# US 12,595,294 B2 · Methods to decrease impurities from recombinant protein manufacturing processes

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- Number: US 12,595,294 B2
- Document ID: US12595294B2
- Status: Granted
- Kind: B2
- Application number: 17/812,145
- Filed: 2022-07-12
- Published: 2023-02-16
- Granted: 2026-04-07
- Earliest claimed: 2021-01-15
- Assignees: Hoffmann-La Roche Inc.
- Applicants: Hoffmann-La Roche Inc.
- Inventors: Marc Pompiati; Christoph Feistl
- Web page: https://patentgrep.com/patent/US12595294B2

## CPC

- C07K 16/065: {Purification, fragmentation}
- C07K 1/34: by filtration, ultrafiltration or reverse osmosis
- C07K 2317/31: multispecific
- C07K 2317/35: Valency
- C07K 2317/515: Complete light chain, i.e. VL + CL
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- 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 invention relates to a method of reducing the amount of non-aggregate produce-related impurities (NAPRIs) in a buffered solution of monoclonal antibodies (mAbs), involving the use of a synthetic depth filter. The invention may be of use in the purification of monoclonal 5 antibodies.

## Claims

### Claim 1

A method of producing a buffered solution of monoclonal antibodies (mAbs) with a reduced amount of non-aggregate product-related impurities (NAPRIs), the method comprising passing an initial buffered solution comprising: (a) mAbs, and (b) NAPRIs, through a synthetic depth filter comprising silica and polyacrylic fiber to produce the buffered solution of mAbs with the reduced amount of NAPRIs,
wherein the NAPRIs comprise incompletely or incorrectly assembled mAb polypeptides,
wherein the NAPRIs comprise high molecular weight (HMW) polypeptides having a molecular weight higher than the mAb,
wherein the NAPRIs do not comprise aggregates, and
wherein the reduced amount of NAPRIs is with respect to the amount of NAPRIs in the initial buffered solution before it has passed through the synthetic depth filter comprising silica and polyacrylic fiber.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

The method of claim 1, wherein the mAb is an antibody fusion protein comprising an antibody or antibody fragment and another biologically active polypeptide.

### Claim 4 (depends on 1)

The method of claim 1, wherein the initial buffered solution of mAbs is passed through the depth filter at a temperature that is between 4° C. and 22° C.

### Claim 5 (depends on 1)

The method of claim 1, wherein the NAPRI is a polypeptide lacking one or more polypeptide chains of the mAb and/or the NAPRI is a polypeptide comprising a different polypeptide chain arrangement than the mAb.

### Claim 6 (depends on 1)

The method of claim 1, wherein the NAPRI comprises two heavy chains having the same amino acid sequence.

### Claim 7 (depends on 1)

The method of claim 1, wherein the depth filter is a dual layer depth filter.

### Claim 8 (depends on 1)

The method of claim 1, wherein the depth filter does not contain diatomaceous earth.

### Claim 9 (depends on 1)

The method of claim 1, further comprising measuring the NAPRI concentration in the buffered solution of mAbs, wherein the NAPRI concentration is measured by Size Exclusion Chromatography (SEC) and/or with Capillary Electrophoresis SDS Page in a non-reducing environment.

### Claim 10 (depends on 1)

The method of claim 1, wherein the initial buffered solution of mAbs has been subjected to chromatography before passing through the synthetic depth filter, and wherein the chromatography comprises one or more of ion exchange chromatography, anion exchange column, a cation exchange column, or multimodal (mixed mode) chromatography.

### Claim 11 (depends on 1)

The method of claim 1, wherein the initial buffered solution of mAbs is passed through the depth filter at a temperature of between 10° C. and 21° C., or between 15° C. and 20° C.

### Claim 12 (depends on 1)

The method of claim 1, wherein the initial buffered solution of mAbs is passed through the depth filter at mass load of in the range of 100 g/m2 to 2500 g/m2, 300 g/m2 to 2000 g/m2, or 500 g/m2 to 1500 g/m2.

### Claim 13 (depends on 1)

The method of claim 1, wherein the initial buffered solution of mAbs has a pH in the range of 4.0 to 7.5, 4.0 to 7.2, or 4.0 to 5.5 when it passes through the depth filter.

### Claim 14 (depends on 1)

The method of claim 1, wherein the initial buffered solution of mAbs is passed through the depth filter at a flow rate in the range of 1 L/min*m2 to 10 L/min*m2, 1.5 L/min*m2 to 8 L/min*m2, or at a flow rate that is 4.3 L/min*m2.

### Claim 15 (depends on 1)

The method of claim 1, further comprising the step of isolating the mAbs from the buffered solution of mAbs.