Methods of assessing antibody-drug conjugates

Abstract

The present disclosure provides methods of assessing DAR of ADC products that provide advantages over known methods. Specifically, methods of the disclosure can be used in high-throughput applications and/or without having to dilute ADC samples during the assessment.

What is claimed: 
     
         1 . A method of determining a ratio of drug to antibody (DAR) in a sample comprising an antibody-drug conjugate, comprising:
 placing the sample in a vessel;   contacting a probe with the bottom of the vessel;   moving the probe from the bottom of the vessel through the sample by a predetermined increment such that a preselected path length through the solution is obtained;   detecting absorbance of the sample at a first light wavelength (λ1), wherein the first wavelength is a predetermined absorbance maxima of the antibody;   repeating steps of moving the probe relative to the sample and taking a measurement at the first wavelength;   generating a regression line from the absorbance at the first wavelength and path length such that a slope of the regression line is obtained;   determining the concentration of the antibody by dividing the slope of the regression line by an extinction coefficient of the antibody at the first wavelength;   detecting absorbance of the sample at a second light wavelength (λ2), wherein the second wavelength is a predetermined absorbance maxima of the drug;   repeating steps of moving the probe relative to the sample and taking a measurement at the second wavelength;   generating a regression line from the absorbance at the second wavelength and path length such that a slope of the regression line is obtained;   determining the concentration of the drug by dividing the slope of the regression line by an extinction coefficient of the drug at the second wavelength; and   calculating a DAR using the determined concentration of drug and the determined concentration of antibody.   
     
     
         2 . The method of  claim 1 , wherein the size exclusion chromatography comprises ultra-performance liquid chromatography (UPLC), a reversed phase (RP) UPLC, or a high performance liquid chromatography (HPLC). 
     
     
         3 . The method of  claim 1 , wherein the sample includes 0.1 μg/μL to 500 μg/μL of antibody-drug-conjugate (ADC). 
     
     
         4 . The method of  claim 1 , wherein the antibody is an anti-tumor antibody. 
     
     
         5 . The method of  claim 1 , wherein the drug is a pro-apoptotic, cytostatic or cytotoxic agent.