 19 , wherein the Domain C sequence includes the amino acid sequence as defined by SEQ ID NO 1.

29 . The process of claim 19 , wherein the Domain C sequence includes the amino acid sequence as defined by SEQ ID NO 2.

30 . The process of claim 19 , wherein the target compound is eluted from the matrix for 2-300 times, optionally with washing steps between.

31 . The process of claim 30 , further comprising alkaline regeneration of the matrix followed optionally by repeating the process of claim 19 .

32 . The process of claim 31 , wherein regeneration is carried out by incubating the matrix with a sodium hydroxide solution.

33 . The process of claim 32 , wherein the sodium hydroxide solution has a concentration of about 0.5 M.

34 . The process of claim 20 , wherein the target compound is a protein, an antibody, or a Fab fragment.

35 . The process of claim 20 , wherein in addition to said at least two Domain C units of the multimer, or at least two functional fragments or variants thereof, also comprises one or more other protein-based units, which are preferably alkaline-stable.

36 . The process of claim 20 , wherein the multimer comprises 2-8 Domain C units, optionally coupled via linker segments.

37 . The process of claim 20 , wherein the ligands present on the matrix has retained at least 95% of its original binding capacity after 5 hours incubation in 0.5 M NaOH.

38 . The process of claim 20 , wherein the Domain C sequence includes the amino acid sequence as defined by SEQ ID NO 1.

39 . The process of claim 20 , wherein the Domain C sequence includes the amino acid sequence as defined by SEQ ID NO 2.

40 . The process of claim 20 , wherein the target compound is eluted from the matrix for 2-300 times, optionally with washing steps between.

41 . The process of claim 40 , further comprising alkaline regeneration of the matrix followed optionally by repeating the process of claim 20 .

42 . The process of claim 41 , wherein regeneration is carried out by incubating the matrix with a sodium hydroxide solution.

43 . The process of claim 42 , wherein the sodium hydroxide solution has a concentration of about 0.5 M.

44 . A process of absorbing one or more immunoglobulins from a liquid, the process comprising,
contacting the liquid comprising said immunoglobulin(s) with an alkaline-stable immunoglobulin adsorbent; allowing said immunoglobulin(s) to adsorb to ligands present on the alkaline-stable immunoglobulin adsorbent, wherein said ligands consist of one or more Staphylococcus protein A (SpA) Domain C, or functional fragments or variant thereof, to remove said immunoglobulin(s) from the liquid.

45 . A process of absorbing one or more a Fab fragments from a liquid, the process comprising,
contacting the liquid comprising said Fab fragment(s) with an alkaline-stable Fab fragment-binding adsorbent; allowing said Fab fragment(s) to adsorb to ligands present on the alkaline-stable Fab fragment-binding adsorbent, wherein said ligands consist of one or more Staphylococcus protein A (SpA) Domain C, or functional fragments or variant thereof, to remove said Fab fragment(s) from the liquid.

Assignees

• Ge Healthcare Bio Sciences Ab

Inventors

• Hall Martin
• Larsson Sture
• Muranyi Andreas
• Rodrigo Gustav
• Zou Jinyu
• Aberg Per-Mikael

Classifications

• B01J20/3219

  involving a particular spacer or linking group, e.g. for attaching an active group · CPC title

• B01J20/3274

  Proteins, nucleic acids, polysaccharides, antibodies or antigens · CPC title

• B01J2220/54

  Sorbents specially adapted for analytical or investigative chromatography · CPC title

• C07K14/31

  from Staphylococcus (G) · CPC title

• C07K1/22

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