# US 10,308,690 B2 · Mutated immunoglobulin-binding polypeptides

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- Number: US 10,308,690 B2
- Document ID: US10308690B2
- Status: Granted
- Kind: B2
- Application number: 14/961,164
- Filed: 2015-12-07
- Published: 2016-06-09
- Granted: 2019-06-04
- Earliest claimed: 2013-07-10
- Assignees: GE Healthcare BioProcess R&D AB
- Applicants: Ge Healthcare Bioprocess R&D AB
- Inventors: Gustav Rodrigo; Mats Ander; Göran Baurén; Tomas Björkman
- Web page: https://patentgrep.com/patent/US10308690B2

## CPC

- C07K 14/31: from Staphylococcus (G)
- B01D 15/3809: {of the antigen-antibody type, e.g. protein A, G or L chromatography}
- B01J 20/286: Phases chemically bonded to a substrate, e.g. to silica or to polymers
- B01J 20/3204: {Inorganic carriers, supports or substrates}
- 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}
- C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes
- C07K 14/745: Blood coagulation or fibrinolysis factors
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- 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)
- 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)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS

## Abstract

The invention discloses a polypeptide with improved alkaline stability, which polypeptide comprises a mutant of a B or C domain of Staphylococcus Protein A (SpA), as specified by SEQ ID NO 1 or SEQ ID NO 2, or of Protein Z, as specified by SEQ ID NO 3, comprising at least the mutation wherein the glutamine residue at position 9 has been mutated to a tryptophan, leucine, glutamic acid, valine or lysine. The invention also discloses multimers of the polypeptide, as well as separation matrices comprising the multimers or polypeptides.

## Claims

### Claim 1

An Fc-binding polypeptide with improved alkaline stability, which polypeptide comprises a mutant of a parental polypeptide, as defined by, or having at least 90% identity to, SEQ ID NO 28, SEQ ID NO 29 or SEQ ID NO 30, comprising at least the mutation wherein the glutamine residue at position 3 has been mutated to leucine, glutamic acid, valine or lysine.

### Claim 2

A multimer with improved alkaline stability comprising or consisting essentially of a plurality of polypeptide units, which polypeptide comprises a mutant of a parental polypeptide, as defined by, or having at least 90% identity to, SEQ ID NO 28, SEQ ID NO 29 or SEQ ID NO 30, comprising at least the mutation wherein the glutamine residue at position 3 has been mutated to tryptophan, leucine, glutamic acid, valine or lysine.

### Claim 3 (depends on 2)

The multimer of claim 2, wherein the polypeptide units are linked by elements comprising up to 15 amino acids.

### Claim 4 (depends on 3)

The multimer of claim 3, further comprising at the C-terminal or N-terminal one or more coupling element, selected from the group consisting of a cysteine residue, a plurality of lysine residues and a plurality of histidine residues.

### Claim 5 (depends on 1)

A separation matrix, comprising a plurality of polypeptides according to claim 1 coupled to a solid support.

### Claim 6 (depends on 4)

A separation matrix, comprising a plurality of multimers according to claim 4 coupled to a solid support.

### Claim 7 (depends on 6)

The separation matrix of claim 6, wherein the multimers have been coupled to the solid support via thioether bonds.

### Claim 8 (depends on 6)

A method of isolating an immunoglobulin, using a separation matrix according to claim 6, the method comprising,
a) contacting a liquid sample comprising an immunoglobulin with the separation matrix according to claim 6,
b) washing said separation matrix with a washing liquid,
c) eluting the immunoglobulin from the separation matrix with an elution liquid, and
d) cleaning the separation matrix with a cleaning liquid.

### Claim 9 (depends on 8)

The method of claim 8, wherein the cleaning liquid is alkaline.

### Claim 10 (depends on 8)

The method of claim 8, wherein the cleaning liquid comprises 0.1-1.0 M NaOH or KOH.

### Claim 11 (depends on 8)

The method of claim 8, wherein steps a)-d) are repeated at least 50 times.

### Claim 12

An Fc-binding polypeptide with improved alkaline stability, which polypeptide comprises a mutant of a B or C domain of Staphylococcus Protein A (SpA), as defined by, or having at least 90% identity to, SEQ ID NO 1 or SEQ ID NO 2, of Protein Z, as defined by, or having at least 90% identity to, SEQ ID NO 3, or of a Protein Z variant as defined by, or having at least 90% identity to, SEQ ID NO 4, comprising at least the mutation wherein the glutamine residue at position 9 has been mutated to leucine, glutamic acid, valine or lysine.

### Claim 13

A multimer comprising or consisting essentially of a plurality of polypeptide units, which polypeptide comprises a mutant of a B or C domain of Staphylococcus Protein A (SpA), as defined by, or having at least 90% identity to, SEQ ID NO 1 or SEQ ID NO 2, of Protein Z, as defined by, or having at least 90% identity to, SEQ ID NO 3, or of a Protein Z variant as defined by, or having at least 90% identity to, SEQ ID NO 4, comprising at least the mutation wherein the glutamine residue at position 9 has been mutated to tryptophan, leucine, glutamic acid, valine or lysin.

### Claim 14 (depends on 13)

A separation matrix, having a plurality of multimers according to claim 13 coupled to a solid support.

### Claim 15 (depends on 14)

A method of isolating an immunoglobulin, comprising the steps of:
a) contacting a liquid sample comprising an immunoglobulin with a separation matrix according to claim 14,
b) washing said separation matrix with a washing liquid,
c) eluting the immunoglobulin from the separation matrix with an elution liquid, and
d) cleaning the separation matrix with a cleaning liquid.

### Claim 16 (depends on 15)

The method of claim 15, wherein the cleaning liquid comprises 0.1-1.0 M NaOH or KOH.

### Claim 17

A polypeptide, comprising or consisting essentially of an amino acid sequence selected from the group consisting of: SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34 and SEQ ID NO 35.

### Claim 18 (depends on 17)

A separation matrix, having a plurality of polypeptides according to claim 17 coupled to a solid support.

### Claim 19 (depends on 18)

A method of isolating an immunoglobulin, comprising the steps of:
a) contacting a liquid sample comprising an immunoglobulin with a separation matrix according to claim 18,
b) washing said separation matrix with a washing liquid,
c) eluting the immunoglobulin from the separation matrix with an elution liquid, and
d) cleaning the separation matrix with a cleaning liquid.

### Claim 20 (depends on 17)

A multimer with improved alkaline stability comprising or consisting essentially of a plurality of polypeptide units as defined by claim 17.

### Claim 21 (depends on 20)

The multimer of claim 20, wherein the polypeptide units are linked by elements comprising up to 15 amino acids.

### Claim 22 (depends on 21)

The multimer of claim 21, further comprising at the C-terminal or N-terminal one or more coupling element, selected from the group consisting of a cysteine residue, a plurality of lysine residues and a plurality of histidine residues.

### Claim 23 (depends on 20)

A separation matrix, comprising a plurality of multimers according to claim 20 coupled to a solid support.

### Claim 24 (depends on 23)

The separation matrix of claim 23, wherein the multimers have been coupled to the solid support via thioether bonds.

### Claim 25 (depends on 23)

A method of isolating an immunoglobulin, using a separation matrix according to claim 23, the method comprising,
a) contacting a liquid sample comprising an immunoglobulin with the separation matrix according to claim 23,
b) washing said separation matrix with a washing liquid,
c) eluting the immunoglobulin from the separation matrix with an elution liquid, and
d) cleaning the separation matrix with a cleaning liquid.

### Claim 26 (depends on 25)

The method of claim 25, wherein the cleaning liquid comprises 0.1-1.0 M NaOH or KOH.

### Claim 27 (depends on 23)

The method of claim 23, wherein steps a)-d) are repeated at least 50 times.