Granted Patent B2

US 12,421,322 · App. 16/758,128 · Granted Sep 23, 2025

Inventors: Kensaku Hosoguchi (Tokyo, JP); Maki Kuwayama (Tokyo, JP); Chifumi Seida (Tokyo, JP); Yosuke Watanabe (Tokyo, JP); Nobuyuki Tanaka (Tokyo, JP); Satoshi Saitoh (Tokyo, JP); Masakazu Fukuda (Tokyo, JP)

Assignee: Chugai Seiyaku Kabushiki Kaisha

C07K16/468G01N33/6854A61K2039/505C07K2317/31C07K2317/51C07K2317/565C07K2317/71C07K2317/94

Abstract

Claims (39)

1. A purified variant of Emicizumab, wherein the variant is identical to Emicizumab except for modifications consisting of, in a heavy chain variable region that corresponds to SEQ ID NO: 13:

(1) the heavy chain variable region has a deletion of either (a) or (b):

(a) Arg at position 61; or

(b) Tyr-Tyr-Arg at positions 59-61,

and

(2) the heavy chain variable region is split into two fragments:

(i) an N-terminal fragment with a C-terminus that is either Tyr at position 60 or Thr at position 58, and

(ii) a C-terminal fragment with an N-terminus that is Arg at position 62,

wherein the two fragments are linked by a disulfide bond, and wherein all position numbers are based on numbering in SEQ ID NO: 13.

2. A method for determining whether a variant of Emicizumab is present in a sample containing Emicizumab, the method comprising:

using a purified variant of Emicizumab as a reference standard for a chromatography column by subjecting the purified variant to a chromatography process on the chromatography column, thereby identifying the position where a peak corresponding to the variant elutes from the chromatography column,

wherein the variant is identical to Emicizumab except for modifications consisting of, in a heavy chain variable region that corresponds to SEQ ID NO: 13:

(1) the heavy chain variable region has a deletion of either (a) or (b):

(a) Arg at position 61; or

(b) Tyr-Tyr-Arg at positions 59-61,

 and

(2) the heavy chain variable region is split into two fragments, linked by a disulfide bond:

(i) an N-terminal fragment with a C-terminus that is either Tyr at position 60 or Thr at position 58, and

(ii) a C-terminal fragment with an N-terminus that is Arg at position 62,

wherein all position numbers are based on numbering in SEQ ID NO: 13, and

wherein the chromatography process comprises ion exchange chromatography or separation based on charge; and

subjecting the sample to the chromatography process on the chromatography column, to determine whether a peak at the position identified as corresponding to the variant appears, as an indication that a variant identical to the purified variant is present in the sample.

3. A method for purifying Emicizumab from a sample containing Emicizumab and an antibody variant, wherein the variant is identical to Emicizumab except for modifications consisting of, in a heavy chain variable region that corresponds to SEQ ID NO: 13:

(1) heavy chain variable region has a deletion of either (a) or (b):

(a) Arg at position 61, or

(b) Tyr-Tyr-Arg at positions 59-61;

and

(2) the heavy chain variable region is split into two fragments:

(i) an N-terminal fragment with a C-terminus that is either Tyr at position 60 or Thr at position 58, and

(ii) a C-terminal fragment with an N-terminus that is Arg at position 62,

wherein the two fragments are linked by a disulfide bond, and wherein all position numbers are based on numbering in SEQ ID NO: 13,

 the method comprising:

(A) applying a purified preparation of the antibody variant to an ion exchange chromatographic column as a reference standard to identify the position where the antibody variant elutes from the column compared to the position where Emicizumab elutes from the column;

(B) subjecting the sample to chromatography on the column to separate a fraction or fractions corresponding to the position where Emicizumab elutes from the column from a fraction or fractions corresponding to the position where the antibody variant elutes from the column; and

(C) collecting a fraction or fractions containing Emicizumab from the column, wherein the collected fraction or fractions contain a higher ratio of Emicizumab to the antibody variant than did the sample prior to (B).

4. The method of claim 3 , wherein the column utilizes cation exchange chromatography.

5. The method of claim 2 , further comprising conducting quantitative analysis to analyze the ratio of the variant to total antibody molecules in the sample.

6. The method of claim 2 , wherein the chromatography process comprises ion exchange chromatography.

7. The method of claim 2 , wherein the chromatography process comprises separation based on charge.

Assignments (1)

Priority Claims (1)

Continuity (1)

References Cited (400)