# US 10,118,962 B2 · Methods for purification of single domain antigen binding molecules

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- Number: US 10,118,962 B2
- Document ID: US10118962B2
- Status: Granted
- Kind: B2
- Application number: 12/608,964
- Filed: 2009-10-29
- Published: 2010-07-08
- Granted: 2018-11-06
- Earliest claimed: 2008-10-29
- Assignees: Ablynx N.V.
- Applicants: Paul R. Brown; Scott Andreas Tobler; Andrew M. Wood; Austin Wayne Boesch
- Inventors: Paul R. Brown; Scott Andreas Tobler; Andrew M. Wood; Austin Wayne Boesch
- Web page: https://patentgrep.com/patent/US10118962B2

## CPC

- C07K 16/241: {Tumor Necrosis Factors}
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 17/06: Antipsoriatics
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 1/18: Ion-exchange chromatography
- C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes
- C07K 16/065: {Purification, fragmentation}
- C07K 16/18: against material from animals or humans
- C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Processes and methods of purifying or separating Single Domain Antigen Binding (SDAB) molecules that include one or more single binding domains (e.g., one or more nanobody molecules), substantially devoid of a complementary antibody domain and an immunoglobulin constant region, using Protein A-based affinity chromatography, are disclosed.

## Claims

### Claim 1

A method of separating or purifying an antigen-binding fusion polypeptide from cell culture medium comprising:
obtaining cell culture medium containing the antigen-binding fusion polypeptide that is produced as a product of a eukaryotic cell culture and secreted into the cell culture medium,
adjusting the cell culture medium containing the antigen-binding fusion polypeptide to a conductivity between about 12 and 9 mS/cm and a pH equal to or less than about 4.5,
contacting the medium containing the antigen-binding fusion polypeptide with a cation exchange-based support, under conditions that allow the antigen-binding fusion polypeptide to bind or absorb to the support, wherein the cation exchange-based support is a cation exchange (CEX) resin, wherein the CEX resin has a capacity of at least about 40 g/L;
and
selectively eluting the antigen-binding fusion polypeptide from the support by washing the support with at least one cation exchange washing buffer, thereby removing one or more contaminants; and eluting the antigen-binding fusion polypeptide with an elution buffer;
wherein said antigen-binding fusion polypeptide is devoid of a complementary antibody domain and an immunoglobulin Fc region, and wherein the antigen-binding fusion polypeptide is not pegylated, wherein said antigen-binding fusion polypeptide comprises one or more VHHs.

### Claim 2 (depends on 1)

The method of claim 1, wherein the step of removing one or more of the contaminants comprises washing the bound support under conditions where the antigen-binding fusion polypeptide remains bound to the support.

### Claim 3 (depends on 1)

The method of claim 1, further comprising one or more of: hydroxyapatite chromatography, affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography, metal affinity chromatography, diafiltration, ultrafiltration, viral inactivation or viral removal filtration.

### Claim 4 (depends on 3)

The method of claim 3, wherein the hydroxyapatite chromatography comprises contacting the antigen-binding fusion polypeptide eluted from the CEX resin with a hydroxyapatite resin and selectively eluting the antigen-binding fusion polypeptide from the hydroxyapatite resin.

### Claim 5 (depends on 3)

The method of claim 3, wherein the hydroxyapatite chromatography comprises ceramic hydroxyapatite chromatography.

### Claim 6 (depends on 1, 4)

The method of claim 1 or 4, further comprising concentrating the eluted antigen-binding fusion polypeptide to a preselected target volume.

### Claim 7 (depends on 6)

The method of claim 6, wherein the concentration step is carried out by performing an ultrafiltration/diafiltration step in the presence of a Histidine buffer or a Tris buffer.

### Claim 8 (depends on 6)

The method of claim 6, wherein the antigen-binding fusion polypeptide is concentrated to at least about 80 g/L to 350 g/L.

### Claim 9 (depends on 1)

The method of claim 1, wherein the antigen-binding fusion polypeptide is a recombinant protein produced in a mammalian host cell.

### Claim 10 (depends on 9)

The method of claim 9, wherein the mammalian host cell is a CHO cell.

### Claim 11 (depends on 1)

The method of claim 1, wherein the contaminants in the medium comprise one or more of high molecular weight protein aggregates, host cell proteins, DNA, or leached Protein A.

### Claim 12 (depends on 1)

The method of claim 1, wherein the antigen-binding fusion polypeptide is purified to at least 90% higher purity.

### Claim 13 (depends on 1)

The method of claim 1, wherein the CEX resin elution buffer comprises about 50 mM sodium chloride or less, and has a pH of about 5.5 to 7.2.

### Claim 14 (depends on 1)

The method of claim 1, further comprising contacting the medium with a hydroxyapatite resin and selectively eluting the antigen-binding fusion polypeptide from the hydroxyapatite resin.

### Claim 15 (depends on 14)

The method of claim 14, further comprising pre-treating the medium with an equilibration buffer and allowing the pre-treated medium to flow through a hydroxyapatite resin.

### Claim 16 (depends on 15)

The method of claim 15, wherein the contacting step of the medium with a hydroxyapatite resin and the elution steps are effected in buffer solutions comprising about 1 to 20 mM sodium phosphate and from about 0.2 to 2.5 M sodium chloride, in a pH from about 6.4 to 7.6.

### Claim 17 (depends on 15)

The method of claim 15, wherein the equilibration buffer comprises about 1 to 20 mM sodium phosphate, from about 0.01 to 2.0 M sodium chloride, from about 0 to 200 mM arginine, from about 0 to 200 mM HEPES, in a pH from about 6.2 to 8.0.

### Claim 18 (depends on 1)

The method of claim 1, wherein the purified antigen-binding fusion polypeptide contains less than 10% high molecular weight aggregates.

### Claim 19 (depends on 1)

The method of claim 1, wherein the antigen-binding fusion polypeptide is a single chain polypeptide, comprising at least one immunoglobulin variable domain.

### Claim 20 (depends on 19)

The method of claim 19, wherein the antigen-binding fusion polypeptide comprises at least one immunoglobulin variable domain from an antibody naturally devoid of light chains.

### Claim 21 (depends on 20)

The method of claim 20, wherein the antibody is a camelid antibody.

### Claim 22 (depends on 19)

The method of claim 19, wherein the antigen-binding fusion polypeptide is a monovalent, bivalent, or trivalent molecule.

### Claim 23 (depends on 19)

The method of claim 19, wherein the antigen-binding fusion polypeptide is a monospecific, bispecific, or trispecific molecule.

### Claim 24 (depends on 19)

The method of claim 19, wherein the antigen-binding fusion polypeptide comprises one or more single domain molecules that are recombinant, CDR-grafted, humanized, camelized, de-immunized, and/or in vitro selected by phage display.

### Claim 25 (depends on 19)

The method of claim 19, wherein the antigen-binding fusion polypeptide binds to one or more target antigens selected from the group consisting of a cytokine, growth factor and a serum protein.

### Claim 26 (depends on 25)

The method of claim 25, wherein the antigen-binding fusion polypeptide binds tumor necrosis factor α (TNF α).

### Claim 27 (depends on 25)

The method of claim 25, wherein the antigen-binding fusion polypeptide binds to human serum albumin (HSA).

### Claim 28 (depends on 19)

The method of claim 19, wherein the antigen-binding fusion polypeptide is a trivalent, bispecific molecule composed of a single chain polypeptide fusion of two camelid variable regions that bind to TNFα, and one camelid variable region that binds to HSA.

### Claim 29 (depends on 28)

The method of claim 28, wherein the antigen-binding fusion polypeptide is arranged in the following order from N- to C-terminus: Anti-TNFα antigen-binding fusion polypeptide—anti-HSA antigen-binding fusion polypeptide−anti-TNFα antigen-binding fusion polypeptide.

### Claim 30 (depends on 29)

The method of claim 29, wherein two or more of the antigen-binding fusion polypeptides are fused with a linking group that comprises at least five, seven eight, nine, ten, twelve, or fifteen glycine and serine residues.

### Claim 31 (depends on 30)

The method of claim 30, wherein two or more of the antigen-binding fusion polypeptides are fused with a linking group comprising the amino acid sequence of SEQ ID NO:9 ((Gly)4-Ser-(Gly)3-Ser).

### Claim 32 (depends on 1)

The method of claim 1, wherein the antigen-binding fusion polypeptide comprises the amino acid sequence of SEQ ID NO:1, or an amino acid sequence at least 90% identical to thereto.

### Claim 33 (depends on 1)

The method of claim 1, wherein the antigen-binding fusion polypeptide comprises at least one antigen-binding fusion polypeptide that binds to TNFα and comprises three CDRs having the amino acid sequence: SEQ ID NO:2 (DYWMY (CDR1)), SEQ ID NO:3 (EINTNGLITKYPDSVKG (CDR2)) and SEQ ID NO:4 (SPSGFN (CDR3)), or having a CDR that differs by fewer than 2 conservative amino acid substitutions from one of said CDRs.

### Claim 34 (depends on 1)

The method of claim 1, wherein the antigen-binding fusion polypeptide comprises at least one antigen-binding fusion polypeptide that binds to TNFα and comprises a variable region having the amino acid sequence from amino acids 1 to 115 of SEQ ID NO:1, or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:1.

### Claim 35 (depends on 33)

The method of claim 33, wherein the antigen-binding fusion polypeptide comprises at least one antigen-binding fusion polypeptide that binds to HSA and comprises three CDRs having the amino sequence: SFGMS (CDR1; SEQ ID NO:5), SISGSGSDTLYADSVKG (CDR2; SEQ ID NO:6) and/or GGSLSR (CDR3; SEQ ID NO:7), or having a CDR that differs by fewer than 2 conservative amino acid substitutions from one of said CDRs.

### Claim 36 (depends on 35)

The method of claim 35, wherein the antigen-binding fusion polypeptide comprises at least one antigen-binding fusion polypeptide that binds to HSA and comprises a variable region having the amino acid sequence from amino acids 125 to 239 of SEQ ID NO:1, or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:1.

### Claim 37

A method of separating or purifying an antigen-binding fusion polypeptide that comprises one or more VHHs from cell culture medium, comprising:
obtaining cell culture medium containing an antigen-binding fusion polypeptide that is produced as a product of a eukaryotic cell culture and secreted into the cell culture medium,
adjusting the cell culture medium containing the antigen-binding fusion polypeptide to a conductivity between about 12 and 9 mS/cm and a pH equal to or less than about 4.5;
contacting the medium containing the antigen-binding fusion polypeptide and one or more contaminants with a cation exchange support,
allowing the antigen-binding fusion polypeptide to flow through the support,
washing the support with at least one cation exchange washing buffer;
removing one or more contaminants;
selectively eluting the antigen-binding fusion polypeptide from the support, thereby obtaining an eluted antigen-binding fusion polypeptide preparation;
contacting the eluted antigen-binding fusion polypeptide preparation with a hydroxyapatite resin; and
selectively eluting the antigen-binding fusion polypeptide from the hydroxyapatite resin, wherein said antigen-binding fusion polypeptide does not have a complementary antibody variable domain or an immunoglobulin Fc region, and wherein the antigen-binding fusion polypeptide is not pegylated.

### Claim 38

A method or process of providing a recombinant antigen-binding fusion polypeptide that comprises one or more VHHs, comprising:
providing a mammalian host cell comprising a nucleic acid that encodes the recombinant antigen-binding fusion polypeptide;
maintaining the host cell under conditions in which the recombinant antigen-binding fusion polypeptide is expressed;
obtaining a medium of the recombinant antigen-binding fusion polypeptide and one or more contaminants;
purifying or separating the recombinant antigen-binding fusion polypeptide from said medium using cation exchange based chromatography support, wherein said purifying or separating step comprises
adjusting the medium containing the antigen-binding fusion polypeptide to a conductivity between about 12 and 9 mS/cm and a pH equal to or less than about 4.5;
contacting the medium with the support, under conditions that allow the antigen-binding fusion polypeptide to bind or absorb to the support;
removing one or more contaminants; and
selectively eluting the antigen-binding fusion polypeptide from the support, thereby obtaining an eluted antigen-binding fusion polypeptide preparation, wherein said antigen-binding fusion polypeptide is devoid of a complementary antibody domain and an immunoglobulin Fc region, and wherein the antigen-binding fusion polypeptide is not pegylated.

### Claim 39 (depends on 38)

The method of claim 38, further comprising subjecting the eluted antigen-binding fusion polypeptide preparation to one or more of hydroxyapatite chromatography, affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography, metal affinity chromatography, diafiltration, ultrafiltration, or viral removal filtration.

### Claim 40 (depends on 38)

The method of claim 38, further comprising preparing a formulation of the recombinant antigen-binding fusion polypeptide as a pharmaceutical composition.