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US 2026/0298942 A1Published application
Methods for analyzing chain mispairing in multispecific binding proteins
AssigneeSanofi
InventorFatemeh TOUSI
Life of this patent
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Abstract
Provided herein are methods for monitoring production of a multispecific binding protein and one or more mispaired species by a cell line, as well as methods of production and screening related thereto. In some embodiments, the methods comprise detecting an amount (e.g., a relative amount) of a multispecific binding protein and one or more mispaired species in a cell culture medium by size-exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS). In some embodiments, the multispecific binding protein is a multispecific antibody, antibody fragment, or Fc fusion protein.
Claims26
1-32. (canceled)
A method for producing a multispecific antibody, the method comprising:
- (a) culturing a cell line comprising one or more polynucleotides encoding the multispecific antibody in a cell culture medium under conditions suitable for production of the multispecific antibody and one or more mispaired species thereof by the cell line;
- (b) separating, from the cell line, the cell culture medium comprising the multispecific antibody and one or more mispaired species thereof, wherein the separation comprises flow filtration (TFF), depth filtration, and/or centrifugation and does not comprise chromatographic separation or protein A affinity chromatography;
- (c) without prior chromatographic separation or protein A affinity chromatography, separating and detecting an amount of the multispecific antibody and the one or more mispaired species thereof from the cell culture medium by denaturing size-exclusion ultra-performance liquid chromatography and online mass spectrometry (SE-UPLC-MS), wherein the mass spectrometry (MS) is quadrupole time-of-flight (QToF) MS;
- and
- (d) removing at least a portion of one or more of the mispaired species from the multispecific antibody produced by the cell line, or determining one or both of quality and purity of the multispecific antibody produced by the cell line;
- 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.
5 more elements
34-43. (canceled)
45-60. (canceled)
(canceled)
64-71. (canceled)
A method for producing an antibody or antibody fragment, the method comprising:
- (a) culturing a cell line comprising one or more polynucleotides encoding the antibody or antibody fragment in a cell culture medium under conditions suitable for production of the antibody or antibody fragment and one or more weight variant species thereof by the cell line;
- (b) separating, from the cell line, the cell culture medium comprising the antibody or antibody fragment and one or more weight variant species thereof, wherein the separation comprises flow filtration (TFF), depth filtration, and/or centrifugation and does not comprise chromatographic separation or protein A affinity chromatography;
- (c) without prior chromatographic separation or protein A affinity chromatography, separating and detecting an amount of the antibody or antibody fragment and the one or more weight variant species thereof from the cell culture medium by denaturing size-exclusion ultra-performance liquid chromatography and online mass spectrometry (SE-UPLC-MS), wherein the mass spectrometry (MS) is quadrupole time-of-flight (QToF) MS;
- and
- (d) removing at least a portion of one or more of the weight variant species from the antibody or antibody fragment produced by the cell line, or determining one or both of quality and purity of the antibody or antibody fragment produced by the cell line;
- wherein the antibody or antibody fragment and one or more weight variant species differ in molecular weight.
3 more elements
73-111. (canceled)
A method for producing a multispecific binding protein, the method comprising:
- (a) culturing a cell line comprising one or more polynucleotides encoding the multispecific binding protein in a cell culture medium under conditions suitable for production of the multispecific binding protein and one or more mispaired species thereof by the cell line;
- (b) separating, from the cell line, the cell culture medium comprising the multispecific binding protein and one or more mispaired species thereof, wherein the separation comprises flow filtration (TFF), depth filtration, and/or centrifugation and does not comprise chromatographic separation or protein A affinity chromatography;
- (c) without prior chromatographic separation or protein A affinity chromatography, separating and detecting an amount of the multispecific binding protein and the one or more mispaired species thereof from the cell culture medium by denaturing size-exclusion ultra-performance liquid chromatography and online mass spectrometry (SE-UPLC-MS), wherein the mass spectrometry (MS) is quadrupole time-of-flight (QToF) MS;
- and
- (d) removing at least a portion of one or more of the mispaired species from the multispecific binding protein produced by the cell line, or determining one or both of quality and purity of the multispecific binding protein produced by the cell line;
- 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:
- and a second polypeptide chain of the multispecific binding protein comprises a structure represented by the formula:
- and a third polypeptide chain of the multi specific binding protein comprises a structure represented by the formula:
- and a fourth polypeptide chain of the multi specific binding protein comprises a structure represented by the formula:
- wherein:
- VL1 is a first immunoglobulin light chain variable domain;
- VL2 is a second immunoglobulin light chain variable domain;
- VL3 is a third immunoglobulin light chain variable domain;
- VH1 is a first immunoglobulin heavy chain variable domain;
- VH2 is a second immunoglobulin heavy chain variable domain;
- VH3 is a third immunoglobulin heavy chain variable domain;
- CL is an immunoglobulin light chain constant domain;
- CH1 is an immunoglobulin CH1 heavy chain constant domain;
- CH2 is an immunoglobulin CH2 heavy chain constant domain;
- CH3 is an immunoglobulin CH3 heavy chain constant domain;
- hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains;
- and
- L1, L2, L3 and L4 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 VH1 and VL1 form a first antigen binding site,
- wherein VH2 and VL2 form a second antigen binding site, and wherein VH3 and VL3 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.
25 more elements
Dependencies are inferred from the claim text.