# US 12,110,330 B2 · Compositions of programmed death receptor 1 (PD-1) antibodies and methods of obtaining the compositions thereof

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 12,110,330 B2
- Document ID: US12110330B2
- Status: Granted
- Kind: B2
- Application number: 17/586,109
- Filed: 2022-01-27
- Published: 2022-08-11
- Granted: 2024-10-08
- Earliest claimed: 2021-01-29
- Assignees: Merck Sharp & Dohme Corp.
- Applicants: Merck Sharp & Dohme LLC
- Inventors: Mark A. Brower; Chung-Jr Huang; Brian Wai Kwan; William Nicholas Napoli; Bhumit A. Patel; Nuno J. Dos Santos Pinto; Douglas Dennis Richardson, II; Sen Xu
- Web page: https://patentgrep.com/patent/US12110330B2

## CPC

- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/2818: {against CD28 or CD152}
- A61K 39/39591: {Stabilisation, fragmentation}
- A61P 35/00: Antineoplastic agents
- A61K 2039/505: {comprising antibodies}
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/40: characterized by post-translational modification
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2317/90: characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K 2317/94: Stability, e.g. half-life, pH, temperature or enzyme-resistance
- C12N 2015/8518: {expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles}
- 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)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention provides compositions of anti-PD-1 antibodies or antigen-binding fragments thereof with less than or equal to about 3.0% oxidation of Met105 in the CDRH3 heavy chain region, and methods of obtaining the purified compositions. The invention also provides compositions comprising anti-PD-1 antibody main species and acidic species thereof, wherein the amount of acidic species is about 1.0-12.0%.

## Claims

### Claim 1

A method of obtaining a purified composition comprising anti-human PD-1 antibodies or antigen-binding fragments thereof, wherein the anti-human PD-1 antibodies or antigen binding fragments thereof comprise a light chain variable region comprising three light chain CDRs comprising CDRL1 of SEQ ID NO:1, CDRL2 of SEQ ID NO:2 and CDRL3 of SEQ ID NO:3 and a heavy chain variable region comprising three heavy chain CDRs of CDRH1 of SEQ ID NO:6, CDRH2 of SEQ ID NO:7 and CDRH3 SEQ ID NO:8, comprising the steps of:
a) perfusing Chinese Hamster Ovary host cells in cell culture medium in a perfusion bioreactor by providing fresh medium at a rate of at least about 0.25-6.0 vessel volume per day (vvd), wherein the host cell comprises a polynucleotide encoding the light chain variable domain and a polynucleotide encoding the heavy chain variable domain, or a polynucleotide encoding the light chain variable domain and the heavy chain variable domain, of the anti-human PD-1 antibodies or antigen-binding fragments thereof, wherein a cell culture broth is formed in the perfusion bioreactor;
b) continuously harvesting the antibodies or antigen-binding fragments thereof from the cell culture broth to obtain a harvest cell culture fluid medium at a rate of at least about 0.25-6.0 vessel volume per day (vvd); and
c) continuously purifying the harvest cell culture fluid with an affinity chromatography step to obtain the purified composition.

### Claim 2 (depends on 1)

The method of claim 1, wherein the perfusion maintains a capacitance value of 70-90 pF/cm.

### Claim 3 (depends on 1)

The method of claim 1, wherein in step b), the rate is about 0.5 vvd on Day 3, about 1 vvd on Day 4 and about 2 vvd on Day 5, and the purified composition Is obtained from step c) on or after Day 5.

### Claim 4 (depends on 1)

The method of claim 1, wherein in step a) the perfusion is initiated when the cell density reaches about 2 to 10×106 cells/ml.

### Claim 5 (depends on 1)

The method of claim 1, wherein in step a) the perfusion is initiated when the cell density reaches about 4 to 8×106 cells/ml.

### Claim 6 (depends on 1)

The method of claim 1, wherein in step b), the rate is at about 0.5 vvd to 4 vvd.

### Claim 7 (depends on 2)

The method of claim 2, wherein in step b), the rate is at about 0.5 vvd to 4 vvd.

### Claim 8 (depends on 1)

The method of claim 1, wherein in step b), the continuous harvesting is initiated after a perfusion rate reaches about 2 vvd.

### Claim 9 (depends on 2)

The method of claim 2, wherein in step b), the continuous harvesting is initiated after a perfusion rate reaches about 2 vvd.

### Claim 10 (depends on 8)

The method of claim 8, wherein the cell culture medium comprises copper, and the copper concentration in the perfusion bioreactor ranges from 1 to 35 ppb.

### Claim 11 (depends on 1)

The method of claim 1, wherein the continuous harvesting is performed by setting a constant permeate rate to obtain a cell-free permeate through a hollow fiber membrane connected to the perfusion bioreactor.

### Claim 12 (depends on 1)

The method of claim 1, wherein between steps (b) and (c), the method further comprises the step of: transferring the harvest cell culture fluid (HCCF) to a surge vessel for a residence time of about 0.5 to 30 hours.

### Claim 13 (depends on 2)

The method of claim 2, wherein between steps (b) and (c), the method further comprises the step of: transferring the harvest cell culture fluid (HCCF) to a surge vessel for a residence time of about 0.5 to 30 hours.

### Claim 14 (depends on 8)

The method of claim 8, wherein between steps (b) and (c), the method further comprises the step of: transferring the harvest cell culture fluid (HCCF) to a surge vessel for a mean residence time of 15 to 45 minutes.

### Claim 15 (depends on 9)

The method of claim 9, wherein between steps (b) and (c), the method further comprises the step of: transferring the harvest cell culture fluid (HCCF) to a surge vessel for a mean residence time of 15 to 45 minutes.

### Claim 16 (depends on 8)

The method of claim 8, wherein the affinity chromatography step is Protein A affinity chromatography.

### Claim 17 (depends on 9)

The method of claim 9, wherein the affinity chromatography step is Protein A affinity chromatography.

### Claim 18 (depends on 16)

The method of claim 16, wherein between steps (b) and (c), the method further comprises the step of: transferring the harvest cell culture fluid (HCCF) to a surge vessel for a mean residence time of 15 to 45 minutes.

### Claim 19 (depends on 16)

The method of claim 16, wherein the affinity chromatography is operated in a continuous multi-column chromatography system.

### Claim 20 (depends on 17)

The method of claim 17, wherein the affinity chromatography is operated in a continuous multi-column chromatography system.

### Claim 21 (depends on 18)

The method of claim 18, wherein the affinity chromatography is operated in a continuous multi-column chromatography system.

### Claim 22 (depends on 20)

The method of claim 20, wherein the affinity chromatography is fluidly connected to the perfusion bioreactor of step a).

### Claim 23 (depends on 12)

The method of claim 12, wherein a surge vessel is fluidly connected to the perfusion bioreactor and affinity chromatography, and the HCCF flow rate to the surge vessel equals the feed rate from the surge vessel to the continuous multi-column chromatography system.

### Claim 24 (depends on 1)

The method of claim 1, wherein the anti-human PD-1 antibodies or antigen binding fragments thereof comprise a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:4, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9.

### Claim 25 (depends on 8)

The method of claim 8, wherein the purified composition comprises anti-human PD-1 antibodies, wherein the anti-human PD-1 antibodies consist of two light chains and two heavy chains, wherein the two light chains consist of the amino acid sequence set forth in SEQ ID NO:5, wherein the two heavy chains consist of the amino acid sequence set forth in any one of SEQ ID NO: 10-15, or a combination thereof.

### Claim 26 (depends on 9)

The method of claim 9, wherein the purified composition comprises anti-human PD-1 antibodies, wherein the anti-human PD-1 antibodies consist of two light chains and two heavy chains, wherein the two light chains consist of the amino acid sequence set forth in SEQ ID NO:5, wherein the two heavy chains consist of the amino acid sequence set forth in any one of SEQ ID NO: 10-15, or a combination thereof.

### Claim 27 (depends on 21)

The method of claim 21, wherein the purified composition comprises anti-human PD-1 antibodies, wherein the anti-human PD-1 antibodies consist of two light chains and two heavy chains, wherein the two light chains consist of the amino acid sequence set forth in SEQ ID NO:5, wherein the two heavy chains consist of the amino acid sequence set forth in any one of SEQ ID NO:10-15, or a combination thereof.

### Claim 28 (depends on 1)

The method of claim 1, wherein the antibody is a pembrolizumab.

### Claim 29 (depends on 1)

The method of claim 1, wherein the antibody is a pembrolizumab variant.

### Claim 30

A method of obtaining a purified composition comprising anti-human PD-1 antibodies or antigen-binding fragments thereof, wherein the anti-human PD-1 antibodies or antigen binding fragments thereof comprise a light chain variable region comprising three light chain CDRs comprising CDRL1 of SEQ ID NO:1, CDRL2 of SEQ ID NO:2 and CDRL3 of SEQ ID NO:3 and a heavy chain variable region comprising three heavy chain CDRs of CDRH1 of SEQ ID NO:6, CDRH2 of SEQ ID NO:7 and CDRH3 SEQ ID NO:8, comprising the steps of:
a) perfusing Chinese Hamster Ovary host cells in cell culture medium in a perfusion bioreactor by applying a perfusion rate of at least about 0.25-6.0 vessel volume per day (vvd), wherein the host cell comprises a polynucleotide encoding the light chain variable domain and a polynucleotide encoding the heavy chain variable domain, or a polynucleotide encoding the light chain variable domain and the heavy chain variable domain, of the anti-human PD-1 antibodies or antigen-binding fragments thereof, wherein a cell culture broth is formed in the perfusion bioreactor;
b) continuously harvesting the antibodies or antigen-binding fragments thereof from the cell culture broth to obtain a harvest cell culture fluid;
c) transferring the harvest cell culture fluid (HCCF) to a surge vessel for a mean residence time of 15 to 45 minutes, or a residence time of about 0.5 to 30 hours; and
d) continuously purifying the harvest cell culture fluid with an affinity chromatography step to obtain the purified composition.

### Claim 31 (depends on 30)

The method of claim 30, wherein the affinity chromatography is a continuous multi-column Protein A chromatography system.

### Claim 32 (depends on 31)

The method of claim 31, wherein the purified composition comprises anti-human PD-1 antibodies, wherein the anti-human PD-1 antibodies consist of two light chains and two heavy chains, wherein the two light chains consist of the amino acid sequence set forth in SEQ ID NO:5, wherein the two heavy chains consist of the amino acid sequence set forth in any one of SEQ ID NOs:10-15, or a combination thereof.