Methods for quantitating individual antibodies from a mixture

Patent metadata

Field	Value
Publication number	US-11020686-B2
Application number	US-201716322292-A
Country	US
Kind code	B2
Filing date	Aug 8, 2017
Priority date	Aug 16, 2016
Publication date	Jun 1, 2021
Grant date	Jun 1, 2021

Abstract

The present disclosure relates to, inter alia, a method of quantitating an amount of an antibody molecule from a mixture comprising two or more antibody molecules, comprising separating each of the two or more antibody molecules from the mixture by hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC) and quantitating an amount of each antibody molecule, wherein the molecular weight of each antibody molecule is within 15 kDa of any other antibody molecule in the mixture and either each antibody molecule is different from another antibody molecule in the mixture by more than about 0.25 unit on the Kyte & Doolittle hydropathy scale or each of the antibody molecules when nm alone on HIC-HPLC elutes at distinct run time with little overlap from the other antibody molecules in the mixture, or both.

First claim

What is claimed is:

1. A method of quantitating amounts of antibodies from a mixture comprising a plurality of antibodies, the method comprising:
separating each of the plurality of antibodies in the mixture using hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC); and
quantitating an amount of each antibody in the mixture,
wherein a molecular weight of each antibody in the mixture is within 15 kDa of a molecular weight of any other antibodies in the mixture, and either a surface hydrophobicity of each antibody in the mixture is different from a surface hydrophobicity of another antibody in the mixture by more than about 0.25 units on the Kyte & Doolittle hydropathy scale, or each antibody in the mixture, when run on HIC-HPLC individually, elutes at a distinct run time from another antibody in the mixture, or both.

2. The method of claim 1 , wherein the surface hydrophobicity of each antibody in the mixture is different from the surface hydrophobicity of each other antibody in the mixture by about 0.5 to about 1.0 units on the Kyte & Doolittle hydropathy scale.

3. The method of claim 1 , wherein a first antibody in the mixture elutes at a first run time during a HIC-HPLC run, a second antibody in the mixture elutes at a second run time during the HIC-HPLC run, and the first and second run times do not overlap.

4. The method of claim 1 , wherein the HIC-HPLC is performed in a buffer at about pH 5.0 to about pH 7.0.

5. The method of claim 1 , wherein the plurality of antibodies comprises three antibodies.

6. The method of claim 1 , wherein the surface hydrophobicity of each antibody in the mixture is determined by one or more methods selected from the group consisting of: calculated surface hydrophobicity based on protein structure or structural model, rapid screening for solubility in ammonium sulfate or PEG8000, or rapid screening for molecule interaction by affinity capture-self-interaction nanoparticle spectroscopy (AC-SINS).

7. The method of claim 1 , wherein:
a first antibody in the mixture and a second antibody in the mixture have protein sequences that are at least 90% homologous;
the first antibody and the second antibody have protein structures that are at least 90% homologous, as determined by their protein sequences; or
the first antibody and the second antibody have isoelectric points (pI) within about 0.6 of one another, as determined by their protein sequences.

8. The method of claim 1 , wherein one or more of the antibodies in the mixture are monoclonal antibodies.

9. The method of claim 8 , wherein one or more of the monoclonal antibodies in the mixture are human monoclonal antibodies.

10. The method of claim 1 , wherein two or more of the antibodies in the mixture are of the same isotype.

11. The method of claim 1 , wherein two or more of the antibodies in the mixture are variants of each other.

12. The method of claim 1 , wherein two or more of the antibodies in the mixture bind to the same antigen.

13. The method of claim 1 , wherein the mixture is a co-formulated composition.

14. The method of claim 13 , wherein the co-formulated composition is configured to treat MERS in a human patient.

15. The method of claim 13 , wherein the co-formulated composition is configured to treat Ebola hemorrhagic fever in a human patient.

16. The method of claim 13 , wherein the co-formulated composition is configured to treat macular degeneration in a human patient.

17. The method of claim 13 , wherein the two or more antibodies in the co-formulated composition are configured to treat an infectious disease in a human patient.

18. The method of claim 13 , wherein the co-formulated composition is included in a drug product.

19. The method of claim 1 , further comprising generating a chromatograph from the HIC-HPLC, wherein for elution of each antibody in the mixture, the chromatograph shows a peak that does not overlap with other peaks in the chromatograph.

20. A method of quantitating amounts of antibodies from a mixture comprising a plurality of antibodies, the method comprising:
separating each of the plurality of antibodies in the mixture using hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC), wherein a molecular weight of each antibody in the mixture is within 15 kDa of a molecular weight of each other antibody in the mixture;
quantitating an amount of each antibody in the mixture; and
generating a chromatograph from the HIC-HPLC, wherein for elution of each antibody in the mixture, the chromatograph shows a peak that does not overlap with other peaks in the chromatograph.

21. The method of claim 20 , wherein one or more of the plurality of antibodies are human monoclonal antibodies.

22. The method of claim 20 , wherein either a surface hydrophobicity of each antibody in the mixture is different from a surface hydrophobicity of another antibody in the mixture by more than about 0.25 units on the Kyte & Doolittle hydropathy scale, or each antibody in the mixture, when run on HIC-HPLC individually, elutes at a distinct run time from another antibody in the mixture, or both.

Assignees

• Regeneron Pharma

Inventors

• Liu Dingjiang
• Luo Lin
• Xu Long

Classifications

• C07K16/102

  Coronaviridae (F) · CPC title

• G01N2030/8831

  involving peptides or proteins · CPC title

• G01N2030/027

  Liquid chromatography · CPC title

• G01N33/6854

  Immunoglobulins · CPC title

• G01N30/88 Primary

  Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 · CPC title