# US 12,109,273 B2 · Process for preparing antibody-drug conjugates with improved homogeneity

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- Number: US 12,109,273 B2
- Document ID: US12109273B2
- Status: Granted
- Kind: B2
- Application number: 17/410,574
- Filed: 2021-08-24
- Published: 2021-12-09
- Granted: 2024-10-08
- Earliest claimed: 2019-02-15
- Assignees: Wuxi Xdc Singapore Private Limited
- Applicants: Wuxi Xdc Singapore Private Limited
- Inventors: Ao Ji; Chuchu Sun; Wenxu He
- Web page: https://patentgrep.com/patent/US12109273B2

## CPC

- A61K 31/40: having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K 47/6803: {Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates}
- A61K 47/68031: {the drug being an auristatin}
- A61K 47/6851: {the antibody targeting a determinant of a tumour cell}
- A61K 47/6889: {Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment}
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/2887: {against CD20}
- C07K 16/32: against translation products of oncogenes
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/55: Fab or Fab'
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- 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 disclosure relates to an applicable bio-conjugation process for preparing antibody-drug conjugates (ADCs) with improved homogeneity. As compared with conventional conjugation process, the homogeneity of antibody-drug conjugate (ADC) products generated from the bio-conjugation process can be dramatically improved. Specifically, in the ADCs prepared by the process of the present disclosure, the content of D0+D8 is less than 10 wt % and the content of D6 is less than 10 wt %. Moreover, the content of D4 is generally more than 65 wt %, for example, more than 70 wt %, and more than 77 wt %, while the content of D4 is normally less than 40 wt % in the ADCs prepared by conventional conjugation processes.

## Claims

### Claim 1

A method of preparing an antibody-drug conjugate (ADC), comprising the following steps:
(a) incubating a reductant and an antibody in the presence of an effective amount of a transition metal ion in a buffer system to reduce inter-chain disulfide bonds within the antibody to generate reduced thiol groups; and
(b) introducing an excess amount of payload having a reactive group to react with the reduced thiol groups resulted from step (a).

### Claim 2 (depends on 1)

The method of claim 1, wherein the transition metal ion is Zn2+, Cd2+, Hg2+, or a combination thereof.

### Claim 3 (depends on 2)

The method of claim 2, wherein the transition metal ion is Zn2+.

### Claim 4 (depends on 1)

The method of claim 1, wherein the buffer system in step (a) is Hepes, Histidine buffer, phosphate-buffered saline (PBS), or 2-morpholinoethanesulphonic acid (MES), and the pH value of the buffer system is 5.5 to 8.

### Claim 5 (depends on 1)

The method of claim 1, wherein the antibody in step (a) has a concentration of 0.01 to 0.1 mM.

### Claim 6 (depends on 1)

The method of claim 1, wherein step (a) is performed at a temperature of −10° C. to 37° C.

### Claim 7 (depends on 1)

The method of claim 1, wherein the reductant in step (a) is tris(2-carboxyethyl)phosphine (TCEP).

### Claim 8 (depends on 1)

The method of claim 1, wherein the payload comprises a maleimide moiety, bromide, or iodide.

### Claim 9 (depends on 1)

The method of claim 1, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

### Claim 10 (depends on 1)

The method of claim 1, wherein the antibody is a human antibody, a humanized antibody, a chimeric antibody or an antigen-binding moiety thereof.

### Claim 11 (depends on 1)

The method of claim 1, wherein the antibody is an IgG1 or an IgG4.

### Claim 12 (depends on 1)

The method of claim 1, wherein the payload comprises a diagnostic agent, a therapeutic agent or a labelling agent.

### Claim 13 (depends on 1)

The method of claim 1, wherein the resultant ADC are classified into D0, D2, D4, D6 and D8 based on the ratio between the antibody and payload molecules in an ADC molecule, wherein D4 in the resultant ADC has a weight percentage of over 65% on the basis of total weight of DO, D2, D4, D6 and D8, wherein
D0 refers to an antibody molecule that is not coupled to any payload molecules;
D2 refers to an ADC molecule in which two payload molecules are coupled to an antibody molecule;
D4 refers to an ADC molecule in which four payload molecules are coupled to an antibody molecule;
D6 refers to an ADC molecule in which six payload molecules are coupled to an antibody molecule; and
D8 refers to an ADC molecule in which eight payload molecules are coupled to an antibody molecule.

### Claim 14 (depends on 1)

The method of claim 1, wherein the resultant ADC are classified into D0, D2, D4, D6 and D8 based on the ratio between the antibody and payload molecules in an ADC molecule, wherein D0 and D8 together in the resultant ADC have a weight percentage of less than 10%, and D6 has a weight percentage of less than 10% on the basis of total weight of D0, D2, D4, D6 and D8, wherein
D0 refers to an antibody molecule that is not coupled to any payload molecules;
D2 refers to an ADC molecule in which two payload molecules are coupled to an antibody molecule;
D4 refers to an ADC molecule in which four payload molecules are coupled to an antibody molecule;
D6 refers to an ADC molecule in which six payload molecules are coupled to an antibody molecule; and
D8 refers to an ADC molecule in which eight payload molecules are coupled to an antibody molecule.

### Claim 15 (depends on 1)

The method of claim 1, wherein the payload selectively reacts with the reduced thiol groups in an Fab region of the antibody in step (b) in presence of the transition metal ion.

### Claim 16 (depends on 6)

The method claim 6, wherein step (a) is performed at a temperature of 0° C. to 20° C.

### Claim 17

A method of preparing a pharmaceutical composition, comprising the following steps: introducing an excess amount of payload having a reactive group to react with an antibody having one or more reduced thiol groups in presence of an effective amount of a transition metal ion; adding an effective amount of an oxidant to oxidize unreacted thiol groups; purifying the resultant antibody-drug conjugate; and preparing a pharmaceutical composition comprising the antibody-drug conjugate and a pharmaceutically acceptable carrier.

### Claim 18 (depends on 1)

The method of claim 1, further comprising
(c) adding an effective amount of an oxidant to oxidize unreacted thiol groups;
(d) purifying the resultant antibody-drug conjugate; and
(e) preparing a pharmaceutical composition comprising the antibody-drug conjugate and a pharmaceutically acceptable carrier.

### Claim 19 (depends on 18)

The method of claim 18, wherein the oxidant in step (c) is dehydroascorbic acid (DHAA).