Multiplexed total antibody and antibody-conjugated drug quantification assay

A method of quantifying both an antibody-conjugated drug concentration and a total antibody concentration of an antibody-drug conjugate (ADC) in a single analysis sample, the method comprising:

• (a) contacting the ADC comprising an antibody, a drug moiety, and a linker moiety that attaches the antibody to the drug moiety, wherein the linker moiety of the ADC is a disulfide linker, and which is bound to an affinity capture media selected from the group consisting of:
• (1) bead- or resin-supported Protein A/G, and (2) target antigen-paramagnetic bead capture media, with (i) a reductant that reduces the antibody portion of the ADC to form a denatured antibody portion of the ADC, wherein the reductant is selected from the group consisting of dithiolthreitol (DTT), 2-mercaptoethanol, and tris(2-carboxyethyl)phosphine (TCEP), whereby the drug moiety is separated from the denatured antibody portion of the ADC;
• (ii) at least one denaturant selected from the group consisting of formamide, dimethylformamide, acetonitrile, SDS, urea, an acid labile surfactant, a decyl furanyl sulfonic acid salt, and guanidine HCl;
• and (iii) at least one chemical selected from the group consisting of methanol, ethanol, HCl, ammonium bicarbonate, Tris buffer, HEPES, ammonium acetate, and acetonitrile;
• whereby the antibody portion of the ADC is denatured;
• (b) digesting the denatured antibody portion of the ADC bound to the affinity capture media to form a digested ADC peptide mixture from the single analysis sample with a proteolytic enzyme selected from the group consisting of trypsin, chymotrypsin, papain, pepsin, LysN, LysC, AspN, GluC, ArgC, and PNGaseF;
• and
• (c) eluting from the affinity capture media and analyzing the digested ADC peptide mixture by LC-MS/MS to detect at least one antibody signature peptide and the drug moiety in the single analysis sample.