# US 10,501,557 B2 · Mutated immunoglobulin-binding polypeptides

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- Number: US 10,501,557 B2
- Document ID: US10501557B2
- Status: Granted
- Kind: B2
- Application number: 15/526,941
- Filed: 2015-11-16
- Published: 2017-11-23
- Granted: 2019-12-10
- Earliest claimed: 2015-06-12
- Assignees: GE Healthcare BioProcess R&D AB
- Applicants: Ge Healthcare Bioprocess R&D AB
- Inventors: Gustav Rodrigo; Tomas Bjorkman; Mats Ander
- Web page: https://patentgrep.com/patent/US10501557B2

## CPC

- C07K 16/46: Hybrid immunoglobulins (hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00)
- B01D 15/3809: {of the antigen-antibody type, e.g. protein A, G or L chromatography}
- B01J 20/289: bonded via a spacer
- 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 14/31: from Staphylococcus (G)
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/065: {Purification, fragmentation}
- 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

An Fc-binding polypeptide of improved alkali stability, comprising a mutant of an Fc-binding domain of Staphylococcus Protein A (SpA), as defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:22, SEQ ID NO 51 or SEQ ID NO 52 wherein at least the asparagine or serine residue at the position corresponding to position 11 in SEQ ID NO:4-7 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

## Claims

### Claim 1

An Fc-binding polypeptide comprising a mutant of an Fc-binding domain of Staphylococcus Protein A (SpA) wherein the polypeptide is selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48, and wherein the mutant binds the Fc domain of an antibody and has improved alkaline stability compared to SEQ ID NO: 47.

### Claim 2 (depends on 1)

The polypeptide of claim 1, wherein the alkaline stability is improved as measured by the remaining IgG-binding capacity after 24 or 25 h incubation in 0.5 M or 1.0 M aqueous NaOH at 22+/−2° C.

### Claim 3 (depends on 1)

A multimer comprising a plurality of polypeptides as defined by claim 1.

### Claim 4 (depends on 3)

The multimer according to claim 3, which is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer or nonamer.

### Claim 5 (depends on 3)

A nucleic acid or a vector encoding a polypeptide or multimer according to claim 3.

### Claim 6 (depends on 5)

An expression system, which comprises a nucleic acid or vector according to claim 5.

### Claim 7 (depends on 3)

A separation matrix, wherein a plurality of polypeptides or multimers according to claim 3 have been coupled to a solid support.

### Claim 8 (depends on 7)

The separation matrix according to claim 7, wherein the polypeptides or multimers have been coupled to the solid support via thioether bonds.

### Claim 9 (depends on 7)

The separation matrix of claim 7, wherein the solid support is a polysaccharide.

### Claim 10 (depends on 6)

The separation matrix of claim 6, wherein the IgG capacity of the matrix after 24 incubation in 0.5 M NaOH at 22+/−2° C. is at least 80% of the IgG capacity before the incubation.

### Claim 11 (depends on 7)

The separation matrix of claim 7, wherein the IgG capacity of the matrix after 24 incubation in 1.0 M NaOH at 22+/−2° C. is at least 70% of the IgG capacity before the incubation.

### Claim 12 (depends on 7)

A method of isolating an immunoglobulin, wherein a separation matrix according to claim 7 is used.

### Claim 13 (depends on 12)

The method of claim 12, comprising the steps of:
a) contacting a liquid sample comprising an immunoglobulin with the separation matrix,
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 14 (depends on 13)

The method of claim 13, wherein the cleaning liquid is alkaline with a pH of 13-14.

### Claim 15 (depends on 13)

The method of claim 13, wherein the cleaning liquid comprises 0.1-1.0 M NaOH or KOH.

### Claim 16 (depends on 13)

The method of claim 13, wherein steps a)-d) are repeated at least 10 times.

### Claim 17 (depends on 13)

The method of claim 13, wherein steps a)-c) are repeated at least 10 times and wherein step d) is performed after a plurality of instances of step c).

### Claim 18 (depends on 1)

The polypeptide of claim 1, comprising the sequence of SEQ ID NO:8.

### Claim 19 (depends on 1)

The polypeptide of claim 1, comprising the sequence of SEQ ID NO:9.

### Claim 20 (depends on 1)

The polypeptide of claim 1, comprising the sequence of SEQ ID NO:10.

### Claim 21 (depends on 1)

The polypeptide of claim 1, comprising the sequence of SEQ ID NO:11.

### Claim 22 (depends on 1)

The polypeptide of claim 1, comprising the sequence of SEQ ID NO:16.

### Claim 23 (depends on 1)

The polypeptide of claim 1, comprising the sequence of SEQ ID NO:23.

### Claim 24 (depends on 1)

The polypeptide of claim 1, comprising the sequence of SEQ ID NO:24.

### Claim 25 (depends on 1)

The polypeptide of claim 1, comprising the sequence of SEQ ID NO:25.

### Claim 26 (depends on 1)

The polypeptide of claim 1, comprising the sequence of SEQ ID NO:26.

### Claim 27 (depends on 1)

The polypeptide of claim 1, comprising the sequence of SEQ ID NO:27.

### Claim 28 (depends on 1)

The polypeptide of claim 1, comprising the sequence of SEQ ID NO:28.

### Claim 29 (depends on 1)

The polypeptide of claim 1, comprising the sequence of SEQ ID NO:44.

### Claim 30 (depends on 1)

The polypeptide of claim 1, comprising the sequence of SEQ ID NO:46.

### Claim 31 (depends on 1)

The polypeptide of claim 1, comprising the sequence of SEQ ID NO:48.