# US 12,760,063 B2 · Eluate collection during antibody chromatography

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- Number: US 12,760,063 B2
- Document ID: US12760063B2
- Status: Granted
- Kind: B2
- Application number: 17/777,366
- Filed: 2020-11-20
- Published: 2022-12-29
- Granted: 2026-10-13
- Earliest claimed: 2019-11-22
- Assignees: Morphosys AG
- Applicants: Morphosys GmbH
- Inventors: Daniela Daubert; Dominik Geltinger; Karin Felderer
- Web page: https://patentgrep.com/patent/US12760063B2

## CPC

- C07K 1/18: Ion-exchange chromatography
- C07K 16/244: {Interleukins [IL]}
- 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 invention relates to an improved method for peak fractionation and eluate collection during chromatography for purification of a human therapeutic antibody.

## Claims

### Claim 1

A method for the purification of an antibody by chromatography, comprising the following steps:
a) loading a sample comprising an antibody onto a chromatography resin,
b) optionally, washing the resin,
c) applying an elution buffer to the chromatography resin, and
d) starting collection of the eluate,
wherein the collection of the eluate is started at a predetermined interval (D0) after the absorbance signal of the eluate has reached a predetermined value (A0), and wherein the predetermined value (A0) is in the range of 10-50% of the absorbance signal at peak maximum of elution peaks obtained by different elution runs varying in pH conditions within a range of pH 5 to pH 7 and load density within a range of 5 to 50 g/l resin load with the antibody sample to be purified.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the predetermined absorbance value (A0) is in the range of 0 to 1500 mAU measured at 280 nm.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the predetermined absorbance value (A0) is 700 mAU measured at 280 nm.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the predetermined interval (D0) is a flow volume interval that is determined by the following steps:
a) receiving the elution peak chromatograms of at least two different elution runs (ER1, . . . , ERN) with the antibody sample to be purified, wherein the different elution runs vary in pH, load density or salt conditions,
b) specifying an absorbance signal A1, . . . , AN for each of the elution peaks received in step a), wherein each of the corresponding eluate fractions of A1, . . . , AN has an aggregate/impurity content below 4%,
c) determining the flow volumes C1, . . . , CN of the absorbance signals A1, . . . , AN for each of the elution runs in the chromatograms,
d) calculating the difference for each of said flow volumes C1, . . . , CN, to the flow volume C0 of A0, and
e) averaging said differences (i.e. obtaining the mean value), thereby obtaining the predetermined flow volume interval (D0).

### Claim 5 (depends on 4)

The method according claim 4, wherein the distance of A1, . . . , AN to the absorbance signal AMAX at height maximum of the corresponding elution peak is in the range of 0 to 300 mAU measured at 280 nm.

### Claim 6 (depends on 5)

The method according to claim 5, wherein the predetermined interval value (D0) is between 0.4 to 1.2 CV flow volumes.

### Claim 7 (depends on 6)

The method according claim 6, wherein the predetermined interval value (D0) is 0.6 CV flow volumes.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the chromatography is ion exchange (IEX) chromatography.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the chromatography is cation exchange (CEX) chromatography.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the monoclonal antibody comprises a VH of SEQ ID NO: 8 and a VL of SEQ ID NO: 7.

### Claim 11 (depends on 10)

The method according to claim 10, wherein the antibody comprises a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9.