# US 8,772,447 B2 · Chromatography ligand comprising domain C from staphylococcus aureus protein A for antibody isolation

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- Number: US 8,772,447 B2
- Document ID: US8772447B2
- Status: Granted
- Kind: B2
- Application number: 13/559,663
- Filed: 2012-07-27
- Published: 2012-11-15
- Granted: 2014-07-08
- Earliest claimed: 2006-09-29
- Assignees: GE Healthcare Bio-Sciences AB
- Applicants: Martin Hall; Sture Larsson; Andreas Muranyi; Gustav Rodrigo; Jinyu Zou; Per-Mikael Aberg
- Inventors: Martin Hall; Sture Larsson; Andreas Muranyi; Gustav Rodrigo; Jinyu Zou; Per-Mikael Aberg
- Web page: https://patentgrep.com/patent/US8772447B2

## CPC

- B01J 20/24: Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
- B01D 15/3809: {of the antigen-antibody type, e.g. protein A, G or L chromatography}
- B01J 20/28019: {Spherical, ellipsoidal or cylindrical}
- B01J 20/285: based on polymers
- B01J 20/286: Phases chemically bonded to a substrate, e.g. to silica or to polymers
- B01J 20/289: bonded via a spacer
- B01J 20/3212: {consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds}
- B01J 20/3219: {involving a particular spacer or linking group, e.g. for attaching an active group}
- B01J 20/3274: {Proteins, nucleic acids, polysaccharides, antibodies or antigens}
- B01J 20/3293: {Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers}
- C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes
- C07K 14/31: from Staphylococcus (G)
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 17/00: Carrier-bound or immobilised peptides (carrier-bound or immobilised enzymes C12N11/00); Preparation thereof
- C07K 17/10: the carrier being a carbohydrate
- B01J 2220/52: Sorbents specially adapted for preparative chromatography
- B01J 2220/54: Sorbents specially adapted for analytical or investigative chromatography
- C07K 2317/55: Fab or Fab'
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## 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.

## Claims

### Claim 1

A process of isolating one or more target compounds, which process comprises 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 comprise at least one of a Staphylococcus protein A (SpA) Domain C sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO 1 or SEQ ID NO: 2, and, optionally, eluting said compound(s) by the passing across said matrix of a liquid that releases them from ligands, wherein said ligands are coupled to a matrix/carrier and wherein said ligands further comprise a terminal coupling group, said group preferably comprising at least one nitrogen and/or sulphur atom(s), wherein said process further includes an alkaline regeneration step and is carried out at least 80 times.

### Claim 2

A process of isolating one or more target compounds, which process comprises 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; and, optionally, eluting said compound(s) by the passing across said matrix of a liquid that releases them from ligands, wherein the Domain C sequence comprises a sequence of amino acids selected from the group consisting of SEQ ID NO 1 or SEQ ID NO: 2, wherein said process further includes an alkaline regeneration step and is carried out at least 80 times.

### Claim 3 (depends on 1)

The process of claim 1, wherein the target compound is a protein.

### Claim 4 (depends on 1)

The process of claim 1, wherein the alkaline regeneration step comprises incubation in a NaOH solution.

### Claim 5 (depends on 4)

The process of claim 4, wherein the NaOH solution has a concentration of at least 0.5 M.

### Claim 6 (depends on 1)

The process of claim 1, wherein the process is carried out at least 200 times.

### Claim 7 (depends on 1)

The process of claim 1, wherein the target compound is a Fab fragment.

### Claim 8 (depends on 2)

The process of claim 2, wherein the alkaline regeneration step comprises incubation in a NaOH solution.

### Claim 9 (depends on 8)

The process of claim 8, wherein the NaOH solution has a concentration of at least 0.5 M.

### Claim 10 (depends on 9)

The process of claim 9, wherein the process is carried out at least 200 times.

### Claim 11 (depends on 2)

The process of claim 2, wherein the target compound is a Fab fragment.

### Claim 12 (depends on 1)

The process of claim 1, wherein the target compound is an antibody.