Methods for quantitating individual antibodies from a mixture

1. A method of quantitating a first antibody and a second antibody in a mixture, the method comprising:

separating the first antibody from the second antibody by using hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC);

quantitating an amount of the first antibody;

quantitating an amount of the second antibody;

wherein each of the first antibody and the second antibody:

elutes at a distinct run time from other antibodies in the mixture, when individually run on HIC-HPLC; and

is a bispecific antibody.

2. The method of claim 1 , wherein the first antibody binds to two or more proteins selected from the group consisting of: EGFR, CD28, PSMA, PD-1, CD3, CD20, and LAG3.

3. The method of claim 2 , wherein the second antibody binds to two or more target proteins selected from the group consisting of: EGFR, CD28, PSMA, PD-1, CD3, CD20, and LAG3.

4. The method of claim 1 , wherein the first antibody, the second antibody, or both, includes a first antigen-binding arm that specifically binds CD20, and a second antigen-binding arm that specifically binds CD3.

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

6. The method of claim 1 , wherein the first antibody and the second antibody are human monoclonal antibodies.

7. The method of claim 1 , wherein the first antibody and the second antibody have protein sequences that are at least 90% homologous; or

the first antibody and the second antibody have protein structures that are at least 90% homologous, as determined by their protein sequences.

8. The method of claim 1 , wherein the mixture is a co-formulated drug product.

9. The method of claim 1 , wherein the co-formulated drug product further comprises a third antibody that binds to one or more molecules selected from the group consisting of: GITR, LAG3, PD-1, PD-L1, EGFR, CD28, PSMA, CD3, and CD20.

10. A method of quantitating antibodies from a co-formulated drug product comprising a plurality of antibodies, the method comprising:

generating a standard curve for each antibody of the plurality of antibodies;

separating each antibody of the plurality of antibodies by using hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC), wherein each antibody of the plurality of antibodies binds to one or more proteins selected from the group consisting of: GITR, LAG3, PD-1, PD-L1, EGFR, CD28, PSMA, CD3, and CD20; and

after separating each antibody, quantitating an amount of each antibody using the standard curve.

11. The method of claim 10 , wherein one or more antibodies of the plurality of antibodies is a bispecific antibody.

12. The method of claim 10 , wherein each antibody elutes at a distinct run time from other antibodies of the plurality of antibodies, when individually run on HIC-HPLC.

13. The method of claim 10 , wherein the plurality of antibodies comprises three human monoclonal antibodies.

14. The method of claim 10 , wherein each antibody of the plurality of antibodies, has a weight within 15 kDa of every other antibody of the plurality of antibodies.

15. A method of quantitating antibodies in a mixture comprising a plurality of antibodies, the method comprising:

generating a standard curve for each antibody of the plurality of antibodies;

separating each antibody of the plurality of antibodies by using hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC);

generating a chromatograph from the HIC-HPLC; and

quantitating an amount of each antibody of the plurality of antibodies;

wherein each antibody elutes at a distinct run time from other antibodies of the of antibodies, when individually run on HIC-HPLC; and

wherein at least one antibody of the plurality of antibodies is a bispecific antibody.

16. The method of claim 15 , wherein the bispecific antibody comprises a first antigen-binding arm that specifically binds CD20, and a second antigen-binding arm that specifically binds CD3.

17. The method of claim 16 , wherein at least one other antibody of the plurality of antibodies binds to GITR, LAG3, PD-1, PD-L1, EGFR, PSMA, or CD28.

18. The method of claim 15 , wherein a first antibody, of the plurality of antibodies, binds to a first epitope of an antigen; and a second antibody, of the plurality of antibodies, binds to a second epitope of the antigen.

19. The method of claim 15 , wherein the first antibody and the second antibody are human monoclonal antibodies;

the first antibody has a first weight, the second antibody has a second weight, and the first weight differs from the second weight by less than 15 kDa; and

the first antibody and the second antibody have protein sequences that are at least 90% homologous.

20. The method of claim 15 , further comprising, prior to generating a chromatograph, determining a surface hydrophobicity of each antibody of the plurality of antibodies, of the plurality of antibodies, wherein the surface hydrophobicity 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).

21. The method of claim 20 , wherein the surface hydrophobicity is a calculated surface hydrophobicity based on protein structure or structural model, and a surface hydrophobicity of each antibody of the plurality of antibodies is different from a surface hydrophobicity of another antibody in the mixture by more than about 1.0 unit on the Kyte & Doolittle hydropathy scale.

22. The method of claim 15 , wherein, for each antibody of the plurality of antibodies, the chromatograph shows a peak that does not overlap with other peaks in the chromatograph.