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US-12618853-B2 - Methods for analyzing chain mispairing in multispecific binding proteins

US12618853B2US 12618853 B2US12618853 B2US 12618853B2US-12618853-B2

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.

Inventors

  • Fatemeh Tousi

Assignees

  • SANOFI

Dates

Publication Date
20260505
Application Date
20200601
Priority Date
20200528

Claims (18)

  1. 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. 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. 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. 4 . The method of claim 1 , wherein the MS is intact MS.
  5. 5 . The method of claim 1 , wherein SE-UPLC is performed using isocratic elution with a mobile phase.
  6. 6 . The method of claim 1 , wherein detection is accomplished in about 33 minutes or less.
  7. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62/926,313, filed Oct. 25, 2019, and EP Application No. EP20315271.5, filed May 28, 2020, the disclosures of each of which are incorporated herein by reference in their entirety. SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 183952032800SEQLIST.TXT, date recorded: May 29, 2020, size: 2 KB). FIELD The disclosure relates to methods for monitoring production of a multispecific binding protein and one or more mispaired species by a host cell, as well as methods of production and screening related thereto. The disclosure further relates to methods for monitoring production of an antibody or antibody derivative and one or more weight variant species by a host cell, as well as methods of production and screening related thereto. BACKGROUND Multi-specific antibodies that bind two or more different epitopes on the same or different antigens have become attractive therapeutic options in immune-oncology in recent years (Baeuerle, P. A. & Reinhardt, C., Cancer Res 2009, 69 (12), 4941-4). Multi-targeting has been used for different purposes, such as achieving enhanced drug specificity or mimicking natural ligands in signaling pathways, for example in hemophilia treatments through simultaneous binding to receptor pairs on the surface of the same cell (Kitazawa, T. et al. Nat Med 2012, 18 (10), 1570-4). A prominent application is the T-cell engager (TCE) concept where one arm of the molecule activates T-cells via CD3/CD28 receptor binding and the other arm targets a tumor antigen for tumor killing (Krah, S. et al. N Biotechnol 2017, 39 (Pt B), 167-173; Correnti, C. E. et al. Leukemia 2018, 32 (5), 1239-1243). IgG-like tri-specific antibodies (tsAb) are comprised of two different heavy chains and two different light chains, commonly expressed in a single host cell followed by intracellular chain assembly. While this method of production eliminates the need to have two separate cell lines and purification processes, it can generate unwanted mispaired species in addition to the desired tsAb. In the absence of rational design and with random association of the subunits, the theoretical yield of the correctly paired species is only 12.5% (FIG. 1B). Forced heterodimerization of the heavy chains has been successfully accomplished through protein engineering approaches such as the knobs-into-holes design (Ridgway, J. B. et al. Protein Eng 1996, 9 (7), 617-21). However, cognate pairing of the light chains to the correct heavy chains remains a significant challenge in tsAb production. As such, there remains a need for methods for monitoring or analyzing production of a multispecific binding protein and one or more mispaired species. In addition, there remains a need for methods for monitoring or analyzing production of antibody or antibody derivatives that comprise multiple species varying in molecular weight (e.g., with different chemical modifications, such as chemically modified cysteine or other residues, or with different glycoforms). All references cited herein, including patent applications, patent publications, and UniProtKB/Swiss-Prot Accession numbers are herein incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference. BRIEF SUMMARY To meet these and other needs, provided herein are methods for monitoring production of a multispecific binding protein and one or more mispaired species (e.g., by a cell line). These methods provide, inter alia, a high throughput analytical platform based on denaturing SEC-LC-Intact MS for identification and relative quantitation of chain mispairing and other IgG-related species. Advantageously, these methods allow for intact MS analysis of mAb-related species (e.g., multispecific binding proteins, antibodies, Fc fusion proteins, antibody fragments, and so forth) directly from a clarified cell harvest, bypassing time consuming and expensive purification (e.g., protein A affinity chromatography) and buffer exchange steps. As such, these methods can be used to rapidly screen a large number of clones for potential production cell lines. In some embodiments, provided herein are methods for monitoring production of a multispecific binding protein and one or more mispaired species, the methods comprising: detecting, by size-exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS), an amount of a multispecific binding protein and one or more mispaired species in a cell culture medium comprising the multispecific binding protein and the one or more mispaired species. In some embodiments, the multispecific binding protein comprises an association of two or more polypeptide chains comprising a