Granted Patent B2

US 12,618,853 · App. 16/889,404 · Granted May 5, 2026

Inventor: Fatemeh Tousi (Dedham, MA)

Assignee: Sanofi

G01N33/6854C07K16/00C12P21/02G01N33/5005B01D15/34B01D15/3809C07K2317/31

Abstract

Claims (59)

1 . A method for monitoring production of a multispecific antibody and one or more mispaired species thereof, the method comprising:

separating a cell culture harvest that comprises a multispecific antibody and one or more mispaired species thereof from a cell line that produces the multispecific antibody and the one or more mispaired species thereof, wherein the separation does not comprise chromatographic separation or protein A affinity chromatography;

without prior chromatographic separation or protein A affinity chromatography, separating and detecting, by denaturing size-exclusion ultra-performance liquid chromatography with online mass spectrometry (SE-UPLC-MS), the multispecific antibody and the one or more mispaired species thereof from the cell culture harvest; wherein, prior to SE-UPLC-MS, the cell culture harvest has been clarified by tangential flow filtration (TFF), depth filtration, and/or centrifugation; and wherein the mass spectrometry (MS) is quadrupole time-of-flight (QToF) MS, thereby monitoring production of the multispecific antibody and one or more mispaired species thereof;

wherein the multispecific antibody comprises a first antibody heavy chain, a first antibody light chain, a second antibody heavy chain different from the first antibody heavy chain, and a second antibody light chain different from the first antibody light chain; and

wherein the one or more mispaired species comprise two or more polypeptide chains of the multispecific antibody in a species other than that of the multispecific antibody.

2 . The method of claim 1 , wherein the one or more mispaired species comprises one or more of:

(i) a species that comprises two of the first antibody heavy chains of the multispecific antibody;

(ii) a species that comprises two of the second antibody heavy chains of the multispecific antibody;

(iii) a species that comprises two of the first antibody light chains of the multispecific antibody; and

(iv) a species that comprises two of the second antibody light chains of the multispecific antibody.

3 . The method of claim 1 , wherein detecting the amount of the multispecific antibody and the one or more mispaired species thereof comprises deconvoluting one or more MS spectra obtained by the MS.

4 . The method of claim 1 , wherein the MS is intact MS.

5 . The method of claim 1 , wherein SE-UPLC is performed using isocratic elution with a mobile phase.

6 . The method of claim 1 , wherein detection is accomplished in about 33 minutes or less.

7 . The method of claim 1 , wherein the MS is capable of resolving a mass difference of about 300 Da between the multispecific antibody and the one or more mispaired species thereof, or between two mispaired species.

8 . The method of claim 1 , wherein the MS is capable of resolving a mass difference of about 162 Da between the multispecific antibody or mispaired species thereof and one of more glycoforms.

9 . A method for monitoring production of an antibody or antibody fragment and one or more weight variant species thereof, the method comprising:

separating a cell culture harvest that comprises an antibody or antibody fragment and one or more weight variant species thereof from a cell line that produces the antibody or antibody fragment and the one or more weight variant species thereof, wherein the separation does not comprise chromatographic separation or protein A affinity chromatography;

without prior chromatographic separation or protein A affinity chromatography, separating and detecting, by denaturing size-exclusion ultra-performance liquid chromatography with online mass spectrometry (SE-UPLC-MS), the antibody or antibody fragment and the one or more weight variant species thereof from the cell culture harvest; wherein, prior to SE-UPLC-MS, the cell culture harvest has been clarified by tangential flow filtration TFF), depth filtration, and/or centrifugation; and wherein the mass spectrometry (MS) is quadrupole time-of-flight (QToF) MS, thereby monitoring production of the antibody or antibody fragment and one or more weight variant species thereof;

wherein the antibody or antibody fragment and one or more weight variant species thereof differ in molecular weight.

10 . The method of claim 9 , wherein the antibody or antibody fragment and the one or more weight variant species thereof comprise species with a free cysteine that has been cysteinylated, N-acetyl cysteinylated, or glutathionylated.

11 . The method of claim 9 , wherein the antibody or antibody fragment and the one or more weight variant species thereof comprise species comprising a chemically modified cysteine residue.

12 . The method of claim 9 , wherein the antibody or antibody fragment and one or more weight variant species thereof differ in molecular weight by at least 119 Da.

13 . The method of claim 9 , wherein the method is capable of resolving a mass difference among cysteinylated, N-acetyl cysteinylated, and glutathionylated species.

14 . The method of claim 9 , wherein the antibody or antibody fragment and the one or more weight variant species thereof comprise glycoforms of the antibody or antibody derivative.

15 . The method of claim 9 , wherein the antibody or antibody fragment and one or more weight variant species thereof differ in molecular weight by at least 162 Da.

16 . The method of claim 9 , wherein the method is capable of resolving a mass difference between the antibody or antibody fragment and one of more weight variant species thereof that represent glycoforms of the antibody or antibody fragment.

17 . A method for monitoring production of a multispecific binding protein and one or more mispaired species thereof, the method comprising:

separating a cell culture harvest that comprises a multispecific binding protein and one or more mispaired species thereof from a cell line that produces the multispecific binding protein and the one or more mispaired species thereof, wherein the separation does not comprise chromatographic separation or protein A affinity chromatography;

without prior chromatographic separation or protein A affinity chromatography, separating and detecting, by denaturing size-exclusion ultra-performance liquid chromatography with online mass spectrometry (SE-UPLC-MS), the multispecific binding protein and the one or more mispaired species thereof from the cell culture harvest; wherein, prior to SE-UPLC-MS, the cell culture harvest has been clarified by tangential flow filtration (TFF), depth filtration, and/or centrifugation; and wherein the mass spectrometry (MS) is quadrupole time-of-flight (QToF) MS, thereby monitoring production of the multispecific binding protein and one or more mispaired species thereof;

wherein the multispecific binding protein comprises four polypeptide chains that form the three antigen binding sites,

wherein a first polypeptide chain of the multispecific binding protein comprises a structure represented by the formula:

V L2 -L 1 -V L1 -L 2 -C L   [I]

and a second polypeptide chain of the multispecific binding protein comprises a structure represented by the formula:

V H1 -L 3 -V H2 -L 4 -C H1 -hinge-C H2 -C H3   [II]

and a third polypeptide chain of the multispecific binding protein comprises a structure represented by the formula:

V H3 -C H1 -hinge-C H2 -C H3   [III]

and a fourth polypeptide chain of the multispecific binding protein comprises a structure represented by the formula:

V L3 -C L   [IV]

wherein:

V L1 is a first immunoglobulin light chain variable domain;

V L2 is a second immunoglobulin light chain variable domain;

V L3 is a third immunoglobulin light chain variable domain;

V H1 is a first immunoglobulin heavy chain variable domain;

V H2 is a second immunoglobulin heavy chain variable domain;

V H3 is a third immunoglobulin heavy chain variable domain;

C L is an immunoglobulin light chain constant domain;

C H1 is an immunoglobulin C H1 heavy chain constant domain;

C H2 is an immunoglobulin C H2 heavy chain constant domain;

C H3 is an immunoglobulin C H3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the C H1 and C H2 domains; and

L 1 , L 2 , L 3 and L 4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair, wherein V H1 and V L1 form a first antigen binding site, wherein V H2 and V L2 form a second antigen binding site, and wherein V H3 and V L3 form a third antigen binding site; and

wherein the one or more mispaired species comprise two or more polypeptide chains of the multispecific binding protein in a species other than that of the multispecific binding protein.

18 . The method of claim 17 , wherein the one or more mispaired species comprises one or more of:

(i) a species that comprises two of the first polypeptide chains of the multispecific binding protein;

(ii) a species that comprises two of the second polypeptide chains of the multispecific binding protein;

(iii) a species that comprises two of the third polypeptide chains of the multispecific binding protein; and

(iv) a species that comprises two of the fourth polypeptide chains of the multispecific binding protein.

Assignments (1)

Priority Claims (1)

Continuity (2)

References Cited (80)