Dual wavelength isoelectric focusing for determining drug load in antibody drug conjugates

A method for determining a number of specific binding pair members bound to a target specific binding pair member in a sample comprising at least one specific binding pair, comprising:

• performing free solution isoelectric focusing on the sample in a capillary tube to obtain a focused sample;
• measuring an absorbance of the focused sample over a range of isoelectric points at a plurality of wavelengths;
• comparing a first absorbance value associated with a first wavelength from the plurality of wavelengths at an isoelectric point to a second absorbance value associated with a second wavelength from the plurality of wavelengths at the isoelectric point, the second wavelength different from the first wavelength, the isoelectric point corresponding to a pI value of the specific binding pair;
• and
• determining the number of specific binding pair members bound to each target specific binding pair member in the at least one specific binding pair based on the comparison.

A method for determining a number of drug molecules in at least two ADC subpopulations in a sample comprising at least one ADC species population, comprising:

• performing free solution isoelectric focusing on the sample to obtain a focused sample;
• measuring absorbance of the focused sample over a range of isoelectric points at two different wavelengths;
• comparing the absorbance values at the two different wavelengths at at least two corresponding pI values, wherein the at least two corresponding pI values are the pIs of the at least two ADC subpopulations;
• and
• determining the number of drug molecules conjugated to each antibody in each of the at least two ADC subpopulation based on the comparison.